[No public or meaningful name is available]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-12-16 - 2016-01-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitization: Local Lymph Node Assay" (adopted 22 July 2010)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

Method B42 Skin Sensitization (Local Lymph Node Assay) of Commission Regulation (EC) No. 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C16-18 (even numbered), reaction products with tetraethylenepentamine

Molecular formula:

not applicable (UVCB substance)

IUPAC Name:

Fatty acids, C16-18 (even numbered), reaction products with tetraethylenepentamine

Test material form:

solid: pellets

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., Inc., Horst, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 to 12 weeks
- Weight at study initiation: 15 to 23 g
- Housing: The animals were 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): 2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK, ad libitum
- Water (e.g. ad libitum): mains tap water, ad libitum
- Acclimation period: at least 5 days
- Indication of any skin lesions: none stated

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

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

25%, 10% or 5% w/w in propylene glycol

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: yes, propylene glycol was chosen as it produced the highest concentration that was suitable for dosing.
- Irritation: 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.
- Systemic toxicity: Any clinical signs of toxicity, if present, were also recorded.
- Ear thickness measurements: 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.
- Erythema scores: as set out in OECD 429

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The test item will be regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in3HTdR incorporation will be classified as a "non-sensitizer".

TREATMENT PREPARATION AND ADMINISTRATION:

Test Item Preparation and Analysis
For the purpose of the study, the test item was freshly prepared as a suspension in propylene glycol. This vehicle was chosen as it produced the highest concentration that was suitable for dosing. The test item was formulated within 2 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.

Test Item Administration
Groups of four mice were treated with the test item at concentrations of 25%, 10% or 5% w/w in propylene glycol. 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 uL 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 mice were injected via the tail vein with 250 uL of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 uCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 \xCi to each mouse.

Positive control substance(s):

other: Phenylacetaldehyde (>90%)

Results and discussion

Positive control results:

Phenylacetaldehyde, 5% v/v in proylene glycol, Stimulation Index = 9.89, Result = positive
Phenylacetaldehyde (>90%) was considered to be a sensitizer under the conditions of the test.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.21

Test group / Remarks:

5% w/w of the test item in propylene glycol

Remarks on result:

other:

Remarks:

negative

Results 2

Parameter:

SI

Value:

1.17

Test group / Remarks:

10% w/w of the test item in propylene glycol

Remarks on result:

other:

Remarks:

negative

Results 3

Key result

Parameter:

SI

Value:

2.43

Test group / Remarks:

25% w/w of the test item in propylene glycol

Remarks on result:

other:

Remarks:

negative

Cellular proliferation data / Observations:

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

BODY WEIGHTS
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.

Any other information on results incl. tables

RESULTS

 

Preliminary Screening Test

 

No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted.

Based on this information the dose levels selected for the main test were 25%, 10% and 5% w/w in propylene glycol.

 

 

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:

 

Concentration (%w/w) in propylene glycol	Stimulation Index	Result
5	1.21	Negative
10	1.17	Negative
25	2.43	Negative

 

 

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.

 

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.

 

 

Tables

 

Clinical Observations, Body Weight and Mortality Data - Preliminary Screening Test

Concentration(%w/w) in propylene glycol	Animal Number	Body Weight (g)		Day
				1		2		3		4	S	6
		Day1	Day6	Pre-Dose	Post Dose	Pre-Dose	Post Dose	Pre-Dose	Post Dose
25	S-l	19.4	20.2	0	0	0	0	0	0	0	0	0

0 =No signs of systemic toxicity

 

Local Skin Irritation-Preliminary Screening Test

Concentration(%w/w) in propylene glycol

Animal Number

Local Skin Irritation

Day1

Day2

Day3

Day4

Day5

Day6

left

right

left

right

left

right

left

right

left

right

left

right

25

S-l

0

0

0

0

0

0

0

0

0

0

0

0

 

Measurement of Ear Thickness and Mean Ear Thickness Changes-Preliminary Screening Test

Concentration(%w/w) in propylene glycol	Animal Number	Ear Thickness Measurement (mm)
		Day1		Day3		Day6
		pre-dose		post dose
		left	right	left	right	left	right
25	S-l	0.24	0.23	0.25	0.24	0.23	0.24
overall mean (mm)		0.24		0.25		0.24
overall mean ear thickness change(%)		na		4.26		0.00

na=Not applicable

 

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

Concentration (% w/w) in propylene glycol	dpm	dpm/Node^a	Stimulation Index^b	Result
Vehicle	10259.85	1282.48	na	na
5	12409.68	1551.21	1.21	Negative
10	11994.82	1499.35	1.17	Negative
25	24971.41	3121.43	2.43	Negative

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

 

Individual Clinical Observations and Mortality Data

Concentration (%w/w) in propylene glycol	Animal Number	Day1		Day2		Day3		Day4	Day5	Day6
		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
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
10	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
25	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

 

Individual Body Weights and Body Weight Change

Concentration (%w/w) in propylene glycol	Animal Number	Body Weight (g)		Body Weight Change (g)
		Day1	Day6
Vehicle	1-1	19.0	19.9	0.9
	1-2	20.1	20.5	0.4
	1-3	21.8	22.2	0.4
	1-4	21.6	21.4	-0.2
5	2-1	20.5	20.0	-0.5
	2-2	18.2	18.9	0.7
	2-3	19.0	19.4	0.4
	2-4	21.0	21.3	0.3
10	3-1	20.0	20.9	0.9
	3-2	21.8	21.7	-0.1
	3-3	18.7	19.4	0.7
	3-4	20.7	20.7	0.0
25	4-1	20.3	20.9	0.6
	4-2	19.4	21.2	1.8
	4-3	21.1	21.1	0.0
	4-4	18.7	19.9	1.2

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

not sensitizing

Conclusions:

The study was conducted under GLP according to OECD guideline 429 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation. Positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the skin sensitization potential of Octadecanoic acid, reaction products with tetraethylenepentamine. Under the experimental conditions reported, the test item did not induce a stimulation index over 3 in the test concentrations suitable for dosing. Therefore, Octadecanoic acid, reaction products with tetraethylenepentamine is considered not to be a skin sensitiser under the test conditions of this study.

Executive summary:

A OECD TG 429 study was performed under GLP to assess the skin sensitization potential Octadecanoic acid, reaction products with tetraethylenepentamine in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

 

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 25% 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 item as a suspension in propylene glycol at concentrations of 25%, 10% or 5% w/w. A further group of four animals was treated with propylene glycol 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 (%w/w) in propylene glycol	Stimulation Index	Result
5	1.21	Negative
10	1.17	Negative
25	2.43	Negative

 

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