Reaction product of D-Glucopyranoside, methyl; esterified with oleic acid, methyl ester

Endpoint summary

Description of key information

OECD Guideline 429 (LLNA) in 5 CBA/Ca strain mice, result: non sensitizing, positive control a-Hexylcinnamaldehyde, tech., 85 % gave a Stimulation Index of greater than 3

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Remarks:

Justification for non-vitro: study was conducted before REACH obligation for in-vitro testing come into force

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-04-28 - 2015-05-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

adopted 22 July 2010

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

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

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)

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V.,Horst, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known:
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 15 - 23 g
- Housing: suspended solid floor polypropylene cages furnished with softwood woodflakes
- Diet: ad libitum (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK)
- Water: ad libitum
- Acclimation period: at least 5 days
- Indication of any skin lesions: not mentioned

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25 °C
- Humidity (%): ~70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours light (06:00 to 18:00) and 12 hours darkness

Vehicle:

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

Concentration:

10% or 1% v/v
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. of animals per dose:

5 mice at each concentration

Details on study design:

PRE-SCREEN TESTS:
- animals: one mouse
- Compound solubility: 25 µL of the undiluted test item
- Irritation: 0 (day 1 to 6)
- Systemic toxicity: 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.
- Ear thickness measurements: daily, 11,364 % at day 6 (overall mean ear thickness change)
- Erythema scores: 0

MAIN STUDY
Based on this information the undiluted test item and the test item at concentrations of 10% and
1% v/v in acetone/olive oil 4:1 were selected for the main test.

ANIMAL ASSIGNMENT AND TREATMENT
- 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 animal and as the ratio or HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).
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 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitizer".

Positive control substance(s):

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

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. Data was first assessed for suitability by analysis of normality and homogeneity of variance. If the assumptions that the data are both normally distributed and has homogeneity of variances, then parametric one way analysis of variance
(ANOVA) and Dunnett’s multiple comparison procedure were used to determine statistical significance. If the assumptions were not met, non-parametric Kruskal-Wallis Rank Sum and Mann-Whitney U test procedures were used.
Probability values (p) are presented as follows:
P<0,001
P<0,01
P<0.05
P>=0.05
***
(not significant)

Positive control results:

Stimulation index: 5.13
Result: positive

Key result

Parameter:

SI

Value:

1.17

Test group / Remarks:

1% v/v in acetone/olive oil 4:1

Remarks on result:

other: negative

Key result

Parameter:

SI

Value:

2.39

Test group / Remarks:

10% v/v in acetone/olive oil 4:1

Remarks on result:

other: negative

Key result

Parameter:

SI

Value:

2.99

Test group / Remarks:

100 %

Remarks on result:

other: negative

Cellular proliferation data / Observations:

The Stimulation Index (SI) is expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group.
Clinical Observations:
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 were comparable to that observed in the corresponding control group animals over the same period.
Skin irritation:
No local skin irritation occured at any of the test animals in any treatment group.

Table1: Individual Disintegrations per Minute and Stimulation Indices

Treatment Group	Animal Number	dpm/ Animal"	Mean dpm/Animal (Standard Deviation)	Stimulation Index^b	Result
Vehicle acetone/olive oil 4:1	1-1	828.65	197829 (±696.57)	na	na
	1-2	2631.73
	1-3	2027.01
	1-4	199023
	1-5	2413.82
Test Item 1% v/v in acetone/olive oil 4:1	2-1	2210.36	2312.76 (±457.96)	1.17	Negative
	2-2	1606.86
	2-3	2885.85
	2-4	2447.13
	2-5	2443.62
Test Item 10% v/v in acetone/olive oil 4:1	3-1	3874.84	4727.73** (±1360.10)	2.39	Negative
	3-2	3053.99
	3-3	6330.76
	3-4	5850.75
	3-5	4528.33
Test Item 100%	4-1	666840	5910.43** (±2259.56)	2.99	Negative
	4-2	8629.09
	4-3	2843.36
	4-4	6923.45
	4-5	4487.84
Positive Control Item 25% v/v in acetone/olive oil 4:1	5-1	6213.81	10148,17** (±3212.47)	5.13	Positive
	5-2	10662.96
	5-3	14919.57
	5-4	8508.99
	5-5	10435.53

dpm - Disintegrations per minute

a = Total number of lymph nodes per animal is 2

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

na= Not applicable

** = Significantly different from vehicle control group p<0.01

Table 2: Measurement of ear thickness and mean ear thickness changes (attached)

Table 3: Individual Body Weights and Body Weight Change

Treatment Group	Animal Number	Body Weight (g)		Body Weight Change (g)
		Day 1	Day 6
Vehicle acetone/olive oil 4:1	1-1	19.6	19.3	-0.3
	1-2	20.4	20.6	0.2
	1-3	18.5	19.1	0.6
	1-4	21.2	18.4	-2.8
	1-5	18.4	18.7	0.3
Test Item 1% v/v in acetone/olive oil 4:1	2-1	20.8	21.8	1.0
	2-2	18.5	19.7	1.2
	2-3	20.8	21.6	0.8
	2-4	18.9	19.3	0.4
	2-5	18.3	20.1	1.8
Test Item 10% v/v in acetone/olive oil 4:1	3-1	19.1	19.7	0.6
	3-2	21.1	19.6	-1.5
	3-3	19.1	20.2	1.1
	3-4	19.4	20.0	0.6
	3-5	18.8	20.4	1.6
Test Item100%	4-1	19.0	19.5	0.5
	4-2	19.4	18.8	-0.6
	4-3	19.0	18.7	-0.3
	4-4	20.9	20.0	-0.9
	4-5	18.0	19.6	1.5
Positive Control Item 25% v/v in acetone/olive oil 4:1	5-1	19.6	19.6	0.0
	5-2	18.8	19.5	0.7
	5-3	20.0	19.2	-0.8
	5-4	19.0	19.7	0.7
	5-5	18.6	19.1	0.5

Interpretation of results:

GHS criteria not met

Conclusions:

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

a-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 when tested at a concentration of 25% v/v in acetone/olive oil 4:1 thus demonstrating the sensitivity and reliability of the test system.

Executive summary:

A study in compliance with OECD Guideline 429 and EU Method B.42 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. The study was conducted in accordance with GLP criteria.

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 100%, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of five animals, were treated with 50 µL (25 µL per ear) of the undiluted test item or the test item as a solution in acetone/olive oil 4:1 at concentrations of 10% or 1% v/v. A further group of five animals was treated with acetone/olive oil 4:1 alone. A concurrent positive control test, using a group of five animals, was also performed with the known sensitizer, α-Hexylcinnamaldehyde tech., 85%, at a concentration of 25% v/v in acetone/olive oil 4:1.

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

Treatment Group	Concentration	Stimulation Index (SI)	Result
Test Item	1% v/v in acetone/olive oil 4:1	1.17	Negative
	10 % v/v in acetone/olive oil 4:l	2.39	Negative
	100%	2.99	Negative
Positive Control Item	25% v/v in acetone/olive oil 4:1	5.13	Positive

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

α-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 when tested at a concentration of 25% v/v in acetone/olive oil 4:1 thus demonstrating the sensitivity and reliability of the test system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The key study (Sanders, 2015 (C)) in compliance with OECD Guideline 429 and EU Method B.42 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. The study was conducted in accordance with GLP criteria.

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 100 %, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of five animals, were treated with 50 µL (25 µL per ear) of the undiluted test item or the test item as a solution in acetone/olive oil 4:1 at concentrations of 10 % or 1 % v/v. A further group of five animals was treated with acetone/olive oil 4:1 alone. A concurrent positive control test, using a group of five animals, was also performed with the known sensitizer, α-Hexylcinnamaldehyde tech., 85 %, at a concentration of 25 % v/v in acetone/olive oil 4:1.

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

Treatment Group	Concentration	Stimulation Index (SI)	Result
Test Item	1 % v/v in acetone/olive oil 4:1	1.17	Negative
	10 % v/v in acetone/olive oil 4:l	2.39	Negative
	100%	2.99	Negative
Positive Control Item	25 % v/v in acetone/olive oil 4:1	5.13	Positive

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

α-Hexylcinnamaldehyde, tech., 85 % gave a Stimulation Index of greater than 3 when tested at a concentration of 25 % v/v in acetone/olive oil 4:1 thus demonstrating the sensitivity and reliability of the test system.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The classification and labelling for the test item is based in the results of a recent skin sensitisation study according to LLNA (OECD 429) in mice with GLP compliance. The Stimulation Index in all mice was below 3 and the result for sensitisation negative.

In accordance with Regulation (EC) No 1272/2008 (CLP Regulation) the test item does not meet the classification criteria and is therefore considered as non sensitising.