Bentonite, acid-leached

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Performed between 08 March 2010 and 30 March 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to GLP and internationally accepted guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Bentonite, acid-leached

EC Number:

274-324-8

EC Name:

Bentonite, acid-leached

Cas Number:

70131-50-9

Molecular formula:

unspecified for this UVCB substance

IUPAC Name:

Bentonite, acid-leached

Details on test material:

- Name of test material (as cited in study report): Bentonite acid leached
- Physical state: Solid
- Expiration date: 31 December 2010
- Storage condition of test material: Room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd, Bicester, Oxon, UK
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 15 to 23 g
- Housing: Individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes
- Diet: 2014 Tekland Global Rodent diet ad libitum
- Water: ad libitum
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 25°C
- Humidity: 30 - 70 %
- Air changes: Approximately 15 per hr
- Photoperiod: 12 hrs dark / 12 hrs light

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Remarks:

Please see below for Vehicle Determination Record

Concentration:

Groups of mice were treated with the test material at concentrations of 10%, 5% or 2.5% w/w in dimethyl sulphoxide

No. of animals per dose:

4 mice per dose

Details on study design:

PRELIMINARY SCREENING TEST:
As no information was available regarding the systemic toxicity/irritancy potential of the test material, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25µL of the test material at a concentration of 10% w/w in dimethyl sulphoxide to the dorsal surface of each ear for three consecutive days (days 1, 2, 3). The mouse was observed twice daily on days 1, 2 and 3 and once daily on days 4, 5 and 6. Any signs of toxicity or excessive local irritation noted during this period were recorded. The bodyweight was recorded on day 1 (prior to dosing) and on day 6.

MAIN STUDY
- Criteria used to consider a positive response:
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node(dpm/node) and as the ratio of 3HTdR incorporation in lymph node cells of test nodes relative to that recorded for the control nodes (stimulation Index).

The test material will be regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitier".


TREATMENT PREPARATION AND ADMINISTRATION:
For the purpose of the study, the test material was freshly prepared as a suspension in dimethyl sulphoxide. Four mice were treated by daily application of 25µL of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (days 1, 2, 3). The test material formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.

3H-Methyl Thymidine Administration:
Five days following the first topical application of the test material (Day 6) all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 µCi/mL, specific activity 2.0 Ci/mmol, GE Healthcare UK Ltd) giving a total of 20 µCi to each mouse.

Positive control substance(s):

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

Results and discussion

Positive control results:

A group of five animals was treated with 50 µL (25 µL per ear) of α Hexylcinnamaldehyde, tech., 85% as a solution in dimethylsulphoxide at a concentration of 15% v/v. A further control group of five animals was treated with dimethylsulphoxide alone.

The Stimulation Index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group was 6.07.

α Hexylcinnamaldehyde, tech., 85% was considered to be a sensitizer under the conditions of the test

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: The stimulation index is provided in table 2.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: The radioactive disintegrations per minute per lymph node are given in table 2.

Any other information on results incl. tables

Table 1: Clinical observations, bodyweight and mortality data - preliminary screening test

Concentration (%w/w) in dimethyl sulphoxide	Animal Number	Bodyweight (g)		Day
				1		2		3		4	5	6
		Day 1	Day 6	Pre-Dose	Post Dose	Pre-Dose	Post Dose	Pre-Dose	Post Dose
10	S-1	20	21	0	0	0	0	0	0	0	0	0

0 = No signs of systemic toxicity

Table 2: Disintegrations per minute, disintegrations per minute/node and stimulation index

Concentration (% w/w) in dimethyl sulphoxide	dpm	dpm/node^a	Stimulation index^b	Result
Vehicle	5305.55	663.19	na	na
2.5	7044.50	880.56	1.33	negative
5	6563.54	820.44	1.24	negative
10	5162.93	645.37	0.97	negative

dpm = disintegrations per minute

a = disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 (total number of lymph nodes)

b = Stimulation index of 3.0 or greater indicates a positive results

na = not applicable

Table 3: Individual clinical observations and mortality

Concentration (% w/w) in dimethyl sulphoxide	Animal Number	Day 1		Day 2		Day 3		Day 4	Day 5	Day 6
		Pre-Dose	Post Dose	Pre-Dose	Post Dose	Pre-Dose	Post Dose
Vehicle	1-1	0	0	0	0	0	0	0	0	0
	1-2	0	0	0	0	0	0	0	0	0
	1-3	0	0	0	0	0	0	0	0	0
	1-4	0	0	0	0	0	0	0	0	0
2.5	2-1	0	0	0	0	0	0	0	0	0
	2-2	0	0	0	0	0	0	0	0	0
	2-3	0	0	0	0	0	0	0	0	0
	2-4	0	0	0	0	0	0	0	0	0
5	3-1	0	0	0	0	0	0	0	0	0
	3-2	0	0	0	0	0	0	0	0	0
	3-3	0	0	0	0	0	0	0	0	0
	3-4	0	0	0	0	0	0	0	0	0
10	4-1	0	0	0	0	0	0	0	0	0
	4-2	0	0	0	0	0	0	0	0	0
	4-3	0	0	0	0	0	0	0	0	0
	4-4	0	0	0	0	0	0	0	0	0

0 = No signs of systemic toxicity

Table 4: Individual bodyweights and bodyweight changes

Concentration (% w/w) in dimethyl sulphoxide	Animal Number	Bodyweight (g)		Bodyweight Change (g)
		Day 1	Day 6
Vehicle	1-1	19	18	-1
	1-2	20	20	0
	1-3	21	20	-1
	1-4	20	20	0
5	2-1	19	19	0
	2-2	18	17	-1
	2-3	16	17	1
	2-4	20	19	-1
10	3-1	21	19	-2
	3-2	21	20	-1
	3-3	23	23	0
	3-4	20	20	0
25	4-1	21	19	-2
	4-2	23	21	-2
	4-3	19	17	-2
	4-4	19	18	-1

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

Bentonite acid leached is not classified as a skin sensitiser in accordance with CLP Regulation (EC) no. 1272/2008