Reaction mass of trientine and trientine, mono- and di-propoxylated

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 conducted between 21 August 2014 and 11 September 2014.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

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 trientine and trientine, mono- and di-propoxylated

EC Number:

942-835-1

Molecular formula:

(C6 H18 N4 . C3 H6 O)x

IUPAC Name:

Reaction mass of trientine and trientine, mono- and di-propoxylated

Test material form:

other: Liquid

Details on test material:

Identification: Triethylenetetramine, propoxylated (triethylene-tetramine adduct)
Purity: not supplied
Physical state / Appearance: very pale green liquid
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:

Animals and Animal Husbandry
Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Harlan Laboratories UK Ltd., Oxon, UK. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non pregnant. After an acclimatization period of at least five days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals were in the weight range of 15 to 23 g, and were eight to twelve weeks old.

The animals were group housed in suspended solid floor polypropylene cages furnished with softwood woodflakes. Free access to mains tap water and food (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study.

The temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70%, respectively. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.

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.


Justification
Mice are the preferred species of choice since quantitative methods have been developed for the measurement of skin sensitization responses in the mouse and are specified in the appropriate test guidelines.

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

50% or 25% v/v

No. of animals per dose:

Four per dose

Details on study design:

Test Item Formulation and Experimental Preparation
For the purpose of the study, the test item was used undiluted and freshly prepared as a solution in acetone/olive oil 4:1. This vehicle was the only vehicle tried as it produced a suitable formulation at the required concentration. The concentration used is given in the procedure section.

The test item was formulated within two hours of being applied to the test system. It is assumed that the formulation was stable for this duration.

No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Procedure
Animals in which any adverse effects were noted that were considered to approach the moderate severity limit set forth in the UK Home Office Project Licence, were humanely killed.


Preliminary Screening Test
Using available information regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µL of the undiluted test, 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. Local skin irritation was scored daily according to the scale included as Appendix 5. Any clinical signs of toxicity, if present, were also recorded. The body weight was recorded on Day 1 (prior to dosing) and on Day 6.

The thickness of each ear was measured using a Mitutoyo 547 300S gauge (Mitutoyo Corporation), pre dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization.

Main Test
Test Item Administration
Groups of four mice were treated with the undiluted test item or the test item at concentrations of 50% or 25% v/v in acetone/olive oil 4:1. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item 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 item or vehicle (Day 6) all surviving 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, ARC UK Ltd) giving a total of 20 µCi to each mouse.

Observations
Clinical Observations: All surviving animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Animals that had to be humanely killed or removed from the study were observed twice daily on Days 1, 2 and 3.Any signs of toxicity or signs of ill health during the test were recorded.

Body Weights: The body weight of each surviving mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination). Animals that had to be humanely killed or removed form the study were recorded for body weights on Day 1 (prior to dosing) and Day 3 (prior to termination).


Terminal Procedures
Termination: Five hours following the administration of 3HTdR all surviving mice were killed by carbon dioxide asphyxiation. The draining auricular lymph nodes from the four mice were excised and pooled for each surviving experimental group. For each group 1 mL of PBS was added to the pooled lymph nodes.

Preparation of Single Cell Suspension: A single cell suspension of pooled lymph node cells 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 labeled 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 pooled lymph node cells were pelleted at 1400 rpm (approximately 190 g) for 10 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  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 20 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 (Beckman Instruments Inc, Fullerton, CA, USA).

Positive control substance(s):

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

Statistics:

No data

Results and discussion

Positive control 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:
At a concentration of 25 % (v/v) in acetone/olive oil (4:1) the stimulation index was 12.76 (positive)
α Hexylcinnamaldehyde, tech., 85% was considered to be a sensitizer under the conditions of the test.

In vivo (LLNA)

Results

Results 1

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: No data

Results 2

Parameter:

SI

Value:

12.76

Test group / Remarks:

At a concentration of 25 % (v/v) in acetone/olive oil (4:1)

Remarks on result:

other: see Remark

Remarks:

Positive

Results 3

Parameter:

SI

Value:

2.98

Test group / Remarks:

At a concentration of 50 % (v/v) in acetone/olive oil (4:1)

Remarks on result:

other:

Remarks:

Negative

Results 4

Parameter:

SI

Variability:

Concentration 100 (%v/v) in acetone/olive oil 4:1

Test group / Remarks:

No results were obtained with the undiluted test item as all the animals were humanely killed on Day 3 (post dose) due to the appearance of clinical observations which approached the moderate severity limit. The remaining mouse was removed from the study.

Remarks on result:

not determinable

Cellular proliferation data / Observations:

Main Test Estimation of the Proliferative Response of Lymph Node Cells 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: At a concentration of 25 % (v/v) in acetone/olive oil (4:1) the stimulation index was 3.72 (positive) At a concentration of 50 % (v/v) in acetone/olive oil (4:1) the stimulation index was 2.98 (negative) No results were obtained with the undiluted test item as all the animals were humanely killed on Day 3 (post dose) due to the appearance of clinical observations which approached the moderate severity limit. The remaining mouse was removed from the study

Any other information on results incl. tables

Preliminary Screening Test

No signs of systemic toxicity, or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted. Very slight erythema was noted on Days 3 and 4.

 

Based on this information the undiluted test material and the test material at concentrations of50% and25% v/vinacetone/olive oil 4:1were selected for the main test.

Main Test

Estimation of the Proliferative Response of Lymph Node Cells

Concentration (%v/v) in acetone/olive oil 4:1	Stimulation Index	Result
25	3.72	Positive
50	2.98	Negative
100	*	*

*=   No results were obtained with the undiluted test item as all the animals were humanely killed on Day 3 (post dose) due to the appearance of clinical observations which approached the moderate severity limit. The remaining mouse was removed from the study.

Clinical Observations and Mortality Data

The animals treated with the undiluted test material were humanely killed, on Day 3, due to the occurrence of clinical signs of toxicity that exceededthe moderate severity limit set forth in the UK Home Office Project Licence or removed from the study due to insufficient animals to produce a Stimulation Index. 

 

There were no deaths or any signs of systemic toxicity noted in the surviving test or control animalsduring the test. Fur loss was noted on Days 3 to 6 in some of the animals treated with the test item at concentrations of 25% and 50% v/v in acetone /olive oil 4:1 and a translucent viscous residual test material was noted on all the test animals on Day 3. 

 

 

Body Weight

Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

 

Ear Thickness Measurements and Ear Thickness Changes, Local Skin Irritation

There was no increase in ear thickness (≥25%) in any of the surviving test or control animals on Days 3 and 6. A greater than 25% mean increase in ear thickness measurements was observed on Day 3 with animals treated with the undiluted test item prior to termination. 

 

Very slight erythema was noted on Days 2 to 4 with all animals treated at a concentration of 25% and Days 2 to 5 with all animals treated at a concentration of 50% v/v in acetone/olive oil 4:1. 

 

Very slight erythema was noted on Day 2 with animals treated with the undiluted test item. Well defined erythema was noted with one animal and three animals showed moderate to severe erythema on Day 3, prior to termination with animals treated with the undiluted test item.

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

Concentration (%v/v) in acetone/olive oil 4:1	dpm	dpm/Nodea	Stimulation Indexb	Result
Vehicle	19988.91	2498.61	na	na
25	74266.09	9283.26	3.72	Positive
50	59549.48	7443.69	2.98	Negative
100	*	*	*	*

dpm = Disintegrations per minute

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

*= No results were obtained with the undiluted test item as all the animals were humanely killed on Day 3 (post dose) due to the appearance of clinical observations which approached the moderate severity limit. The remaining mouse was removed from the study.

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	0Rt	0	0	0
	2-2	0	0	0	0	0Fl	0Rt Fl	0Fl	0Fl	0Fl
	2-3	0	0	0	0	0	0Rt Fl	0Fl	0Fl	0Fl
	2-4	0	0	0	0	0Fl	0Rt Fl	0Fl	0Fl	0Fl
50	3-1	0	0	0	0	0	0Rt	0	0Fl	0Fl
	3-2	0	0	0	0	0	0Rt	0	0Fl	0Fl
	3-3	0	0	0	0	0	0Rt	0	0Fl	0Fl
	3-4	0	0	0	0	0Fl	0Rt Fl	0Fl	0Fl	0Fl
100	4-1	0	0	0	0	0	0Rt+	*	*	*
	4-2	0	0	0	0	0Fl	H Fl Fa Rt K	*	*	*
	4-3	0	0	0	0	0Fl	H Fl Rt K	*	*	*
	4-4	0	0	0	0	0Fl	H Fl Fa Rt K	*	*	*

0=    No signs of systemic toxicity

H=   Hunched Posture

Fl=   Fur loss

Rt=  Translucent viscous residual test item on the back of the ears

Fa= Fasciculations

K =  Animals humanely killed due to the occurrence of clinical signs of toxicity that exceeded the moderate severity limit set forth in the UK Home Office Project Licence. Animal body weights were 17.4g (4-2), 19.3g (4-3) and 17.7g (4-4)

+=    Animal removed from study. The bodyweight was 20.5g before termination

*=    No data animals dead

Local Skin Irritation – Main Test 

Treatment	Animal Number	Local Skin Irritation
		Day 1		Day 2		Day 3		Day 4		Day 5		Day 6
		left	right	left	right	left	right	left	right	left	right	left	right
Vehicle acetone/olive oil 4:1	1-1	0	0	0	0	0	0	0	0	0	0	0	0
	1-2	0	0	0	0	0	0	0	0	0	0	0	0
	1-3	0	0	0	0	0	0	0	0	0	0	0	0
	1-4	0	0	0	0	0	0	0	0	0	0	0	0
Test Item 25% v/v in acetone/olive oil 4:1	2-1	0	0	1	1	1	1	1	1	0	0	0	0
	2-2	0	0	1	1	1	1	1	1	0	0	0	0
	2-3	0	0	1	1	1	1	1	1	0	0	0	0
	2-4	0	0	1	1	1	1	1	1	0	0	0	0
Test Item 50% v/v in acetone/olive oil 4:1	3-1	0	0	1	1	1	1	1	1	1	1	0	0
	3-2	0	0	1	1	1	1	1	1	1	1	0	0
	3-3	0	0	1	1	1	1	1	1	1	1	0	0
	3-4	0	0	1	1	1	1	1	1	1	1	0	0
Test Item 100%	4-1	0	0	1	1	2	2	*	*	*	*	*	*
	4-2	0	0	1	1	3	3	*	*	*	*	*	*
	4-3	0	0	1	1	3	3	*	*	*	*	*	*
	4-4	0	0	1	1	3	3	*	*	*	*	*	*

*=    No data animals dead

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

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

Executive summary:

Introduction

A study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

 

Methods

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 100% v/v, 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) ofthe undiluted test item or the test item as asolutioninacetone/olive oil 4:1at concentrations of50% or25% v/v. A further group offour animals was treated withacetone/olive oil 4:1alone.

 

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	3.72	Positive
50	2.98	Negative
100	*	*

*=   No results were obtained with the undiluted test item as all the animals were humanely killed on Day 3 (post dose) due to the appearance of clinical observations which approached the moderate severity limit. The remaining mouse was removed from the study.

Conclusion

The test item was considered to be a sensitizer under the conditions of the test.