branched and linear mono-C20,22,24 and di-C10-C13 alkyl benzenesulfonic acids and petroleum sulphonic acids, barium salts

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 July 2012 to 02 August 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD, EU, US EPA and JMAFF test guidelines in compliance with GLP and reported with a valid GLP certificate.

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)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

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.

GLP compliance:

yes (incl. QA statement)

Type of study:

Buehler test

Justification for non-LLNA method:

The LLNA test method was not suitable for the test article.

Test material

Test material information

Constituent 1

Reference substance name:

branched and linear mono-C20,22,24 and di-C10-C13 alkyl benzenesulfonic acids and petroleum sulphonic acids, barium salts

EC Number:

701-461-7

Molecular formula:

BaC32H50S2O6 to BaC104H186S2O6 General series and/or BaC66H98S4O6 to BaC84H142S4O6 Dibenzothiophene series

IUPAC Name:

branched and linear mono-C20,22,24 and di-C10-C13 alkyl benzenesulfonic acids and petroleum sulphonic acids, barium salts

Constituent 2

Reference substance name:

Benzenesulfonic Acid, di C10-18-alkyl derivs barium salts, neutral, in diluents oil and TBN = <10

IUPAC Name:

Benzenesulfonic Acid, di C10-18-alkyl derivs barium salts, neutral, in diluents oil and TBN = <10

Test material form:

liquid: viscous

Details on test material:

Test substance information
Identification: 93820-55-4 Benzenesulfonic Acid, di C10-18-alkyl derivs barium salts, neutral, in diluents oil and TBN = <10
Molecular formula: UVCB
Molecular weight: UVCB
CAS Number: 93820-55-4
Description: Clear brown viscous liquid (determined at WIL Research Europe)
Batch: Not indicated
Purity/Composition: UVCB 100%
Test substance storage: At room temperature in the dark
Stability under storage conditions: Stable
Expiry date: 31 May 2013

Study specific test substance information
Hygroscopic: No
Volatile: No
Stability in vehicle:
Kaydol White Mineral Oil: Stable
Solubility in vehicle:
Kaydol White Mineral oil: Miscible

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

Test system
Species: Albino guinea pig, Dunkin Hartley strain, (SPF-quality). Recognized by international guidelines as the recommended test system (e.g. OECD, EC).
Source: Charles River Deutschland, Kisslegg, Germany.
Number of animals
Experimental group: 20 females.
Control group: 10 females.
(females were nulliparous and non-pregnant).
Age: Young adult animals (approx. 5 weeks old) were selected.
Identification: Ear tattoo.
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 skin to be treated, which was intact and free from any abnormality.

Animal husbandry
Conditions: Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.
Accommodation: Group housing of maximally 5 animals per labeled 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.
The acclimatization period was at least 5 days before the start of treatment under laboratory conditions.
Diet: Complete breeding diet for guinea pigs (SSNIFF® MS-Z, V2273; SSNIFF® Spezialdiäten GmbH, Soest, Germany). Hay (TecniLab-BMI BV, Someren, The Netherlands) was provided at least twice a week.
Water: Free access to tap water.
Results of analysis for diet, sawdust, shelters and water were assessed and did not reveal any findings that were considered to have affected the study integrity. All certificates and results of analysis are retained in the WIL Research Europe archives.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

epicutaneous, occlusive

Vehicle:

other: Kaydol White Mineral Oil

Induction 2

Concentration / amount:

Induction: 0.5 mL of the undiluted test substance concentration
Challenge: undiluted test substance and the vehicle (0.1 mL of each)

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

other: Kaydol White Mineral Oil

Challenge 2

Concentration / amount:

Induction: 0.5 mL of the undiluted test substance concentration
Challenge: undiluted test substance and the vehicle (0.1 mL of each)

No. of animals per dose:

Control: 10 animals
Experimental test group: 20 animals

Details on study design:

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. The starting- and subsequent concentrations were taken from the series: 100% (undiluted), 50%, 20%, 10%, and, if needed, further lower concentrations using the same steps. 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 bodyweights 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 exposure.

Main study
INDUCTION - Experimental animals
Days 1, 8 and 15: The left side of the scapular region was clipped and subsequently epidermally treated with 0.5 mL of the undiluted test substance concentration, using Metalline patches (2x3 cm) mounted on Medical tape, which was 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 using Kaydol White Mineral Oil. Immediately after removal of the last induction application on Day 15, the treated skin area was assessed for irritation.

INDUCTION - Control animals
The control animals were treated as described for the experimental animals, except that, instead of the test substance, vehicle alone was administered.

CHALLENGE - All animals
Day 29: 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.
After 6 hours, the dressings were removed and the skin cleaned of residual test substance and vehicle using Kaydol White Mineral Oil. The treated sites were assessed for challenge reactions 24 and 48 hours after removal of the dressings.

After termination, animals were sacrificed using isoflurane and an intra-peritoneal injection of Euthasol® 20%.

Observations:
Mortality/Viability: Twice daily.
Toxicity: At least once daily.
Bodyweights: Prior to start and at termination of the study.
Necropsy: No necropsy was performed according to protocol.
Irritation: Skin reactions were graded according to the following numerical scoring systems.
Furthermore, a description of all other (local) effects was recorded. To facilitate scoring, the epidermally treated skin-areas were clipped at least 3 hours before the 48-hour reading of these areas in the preliminary irritation study and challenge phase.

Challenge controls:

The control animals were treated as described for the experimental animals, except that, instead of the test substance, vehicle alone was administered.

Positive control substance(s):

no

Results and discussion

Positive control results:

Postive control not utilised in this study.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100%

No. with + reactions:

7

Total no. in group:

20

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 100%. No with. + reactions: 7.0. Total no. in groups: 20.0.

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100%

No. with + reactions:

3

Total no. in group:

20

Clinical observations:

Scaliness in 2/20

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100%. No with. + reactions: 3.0. Total no. in groups: 20.0. Clinical observations: Scaliness in 2/20 .

Any other information on results incl. tables

Table 1: PRELIMINARY IRRITATION STUDY

SKIN REACTIONS AFTER EPIDERMAL EXPOSURE

Animal number	Conc. %	24 hours after exposure		48 hours after exposure
		Erythema	Oedema	Erythema	Oedema
		(grade)	(grade)	(grade)	(grade)
5	100	0	0	0	0
	50	0	0	0	0
6	100	0	0	0	0
	50	0	0	0	0
7	20	0	0	0	0
	10	0	0	0	0
8	20	0	0	0	0
	10	0	0	0	0

 

Table 2: INDUCTION AND CHALLENGE READINGS

Animal No	Induction		Challenge
	Day 15		Day 30		Day 31
	Readings		24 Hour Readings		48 Hour Readings
	100% #		100%	Vehicle	100%	Vehicle
	Er	Oe
Control
61	0	0	0	0	0	0
62	0	0	0	0	0	0
63	0p	0	0	0	0	0
64	0	0	0	0	0	0
65	0p	0	0	0	0	0
66	0	0	0	0	0	0
67	0	0	0	0	0	0
68	0	0	0	0	0	0
69	0	0	0	0	0	0
70	0	0	0	0	0	0
Experimental
71	1	0	0	0	0	0
72	1p	0	1	0	1	0
73	0	0	0	0	0	0
74	0	0	0	0	0	0
75	1	0	0	0	0	0
76	1	0	0	0	0	0
77	1	0	1	0	0	0
78	0	0	0	0	0	0
79	1	0	1	0	0	0
80	0	0	0	0	0	0
81	1	0	1	0	1p	0
82	1	0	1	0	0	0
83	1	0	0	0	0	0
84	0	0	0	0	0	0
85	1	0	0	0	0	0
86	1p	0	0	0	0	0
87	1p	0	1	0	1	0
88	1	0	1	0	0p	0
89	0p	0	0	0	0	0
90	1	0	0	0	0	0

#. Test substance concentration (experimental animals) or vehicle (control animals).

Er: Erythema

Oe: Oedema

p. Scaliness

Vehicle: Kaydol White Mineral Oil.

 

Table 3: BODYWEIGHTS (GRAM)

SEX/DOSE LEVEL	ANIMAL	DAY 1	DAY 31
FEMALES CONTROL
	61	320	518
	62	303	439
	63	322	502
	64	348	515
	65	332	513
	66	326	495
	67	315	475
	68	310	486
	69	301	425
	70	307	443
	MEAN	318	481
	ST.DEV.	14	34
	N	10	10
FEMALES EXPERIMENTAL
	71	325	491
	72	316	471
	73	341	518
	74	324	519
	75	310	416
	76	324	448
	77	327	497
	78	341	495
	79	295	491
	80	311	422
	81	320	447
	82	311	473
	83	306	437
	84	314	446
	85	340	495
	86	294	448
	87	303	471
	88	345	524
	89	325	468
	90	355	539
	MEAN	321	476
	ST.DEV.	17	35
	N	20	20

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The skin reactions observed in response to the undiluted test substance in seven of the twenty, (35%) experimental animals in the challenge phase were considered indicative of sensitization, based on the ≥15% criteria for evidence of skin sensitisation and the absence of any response in the control animals.

Executive summary:

Assessment of Contact Hypersensitivity to 93820-55-4 Benzenesulfonic Acid, di C10-18-alkyl derivs barium salts, neutral, in diluents oil and TBN = <10 in the Albino Guinea Pig (Buehler Test).

 

The study was carried out based on the guidelines and test method described in:

OECD No. 406 (1992), "Skin Sensitization"

EC No 440/2008; B6: "Skin Sensitization: Buehler Test”.

EPA OPPTS 870.2600 (2003) “Skin Sensitization”

JMAFF: Japanese Test Guidelines (2000) including the most recent partial revisions.

 

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 preferred sensitization test (Local Lymph Node Assay) over predicts sensitization potential of these substances

 

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 (Days 1, 8 and 15) with the undiluted test substance and ten control animals were similarly treated, but with vehicle alone (Kaydol white mineral oil). Two weeks after the last induction exposure, all animals were challenged with the undiluted test substance.

 

RESULTS: In the challenge phase, skin reactions of grade 1 were observed in seven experimental animals in response to the undiluted test substance. No skin reactions were evident in the control animals. Scaliness was seen in two of the experimental animals with grade 1 skin reactions.

 

CONCLUSION: The skin reactions observed in response to the undiluted test substance in seven of the twenty (35%) experimental animals in the challenge phase were considered indicative of sensitization, based on the ≥ 15% criteria for evidence of skin sensitisation and the absence of any response in the control animals.