Paraffin waxes and Hydrocarbon waxes, chloro, sulfochlorinated

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 performed between 04 May 2010 and 08 June 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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:

SULFOCLOR LF 1013

IUPAC Name:

SULFOCLOR LF 1013

Constituent 2

Reference substance name:

Paraffin waxes and Hydrocarbon waxes, chloro, sulfochlorinated

EC Number:

269-145-7

EC Name:

Paraffin waxes and Hydrocarbon waxes, chloro, sulfochlorinated

Cas Number:

68188-19-2

Molecular formula:

CnHxCly(SO2Cl)z n= 14 - 17 or n=17 - 30 ; x=2n+2-y-z

IUPAC Name:

Paraffin waxes and Hydrocarbon waxes, chloro, sulfochlorinated

Details on test material:

Sponsor's identification : SULFOCLOR LF 1013
CAS No : 68188-19-2
Description : pale yellow slightly viscous liquid
Purity : >99%
Batch number : not supplied
Label : Sulfoclor LF 1013 TANQUE 25.09.2009
Date received : 05 February 2010
Expiry date : 23 September 2010
Storage conditions : room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/Ca (CBA/CaOlaHsd)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source:
Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Harlan UK Limited, Oxon, UK.

- Age at study initiation:
At the start of the study the animals were eight to twelve weeks old.

- Weight at study initiation:
At the start of the study the animals were in the weight range of 15 to 23g.

- Housing:
The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.

- Diet (e.g. ad libitum):
ad libitum (2014 Teklad Global Rodent diet supplied by Harlan UK Limited, Oxon, UK)

- Water (e.g. ad libitum):
ad libitum.

- Acclimation period:
At least five days.


ENVIRONMENTAL CONDITIONS

- Temperature (°C):
The temperature was controlled to remain within the target ranges of 19 to 25 deg C.

- Humidity (%):
The humidity was controlled to remain within the target ranges of 30 to 70%.

- Air changes (per hr):
The rate of air exchange was approximately fifteen changes per hour.

- Photoperiod (hrs dark / hrs light):
The lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.

IN-LIFE DATES:
From: Day 1 To: Day 6

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Remarks:

Please see below for Vehicle Determination Record

Concentration:

Each group was exposed to concentrations of 100% (undiluted), 50% or 25% v/v (in acetone/olive oil 4:1)

No. of animals per dose:

Groups of four mice were treated

Details on study design:

RANGE FINDING TESTS:
Using available information regarding the systemic toxicity/irritancy potential of the test material, a preliminary screening test was performed using two mice. The mice were treated by daily application of 25 µl of the undiluted test material or the test material at a concentration of 50% v/v in acetone/olive oil 4:1, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mice were 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 of each mouse was recorded on Day 1 (prior to dosing) and on Day 6.

- Lymph node proliferation response:
Clinical observations, bodyweight and mortality data are give in the results section (table 1).

No signs of systemic toxicity were noted.

Based on this information the dose levels selected for the main test were 25% and 50% v/v in acetone/olive oil 4:1 and 100%.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT

- Name of test method:
Local Lymph Node Assay in the Mouse. The assay has undergone extensive inter-laboratory validation and has been shown to reliably detect test materials that are moderate to strong sensitisers.

- 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 used undiluted and also freshly prepared in acetone/olive oil 4:1. This vehicle was chosen as it produced the most suitable formulation at the required concentration. The concentrations used are given above.

Determination, by analysis, of the concentration, homogeneity and stability of the test material preparations was not appropriate because it was not specified in the Study Plan and is not a requirement of the Test Guidelines.

The preliminary screening test suggested that the test material would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. The mice were treated by daily application of 25 µl of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (Days 1, 2 and 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)

Statistics:

None provided.

Results and discussion

Positive control results:

Introduction.

A study was performed to assess the sensitivity of the strain of mouse used at these laboratories to a known sensitiser. The methodology for the LLNA is detailed in the OECD Guideline for the Testing of Chemicals, No. 429, and Method B.42 of Commission Regulation (EC) No. 440/2008. The study described in this document is based on these test methods but has been refined in order to reduce the number of animals required. The reduced LLNA (rLLNA) has been endorsed by the non Commission members of the European Centre for the Validation of Alternative Methods (ECVAM) Scientific Advisory Committee (ESAC) at its 26th meeting held on 26 – 27 April 2007 at ECVAM, Ispra, Italy.

Test Material: α Hexylcinnamaldehyde, tech., 85%
Project number: 0039/1120
Study dates: 05 November 2009 to 11 November 2009

Methods.

A group of five animals was treated with 50 µl (25 µl per ear) of α Hexylcinnamaldehyde, tech., 85% as a solution in acetone/olive oil 4:1 at a concentration of 15% v/v. A further control group of five animals was treated with acetone/olive oil 4:1 alone.
Results. The Stimulation Index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group is as follows:

Concentration (% v/v) in
acetone/olive oil 4:1 Stimulation Index Result
15 3.12 Positive

Conclusion.

α Hexylcinnamaldehyde, tech., 85% was considered to be a sensitiser under the conditions of the test.
For full details please see Appendix1  Current Positive Control Study for the Local Lymph Node Assay in any other details on result section.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: A stimulation index of greater than 3 was recorded for the undiluted test material and the test material at concentrations of 50% and 25% v/v in acetone/olive oil 4:1. The stimulation index (SI) results are given in Table 2.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: The radioactive disintegrations per minute (dpm) per lymph node and the stimulation index (SI) are given in Table 2.

Any other information on results incl. tables

Interpretation of Results.

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).

Table1              Clinical Observations, Bodyweight and Mortality Data – Preliminary Screening Test

Concentration (%v/v) in acetone/olive oil 4:1	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
100	S-1	19	20	0	0	0	0	0	0*	0*	0	0
50	S-2	19	19	0	0	0	0	0	0	0*	0*	0

0=      No signs of systemic toxicity

* =       Mild redness on the ears and neck

Table2              Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration (%v/v) in acetone/olive oil 4:1	dpm	dpm/Node^a	Stimulation Index^b	Result
Vehicle	8267.98	1033.50	na	na
25	121085.30	15135.66	14.65	Positive
50	128441.10	16055.14	15.53	Positive
100	138355.90	17294.49	16.73	Positive

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

Table3              Individual Clinical Observations and Mortality Data

Concentration (% v/v) in acetone/olive oil 4:1	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
25	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
50	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+
100	4-1	0	0	0	0	0	0	0+	0+	0+Fl
	4-2	0	0	0	0	0	0	0+	0+	0+Fl
	4-3	0	0	0	0	0	0	0+	0+	0+Fl
	4-4	0	0	0	0	0	0	0+	0+	0+Fl

0=      No signs of systemic toxicity

+ =      Mild redness to the head, neck and ears

Fl =     Fur loss

Table4              Individual Bodyweights and Bodyweight Changes

Concentration (% v/v) in acetone/olive oil 4:1	Animal Number	Bodyweight (g)		Bodyweight Change (g)
		Day 1	Day 6
Vehicle	1-1	20	18	-2
	1-2	19	20	1
	1-3	20	20	0
	1-4	19	19	0
25	2-1	21	20	-1
	2-2	20	18	-2
	2-3	21	20	-1
	2-4	21	20	-1
50	3-1	18	18	0
	3-2	21	20	-1
	3-3	21	21	0
	3-4	20	21	1
100	4-1	19	20	1
	4-2	19	20	1
	4-3	18	18	0
	4-4	21	19	-2

Appendix 1      Current Positive Control Study for the Local Lymph Node Assay

Introduction. 

A study was performed to assess the sensitivity of the strain of mouse used at these laboratories to a known sensitiser. The methodology for the LLNA is detailed in the OECD Guideline for the Testing of Chemicals, No. 429, and Method B.42 of CommissionRegulation (EC) No. 440/2008. The study described in this document is based on these test methods but has been refined in order to reduce the number of animals required. The reduced LLNA (rLLNA) has been endorsed by the non-Commission members of the European Centre for the Validation of Alternative Methods (ECVAM) Scientific Advisory Committee (ESAC) at its26^thmeeting held on 26 – 27 April 2007 at ECVAM,Ispra, Italy.

Test Material:               α-Hexylcinnamaldehyde, tech., 85%

Project number:           0039/1120

Study dates:                 05 November 2009 to 11 November 2009

Methods. 

A group of five animals was treated with 50 µl (25 µl per ear) ofα‑Hexylcinnamaldehyde, tech., 85% as a solution in acetone/olive oil 4:1ata concentration of 15% v/v. A further control group of five animals was treated withacetone/olive oil 4:1alone.

Results. 

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

Concentration (% v/v) in acetone/olive oil 4:1	Stimulation Index	Result
15	3.12	Positive

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

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information

Conclusions:

SULFOCLOR LF 1013 was considered to be a sensitiser under the conditions of the test.

Executive summary:

Introduction. 

A study was performed to assess the skin sensitisation potential of SULFOCLOR LF 1013 in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed to meet the requirements of the following:

§        OECD Guideline for the Testing of Chemicals No.429 "Skin Sensitisation: Local Lymph Node Assay" (adopted 24 April 2002)

§        Local Lymph Node Assay) of CommissionRegulation (EC) No. 440/2008

Methods. 

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of100%, 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 undiluted test material or the test material as a solution in acetone/olive oil 4:1 at concentrations of 50% or25% v/v. A further group of four animals was treated with acetone/olive oil 4:1 alone.

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 (%v/v) in acetone/olive oil 4:1	Stimulation Index	Result
25	14.65	Positive
50	15.53	Positive
100	16.73	Positive

Conclusion.  SULFOCLORLF 1013 was considered to be a skin sensitiser under the conditions of the test.