Barium, 1,4-bis (C13-rich C11-14-isoalkyl) 2-sulfobutanedioate phosphate complex

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 24 August 2005 and 07 September 2005.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

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)

Remarks:

Date of Inspection: 02/12/2002. Date of Signature: 13/02/2003

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

dibarium(2+) bis(1,5-dioxo-1,5-bis(tridecyloxy)pentane-3-sulfonate) hydrogen phosphate

EC Number:

931-037-9

Molecular formula:

Molecular formula for each combination of chain length. C13,13: (C30H57O7S)2 Ba @ BaHOP4 C11,12: (C27H51O7S)2 Ba @ BaHOP4 C12,12: (C28H53O7S)2 Ba @ BaHOP4 C12,13: (C29H55O7S)2 Ba @ BaHOP4 C13,14: (C31H59O7S)2 Ba @ BaHOP4 C14,14: (C32H61O7S)2 Ba @ BaHOP4

IUPAC Name:

dibarium(2+) bis(1,5-dioxo-1,5-bis(tridecyloxy)pentane-3-sulfonate) hydrogen phosphate

Details on test material:

Identification : Barium di(bistridecylsulfosuccinate) in mixture with barium hydrogen phosphate
Description : colourless waxy solid block
Batch number : Y-T-1
Storage conditions: 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: Female CBA/Ca strain mice were supplied by B & K Universal Ltd, Hull, UK
- Age at study initiation: 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 23 g.
- Housing: The animals were individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes.
- Diet (e.g. ad libitum): Free access to food (Certified Rat and Mouse Diet (Code 5LF2) supplied by BCM IPS Limited, London, UK) was allowed throughout the study.
- Water (e.g. ad libitum): Free access to mains tap water was allowed throughout the study.
- Acclimation period: At least five days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): The temperature was controlled to remain within target ranges of 19 to 25°C.
- Humidity (%): The relative humidity was controlled to remain within target ranges of 19 to 25°C and 30 to 70%.
- Air changes (per hr): 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:

other: Butanone

Concentration:

Test material at concentrations of 10%, 25% or 50% w/w in Butanone.

No. of animals per dose:

Preliminary Screening Test - One mouse
Main Test - Groups of five mice were treated with the test material at concentrations of 10%, 25% or 50% w/w in Butanone

Details on study design:

RANGE FINDING TESTS:
Using all available information 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 25 µl of the test material at a concentration of 50% w/w in Butanone, 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.

- Compound solubility:
For the purpose of the study the test material was freshly prepared in Butanone. This vehicle was chosen as it produced the highest concentration that was suitable for dosing.

- Irritation:
Any signs of toxicity or excessive local irritation noted during this period were recorded. The bodyweight of the mouse was recorded on Day 1 (prior to dosing) and on Day 6.

- Lymph node proliferation response:
Not assessed in preliminary screening test.

See Table 1 for results of Preliminary Screening Test.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay

- 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 nodes from each individual animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index (SI)).
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 sensitiser".



TREATMENT PREPARATION AND ADMINISTRATION:
For the purpose of the study the test material was freshly prepared in Butanone. This vehicle was chosen as it produced the highest concentration that was suitable for dosing. The concentrations used are given in the procedure section.

Groups of five mice were treated with the test material at concentrations of 10%, 25% or 50% w/w in Butanone. 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, 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.
A further group of five mice received the vehicle alone in the same manner.

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:80µCi/ml, specific activity 2.0 Ci/mmol, Amersham Biosciences UK Ltd) giving a total of 20 µCi to each mouse.

Positive control substance(s):

other: alpha-Hexylcinnamaldehyde, Tech, 85%

Statistics:

Statistical Analysis:
Data was processed to give group mean values for dpm and standard deviations where appropriate. Individual and group mean dpm values were assessed for dose response relationships by analysis of homogeneity of variance followed by one way analysis of variance (ANOVA). In the event of a significant result from the ANOVA, pairwise comparisons were performed between control and treated groups. For homogenous datasets Dunnett’s Multiple Comparison test was used and for non homogenous datasets Dunnett’s T3 Multiple Comparison Method was used.
Probability values (p) are presented as follows:
P<0.001 ***
P<0.01 **
P<0.05 *
P>0.05 (not significant)

Results and discussion

Positive control results:

Methods.
Three groups, each of five animals, were treated with 50 µl (25 µl per ear) of alpha-Hexylcinnamaldehyde, Tech, 85% as a solution in acetone/olive oil (4:1 v/v) at concentrations of 5%, 10% and 25% v/v. A further control group of five animals was treated with acetone/olive oil (4:1 v/v) alone.

Results:
The Stimulation Index (SI) 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 v/v): 5
Stimulation Index (SI): 2.76
Result: Negative

Concentration (% v/v) in acetone/olive oil (4:1 v/v): 10
Stimulation Index (SI): 3.34
Result: Positive

Concentration (% v/v) in acetone/olive oil (4:1 v/v): 25
Stimulation Index (SI): 8.91
Result: Positive

Conclusion:
alpha-Hexylcinnamaldehyde, Tech, 85% was considered to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: A stimulation index of greater than 3 was recorded for the three concentrations of the test material (10%, 25% and 50% w/w in Butanone).

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: The radioactive disintegrations per minute (dpm) per lymph nodes for each individual animal and the stimulation index (SI) are given in Table 2.

Any other information on results incl. tables

Preliminary Screening Test

Clinical observations, bodyweight and mortality data are given in Table 1.

No signs of systemic toxicity were noted. Fur loss was noted pre-dose on Day 2 and for the remainder of the test.

Based on this information the dose levels selected for the main test were 10%, 25% and 50% w/w in Butanone.

Main Test

Estimation of the Proliferative Response of Lymph Node Cells

The radioactive disintegrations per minute (dpm) per lymph nodes for each individual animal and the stimulation index (SI) are given in Table2.

A stimulation index of greater than 3 was recorded for the three concentrations of the test material (10%, 25% and 50% w/w in Butanone).

Clinical Observations and Mortality Data

Individual clinical observations and mortality data for test and control animals are given in Table 3.

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. Fur loss was noted pre-dose on Day 2 and for the remainder of the test in animals treated with the test material at concentrations of 25% and 50% w/w in Butanone. Mild redness to the head and ears was noted on Days 3 and 4 in animals treated with the test material at a concentration of 10% w/w in Butanone and pre-dose on Day 3 in animals treated with the test material at a concentration of 25% and 50% w/w in Butanone. Moderate redness to the head and ears was noted in animals treated with the test material at concentrations of 25% and 50% w/w in Butanone one hour post dosing on Day 3 and on Days 4 and 5.

Bodyweight

Individual bodyweights and bodyweight changes for test and control animals are given in Table 4.

Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

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

Concentration (% w/w) in Butanone

Animal Number

Bodyweight (g)

Day

1

2

3

4

5

6

Day 1

Day 6

Pre-Dose

1HrPost Dose

Pre-Dose

1 Hr Post Dose

Pre-Dose

1 Hr Post Dose

50

S-1

20

20

0

0

0Fl

0Fl

0Fl

0Fl

0Fl

0Fl

0Fl

Hr= Hour

0= No signs of systemic toxicity

Fl= Fur loss

Table2               Individual Dpm’s and Stimulation Index (SI)

Concentration (% w/w) in Butanone	Animal Number	Dpm/ Animal^a	Mean Dpm/Animal (Standard Deviation)	Stimulation Index (SI)^b	Result
Vehicle	1-1	908.58	850.24 (±123.36)	N/A	N/A
	1-2	766.65
	1-3	784.73
	1-4	1040.65
	1-5	750.57
10	2-1	10222.18	8209.24* (±2507.70)	9.66	Positive
	2-2	11581.39
	2-3	6221.15
	2-4	6444.81
	2-5	6576.68
25	3-1	6679.59	9021.53** (±1377.50)	10.61	Positive
	3-2	8865.15
	3-3	9844.81
	3-4	9847.96
	3-5	9870.13
50	4-1	9784.88	9989.24** (±1817.25)	11.75	Positive

a= Total number of lymph nodes per animal is 2

b= Stimulation Index of 3.0 or greater indicates a positive result

N/A= Not applicable

*= Significantly different from control group p<0.05

**= Significantly different from control group p<0.01

Table3               Individual Clinical Observations and Mortality Data

Concentration (% w/w) in Butanone	Animal Number	Day 1		Day 2		Day 3		Day 4	Day 5	Day 6
		Pre-Dose	1-Hr Post Dose	Pre-Dose	1-Hr Post Dose	Pre-Dose	1-Hr 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
	1-5	0	0	0	0	0	0	0	0	0
10	2-1	0	0	0	0	0x	0x	0x	0	0
	2-2	0	0	0	0	0x	0x	0x	0	0
	2-3	0	0	0	0	0x	0x	0x	0	0
	2-4	0	0	0	0	0x	0x	0x	0	0
	2-5	0	0	0	0	0x	0x	0x	0	0
25	3-1	0	0	0Fl	0Fl	0Flx	0FlÄ	0FlÄ	0FlÄ	0Fl
	3-2	0	0	0Fl	0Fl	0Flx	0FlÄ	0FlÄ	0FlÄ	0Fl
	3-3	0	0	0Fl	0Fl	0Flx	0FlÄ	0FlÄ	0FlÄ	0Fl
	3-4	0	0	0Fl	0Fl	0Flx	0FlÄ	0FlÄ	0FlÄ	0Fl
	3-5	0	0	0Fl	0Fl	0Flx	0FlÄ	0FlÄ	0FlÄ	0Fl
50	4-1	0	0	0Fl	0Fl	0Flx	0FlÄ	0FlÄ	0FlÄ	0Fl
	4-2	0	0	0Fl	0Fl	0Flx	0FlÄ	0FlÄ	0FlÄ	0Fl
	4-3	0	0	0Fl	0Fl	0Flx	0FlÄ	0FlÄ	0FlÄ	0Fl
	4-4	0	0	0Fl	0Fl	0Flx	0FlÄ	0FlÄ	0FlÄ	0Fl
	4-5	0	0	0Fl	0Fl	0Flx	0FlÄ	0FlÄ	0FlÄ	0Fl

Hr= Hour

0= No signs of systemic toxicity

x= Mild redness to head and ears

Fl= Fur loss on head and ears

Ä= Moderate redness to head and ears

Table4               Individual Bodyweights and Bodyweight Changes

Concentration (% w/w) in Butanone	Animal Number	Bodyweight (g)		Bodyweight Change (g)
		Day 1	Day 6
Vehicle	1-1	18	18	0
	1-2	19	20	1
	1-3	20	21	1
	1-4	19	20	1
	1-5	19	19	0
10	2-1	20	20	0
	2-2	20	21	1
	2-3	19	19	0
	2-4	21	21	0
	2-5	21	22	1
25	3-1	22	21	-1
	3-2	20	21	1
	3-3	19	19	0
	3-4	20	20	0
	3-5	19	20	1
50	4-1	20	19	-1
	4-2	19	20	1
	4-3	19	19	0
	4-4	21	21	0
	4-5	19	20	1

The test material 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:

A stimulation index of greater than 3 was recorded for the three concentrations of the test material (10%, 25% and 50% w/w in Butanone).

The test material 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 the test material 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)

§        Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Directive 2004/73/EC

Methods. 

Following a preliminary screening test, three groups, each of five animals, were treated with 50 µl (25 µl per ear) of the test material as a solution in Butanone at concentrations of 10%, 25% or 50% w/w. A further group of five animals was treated with Butanone alone.

Results.

 The Stimulation Index (SI) 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 Butanone	Stimulation Index (SI)	Result
10	9.66	Positive
25	10.61	Positive
50	11.75	Positive

Conclusion. 

The test material was considered to be a sensitiser under the conditions of the test.