Reaction mass of N,N’-bis-(3-methoxypropyl)-C22(branched)-alkyldiamide and N,N’-bis-(3-methoxypropyl)-C17(branched)-alkyldiamide

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 08 October 2007 and 08 November 2007 .

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

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:

Reaction mass of N,N’-bis-(3-methoxypropyl)-C22(branched)-alkyldiamide and N,N’-bis-(3-methoxypropyl)-C17(branched)-alkyldiamide

EC Number:

940-953-8

Molecular formula:

not applicable UVCB

IUPAC Name:

Reaction mass of N,N’-bis-(3-methoxypropyl)-C22(branched)-alkyldiamide and N,N’-bis-(3-methoxypropyl)-C17(branched)-alkyldiamide

Test material form:

other: off white waxy solid block

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: One female CBA/Ca (CBA/Ca CruBR) strain mouse was supplied by Charles River UK Limited and twenty female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Harlan UK Limited.
- Age at study initiation: Eight to twelve weeks old.
- Weight at study initiation: 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) 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): Set to achieve limits of 19 to 25°C.
- Humidity (%): Set to achieve limits of 30 to 70%.
- Air changes (per hr): The rate of air exchange was at least fifteen changes per hour
- Photoperiod (hrs dark / hrs light): Lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

Preliminary Screening Test: 50% w/w
Main Test: 50%, 25% and 10% w/w.

No. of animals per dose:

Main Test: Five mice per group.

Details on study design:

PREPARATION OF TEST MATERIAL:
The test material was freshly prepared in dimethyl formamide, aided by warming in a water bath at 37°C. This vehicle was chosen as it produced the highest concentration that was suitable for dosing.

PROCEDURE:
PRELIMINARY SCREENING TEST:
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 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 . 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.

MAIN TEST:
TEST MATERIAL ADMINISTRATION:
Groups of five mice were treated with the test material at concentrations of 50%, 25% or 10% 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 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: 80 µCi/ml, specific activity 2.0 Ci/mmol) giving a total of 20 µCi to each mouse.

OBSERVATIONS:
Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.

Body Weights: The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

TERMINAL PROCEDURES:
Termination:
Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation. For each individual animal of each group the draining auricular lymph nodes were excised and processed. For each individual animal 1 ml of PBS was added to the lymph nodes.

Preparation of Single Cell Suspension:
A single cell suspension of pooled lymph node cells for each individual animal was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labelled with the study number and dose concentration. The lymph node cell suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The lymph node cells were pelleted at 1400 rpm (approximately 190 g) for ten minutes. The pellet was re-suspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was re-suspended in 3 mL of 5% Trichloroacetic acid (TCA).

Determination of 3HTdR Incorporation:
After approximately eighteen hours incubation at approximately 4°C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, re-suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid (Optiphase 'Trisafe'). 3HTdR incorporation was measured by beta-scintillation counting. The "Poly Q" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately twenty minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately twenty minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system.

Positive control substance(s):

other: α-Hexylcinnamaldehyde, Tech, 85%

Statistics:

Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships by analysis of homogeneity of variance followed by one way analysis of variance (ANOV A). 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:

Three groups, each of five animals, were treated with 50 µL (25 µL per ear) of α-Hexylcinnamaldehyde, tech, 85% as a solution in dimethyl formamide at concentrations of 5%, 10% or 25% v/v. A further group of five animals were treated with dimethyl formamide alone.

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 dimethyl formamide: 5%
Stimulation Index: 2.11
Result: Negative

Concentration (%) v/v in dimethyl formamide: 10%
Stimulation Index: 2.82
Result: Negative

Concentration (%) v/v in dimethyl formamide: 25%
Stimulation Index: 6.37
Result: Positive

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

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: A Stimulation Index of less than 3 was recorded for the three concentrations of the test material.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: See any other information on results.

Any other information on results incl. tables

Preliminary Screening Test:

There was no mortality and no signs of systemic toxicity were noted.

Main Test:

Estimation of the Proliferative Response of Lymph Node Cells

The radioactive disintegrations per minute per lymph nodes for each individual animal and the stimulation index are given in the table below.

Individual Disintegrations per Minute and Stimulation Indices

Concentration (% w/w) in dimethyl formamide	Animal Number	Dpm/Animal^a	Mean dpm/Animal (Standard Deviation)	Stimulation Index^b	Result
Vehicle	1-1	710.42	794.35 (±81.14)	N/A	N/A
	1-2	837.83
	1-3	842.20
	1-4	703.80
	1-5	877.49
10	2-1	1219.66	866.20 (±355.27)	1.09	Negative
	2-2	547.40
	2-3	486.45
	2-4	845.66
	2-5	1231.82
25	3-1	1010.83	1025.11 (±84.61)	1.29	Negative
	3-2	1078.91
	3-3	924.00
	3-4	973.99
	3-5	1137.82
50	4-1	716.75	1037.48 (±625.67)	1.31	Negative
	4-2	948.14
	4-3	2108.94
	4-4	916.87
	4-5	496.69

 

dpm = Disintegrations per minute

a = Total number of lymph nodes per animal is 2

b = Stimulation of 3.0 or greater indicates a positive result

N/A = Not applicable

Clinical Observations and Mortality Data

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

Bodyweight:

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.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

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 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 dimethyl formamide at concentrations of 50%, 25% or 10% w/w. A further group of five 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
10	1.09	Negative
25	1.29	Negative
50	1.31	Negative

Conclusion.

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