Reaction products of bis(4-methylpentan-2-yl)dithiophosphoric acid with phosphorus oxide, propylene oxide and amines, C12-14-alkyl (branched)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 05 May 2009 and 27 May 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in accordance with recognised guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2009

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

Details on test material:

- Name of test material (as cited in study report):
- Physical state: pale yellow viscous liquid (4°C)
- Lot/batch No.:

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Strain: CBA/Ca (CBA/CaOlaHsd)
- Source: Harlan UK Limited, Bicester, Oxon, UK
- Age at study initiation: 8 to 12 weeks
- Weight at study initiation: 15 to 23 g
- Housing: Individually housed in suspended solid floor polypropylene cages furnished with softwood flakes.
- Diet (e.g. ad libitum): 2014 Teklad global rodent diet, ad libitum
- Water (e.g. ad libitum):mains tap water, ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): 30 to 70 %
- Air changes (per hr): at least 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours continuous light/dark cycle


IN-LIFE DATES: No data available

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

10%, 25%, 50%

No. of animals per dose:

4 mice per dose.

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: the substance was considered suitable for dosing in acetone/olive oil (4:1)at a concentration of 50% as upon mixing by vortex mixer the formulation formed a solution which could be administered by micropipette.
- Irritation: None noted
- Lymph node proliferation response: Expressed as the number of radioactive disintegrations per minute per lymph nodes from each individual animal and as the ration of ^3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: “Skin Sensitisation: Local Lymph Node Assay” / “Skin Sensitisation (Local Lymph Node Assay of Commission directive 2004/73/EC”
- Criteria used to consider a positive response: 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.


TREATMENT PREPARATION AND ADMINISTRATION:
For the purpose of the study, the test material was freshly prepared as a solution in acetone/olive oil 4:1. This vehicle was chosen as it produced the highest concentration that was suitable for dosing. (50%)
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 Guideline.

Positive control substance(s):

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

Statistics:

The EC3 value was interpreted using GraphPad PRISM version 3.03.

Results and discussion

Positive control results:

Concentration v/v in acetone/olive oil 4:1: 15%
Stimulation Index: 8.34
Result: Positive

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

3.14

Test group / Remarks:

10 % test item in acetone/olive oil 4:1

Results 2

Parameter:

SI

Value:

7.94

Test group / Remarks:

25 % test item in acetone/olive oil 4:1

Results 3

Parameter:

SI

Value:

19.03

Test group / Remarks:

50 % test item in acetone/olive oil 4:1

Any other information on results incl. tables

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

Concentration (% w/w) in Acetone/olive oil 4:1	dpm	dpm/Node^a	Stimulation Index^b	Result
Vehicle	9376.99	1172.12	na	na
10	29437.64	3679.71	3.14	Positive
25	74447.31	9305.91	7.94	Positive
50	178410.2	22301.28	19.03	Positive

 

Dpm = disintegrations per minutes

a = total number of lymph nodes per animals is 2

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

N/A = Not Applicable

 

Individual clinical Observations and Mortality Data

 

Conc (%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
	1-5	0	0	0	0	0	0	0	0	0
10	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
	2-5	0	0	0	0	0	0	0	0	0
25	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
	3-5	0	0	0	0	0	0	0	0	0
50	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
	4-5	0	0	0	0	0	0	0	0	0

 

0 = No signs of systemic toxicity

 

 

Individual Bodyweights and Bodyweight Changes

 

Conc (%v/v) in acetone/olive oil 4:1	Animal Number	Bodyweight (g)		Bodyweight Change (g)
		Day 1	Day 6
Vehicle	1-1	20	20	0
	1-2	19	19	0
	1-3	21	20	-1
	1-4	20	21	0
10	2-1	20	20	0
	2-2	20	20	0
	2-3	22	23	1
	2-4	20	19	-1
25	3-1	18	17	-1
	3-2	18	18	0
	3-3	22	21	-1
	3-4	18	17	-1
50	4-1	19	18	-1
	4-2	20	19	-1
	4-3	18	17	-1
	4-4	21	20	-1

 

 

 

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information

Conclusions:

In a Local Lymph Node Assay in the Mouse, the substance initiated a Stimulation Index of greater than 3 at concentrations of 10, 25 and 50% and thus is considered to be a skin sensitizer under the conditions of the test.

Executive summary:

Introduction. A study was performed to assess the skin sensitization 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 OECD 429 and Method B42.

Methods. Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration 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 material as a solution in acetone/olive oil 4:1 at concentrations of 50%, 25% or 10% w/w. 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 (% w/w) in acetone/olive oil 4:1	Stimulation Index	Result
10	3.14	Positive
25	7.94	Positive
50	19.03	Positive

 

According to the calculation the concentration of test material expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was found to be 9.39%.

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