[1,1'-Biphenyl]-2,2'-diol, 3,3'-bis(1,1-dimethylethyl)-5,5'-dimethoxy-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

The study was performed between 14 July 2009 and 29 July 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: 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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

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)

Remarks:

Date of inspection: 19/08/08 Date of signature: 04/03/09

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

L17_Precursor

IUPAC Name:

L17_Precursor

Details on test material:

Sponsor's identification : L17_Precursor
Description : pale beige powder
Batch number : DALA046312
Date received : 11 November 2008
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:
Harlan Laboratories UK Limited, Bicester, 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 23 g

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

- Diet (e.g. ad libitum):
ad libitum (2014 Teklad Global Rodent diet supplied by Harlan Teklad, Blackthorn, Bicester, 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:

dimethylformamide

Concentration:

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

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

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 one mouse. The mouse was treated by daily application of 25 µl of the test material at a concentration of 10% 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. 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.

- Lymph node proliferation response:
Clinical observations, bodyweight and mortality data are give in the results section (table 1).
The preliminary screening test suggested that the test material would not produce systemic toxicity or excessive local irritation at the highest suitable concentration.

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 (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).
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:
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.

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

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

Concentration (% v/v) in
dimethyl formamide Stimulation Index Result
15 4.24 Positive

Conclusion. α Hexylcinnamaldehyde was considered to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: 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

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

Concentration (%w/w) in dimethyl formamide		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		19		20		0		0		0		0		0		0		0		0		0

0=      No signs of systemic toxicity

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

Concentration (%w/w) in dimethyl formamide	dpm	dpm/Node^a	Stimulation Index^b	Result
Vehicle	3931.44	491.43	na	na
2.5	6365.39	795.67	1.62	Negative
5	-	-	-	-
10	8666.48	1083.31	2.20	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

-=      Due to a technician error the lymph node cells were dropped

Table3              Individual Clinical Observations and Mortality Data

Concentration (% w/w) in dimethyl formamide		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

Table4              Individual Bodyweights and Bodyweight Changes

Concentration (% w/w) in dimethyl formamide	Animal Number	Bodyweight (g)		Bodyweight Change (g)
		Day 1	Day 6
Vehicle	1-1	23	22	-1
	1-2	18	18	0
	1-3	20	20	0
	1-4	19	18	-1
2.5	2-1	18	19	1
	2-2	21	20	-1
	2-3	22	20	-2
	2-4	21	21	0
5	3-1	20	18	-2
	3-2	19	19	0
	3-3	19	21	2
	3-4	19	19	0
10	4-1	20	22	2
	4-2	21	19	-2
	4-3	21	20	-1
	4-4	20	19	-1

 

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

The test material was considered to be a non-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 CommissionRegulation (EC) No. 440/2008

Methods. 

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 10% 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 dimethyl formamide at concentrations of 10%, 5% or 2.5% w/w. A further group of four animals was treated with dimethyl formamide 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 dimethyl formamide Stimulation Index Result 2.5 1.62 Negative 5 - - 10 2.20 Negative

Conclusion. 

The test material was considered to be anon-sensitiserunder the conditions of the test.

- = Due to a technician error the lymph node cells were dropped