Bis(2-hydroxyethyl) sulphone

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 26 January 2017 and 09 February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

Bis(2-hydroxyethyl) sulphone

EC Number:

219-948-3

EC Name:

Bis(2-hydroxyethyl) sulphone

Cas Number:

2580-77-0

Molecular formula:

C4H10O4S

IUPAC Name:

2-(2-hydroxyethanesulfonyl)ethan-1-ol

Test material form:

solid

Specific details on test material used for the study:

Identification: Bis(2-hydroxyethyl) Sulphone
Batch: 160226
Purity: 99.64%
Physical state/Appearance: white solid block
Expiry Date: 01 March 2017
Storage Conditions: room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Envigo RMS B.V., Inc., Horst, The Netherlands. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non pregnant. After an acclimatization period of at least 5 days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals were in the weight range of 15 to 23 g, and were 8 to 12 weeks old.

Animal Care and Husbandry:
The animals were housed in suspended solid floor polypropylene cages furnished with softwood woodflakes. Free access to mains tap water and food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study.
The temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70%, respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Remarks:

This vehicle was chosen as it produced the highest concentration that was suitable for dosing.

Concentration:

Preliminary screening test:
50 % w/w

Main test:
50, 25 or 10 % w/w

No. of animals per dose:

Preliminary screening test:
1 mouse

Main test:
4 mice per concentration, including vehicle control

Details on study design:

PRE-SCREEN TESTS:
The mouse was treated by daily application of 25 µL of the test item at a concentration of 50% w/w in dimethyl formamide, 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. Local skin irritation was scored daily. Any clinical signs of toxicity, if present, were also recorded. The body weight of the mouse was recorded on Day 1 (prior to dosing) and on Day 6.
The thickness of each ear was measured using a Mitutoyo 547 300S gauge (Mitutoyo Corporation), pre dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization.

MAIN STUDY - Local Lymph Node Assay in the Mouse - Pooled Method
Groups of four mice were treated with the test item at concentrations of 50%, 25% or 10% w/w in dimethyl formamide. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of four mice received the vehicle alone in the same manner.

3H-Methyl Thymidine Administration:
Five days following the first topical application of the test item or vehicle (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, ARC UK Ltd) giving a total of 20 µCi to each mouse.

- Criteria used to consider a positive response:
The test item will be regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non sensitizer".
The results were also evaluated according to the Globally Harmonized Classification System and to Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

Positive control substance(s):

other: α-Hexylcinnamaldehyde, tech., 85%

Statistics:

The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).

Results and discussion

Positive control results:

The stimulation index for a group of 5 mice treated with α-Hexylcinnamaldehyde, tech., 85% was 6.08, thus demonstrating the positive repsonse of this system to a known sensitizer.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.58

Test group / Remarks:

50 % w/w

Remarks on result:

other: Negative

Results 2

Parameter:

SI

Value:

1.51

Test group / Remarks:

25 % w/w

Remarks on result:

other: Negative

Results 3

Parameter:

SI

Value:

1.12

Test group / Remarks:

10 % w/w

Remarks on result:

other: Negative

Cellular proliferation data / Observations:

PRELIMINARY SCREENING TEST:
No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted.

DETAILS ON STIMULATION INDEX CALCULATION
The Stimulation Index is expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group

CLINICAL OBSERVATIONS:
There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

BODY WEIGHTS:
Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

Any other information on results incl. tables

Clinical Observations, Body Weight and Mortality Data – Preliminary Screening Test

Concentration (% w/w) in dimethyl formamide	Animal Number	Body Weight (g)		Day
				1		2		3		4	5	6
		Day 1	Day 6	Pre-Dose	Post Dose	Pre-Dose	Post Dose	Pre-Dose	Post Dose
50	S-1	18.5	17.9	0	0	0	0	0	0	0	0	0

0=    No signs of systemic toxicity

Local Skin Irritation – Preliminary Screening Test

Concentration (% w/w) in dimethyl formamide

Animal Number

Local Skin Irritation

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

left

right

left

right

left

right

left

right

left

right

left

right

50

S-1

0

0

0

0

0

0

0

0

0

0

0

0

Measurement of Ear Thickness and Mean Ear Thickness Changes – Preliminary Screening Test

Concentration (% w/w) in dimethyl formamide	Animal Number	Ear Thickness Measurement (mm)
		Day 1		Day 3		Day 6
		pre‑dose		post dose
		left	right	left	right	left	right
50	S-1	0.22	0.21	0.22	0.20	0.22	0.20
overall mean (mm)		0.215		0.210		0.210
overall mean ear thickness change (%)		na		-2.326		-2.326

na=  Not applicable

Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration (% w/w) in dimethyl formamide	dpm	dpm/Nodea	Stimulation Indexb	Result
Vehicle	5110.53	638.82	na	na
10	5702.71	712.84	1.12	Negative
25	7721.40	965.18	1.51	Negative
50	8071.59	1008.95	1.58	Negative

dpm = Disintegrations per minut

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 result

na = Not applicable

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to be a non-sensitizer under the conditions of the test.
The test item does not meet the criteria for classification according to the Globally Harmonized Classification System and to the Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

Executive summary:

Introduction

A study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

Methods

Following a preliminary screening test in which no clinical signs of toxicity were noted at aconcentration of 50% w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 µL (25 µL per ear) of the test item as asolutionindimethyl formamideat concentrations of 50%, 25% or 10% w/w. A further group offour animals was treated withdimethyl formamidealone.

Results

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (%w/w) in dimethyl formamide	Stimulation Index	Result
10	1.12	Negative
25	1.51	Negative
50	1.58	Negative

 

Conclusion

The test item was considered to be a non-sensitizer under the conditions of the test.

The test item does not meet the criteria for classification according to the Globally Harmonized Classification System and to the Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.