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

Endpoint summary

Description of key information

Available human data indicates that the registered substance is non-sensitising. These data are supported by results from a local lymph node assay, which showed an analogue test item to be non-sensitising(OECD 429 and EU Method B.42).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 April 2015 to 27 May 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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)

Species:

mouse

Strain:

CBA/Ca

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 B.V., Horst, The Netherlands.
- On receipt, the animals were randomly allocated to cages.
- The animals were nulliparous and non-pregnant.
- After an acclimatisation period of at least 5 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 8 to 12 weeks old.
- Animals were 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.
- 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 at least fifteen changes per hour.
- Lighting was controlled by a time switch to give 12 hours continuous light (06:00 to 18:00) and 12 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.

Vehicle:

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

Concentration:

Test item concentration 1 %, 10 % or 100 % in the main test

No. of animals per dose:

Five

Details on study design:

TEST ITEM
- 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 chosen as it produced the most suitable formulation at
the required concentration. The concentrations used are given in the procedure section. The
vehicle determination record is included as Appendix 2 (attached).
- 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.

REFERENCE ITEM PREPARATION
- The positive control item was freshly prepared as a 25 % v/v dilution in acetone/olive oil 4:1.

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 item 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 3 (attached). 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 and post dose on Day 1, post dose on Days 2 and 3 and on Days 4, 5 and 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 sensitisation.

MAIN TEST
- Groups of five mice were treated with the undiluted test item or the test item at concentrations of 10 % or 1 % 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 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 five mice received the vehicle alone in the same manner.
- The positive control animals were similarly treated to the test animals except that 25 µL of the
positive control item, a-Hexylcinnamaldehyde, tech, 85 %, at a concentration of 25 % v/v in acetone/olive oil 4:1 was applied to the dorsal surface of each ear.
- Local skin irritation was scored as described in the preliminary screening test. The thickness of each ear was measured using a Mitutoyo 547-300S gauge (Mitutoyo Corporation), pre-dose and post-dose on Day 1, post dose on Days 2 and 3 and on Days 4, 5 and 6. Mean ear thickness changes were calculated as described in the preliminary screening test.

3H-METHYL THYMIDINE ADMINISTRATION
- Five days following the first topical application of the test item, vehicle control or positive control item (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, ARC UK Ltd) 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 followed by cervical separation. 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 the 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 cells 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 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 18 hours incubation at approximately 4 °C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, re-suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by P-scintillation counting. The "Poly Q" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left to stand in darkness 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 20 minutes, the vials were shaken vigorously. The number ofradioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).

INTERPRETATION OF RESULTS
- The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per animal 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 item will be regarded as a sensitiser 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-sensitiser".

Positive control substance(s):

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

Statistics:

STATISTICAL ANALYSIS
- 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 were met, 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) were presented as *** (p < 0.001); ** (p < 0.0); * (p < 0.05); not significant (p ≥ 0.05).

Positive control results:

- The Stimulation Index expressed as the mean radioactive incorporation for the positive control was reported as 5.13 (25 % v/v positive control item in acetone/olive oil 4:1; positive).

Parameter:

SI

Value:

1.17

Test group / Remarks:

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

Remarks on result:

other: negative

Parameter:

SI

Value:

2.29

Test group / Remarks:

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

Remarks on result:

other: negative

Parameter:

SI

Value:

2.99

Test group / Remarks:

100% v/v in acetone/oilive oil 4:1

Remarks on result:

other: negative

Cellular proliferation data / Observations:

PRELIMINARY SCREENING TEST
- Clinical observations, body weight and mortality data are given in Table 1 (attached).
- Local skin irritation is given in Table 2 (attached).
- The ear thickness measurements and mean ear thickness changes are given in Table 3 (attached).
- 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 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.

ESTIMATION OF PROLIFERATIVE RESPONSE OF LYMPH NODE CELLS IN MAIN TEST
- The radioactive disintegrations per minute per lymph node and the stimulation index are given in Table 4 (attached).
- The Stimulation Index expressed as the mean radioactive incorporation for each treatment
group divided by the mean radioactive incorporation of the vehicle control group was reported as 1.17 (1 % test item v/v in acetone/olive oil 4:1; negative), 2.39 (10 % test item v/v in acetone/olive oil 4:1; negative) and 2.99 (100 % test item v/v in acetone/olive oil 4:1; negative).

CLINICAL OBSERVATIONS AND MORTALITY DATA

- Individual clinical observations and mortality data for test and control animals are given in Table 5 (attached).

- Local skin irritation is shown in Table 6 (attached).

- The ear thickness measurements and mean ear thickness changes are given in Table 7 (attached).

- There were no unplanned animal deaths during the study.

- No signs of systemic toxicity were noted in the test or control animals during the test.

 

BODY WEIGHT

- Individual body weights and body weight change for test and control animals are given in Table 8 (attached).

- 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.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to be a non-sensitiser under the conditions of the study. The positive control 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:

GUIDELINE

An investigation was performed to assess the skin sensitisation 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 compliance with OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted 22 July 2010) and Method B.42 Skin Sensitisation (Local Lymph Node Assay) of Commission Regulation (EC) No. 440/2008.

 

METHODS

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 sensitiser, α-Hexylcinnamaldehyde tech, 85%, at a concentration of 25% v/v in acetone/olive oil 4:1.

 

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 was reported as 1.17 (1 % test item v/v in acetone/olive oil 4:1; negative), 2.39 (10 % test item v/v in acetone/olive oil 4:1; negative) and 2.99 (100 % test item v/v in acetone/olive oil 4:1; negative). The Stimulation Index expressed as the mean radioactive incorporation for the positive control was reported as 5.13 (25 % v/v positive control item in acetone/olive oil 4:1; positive).

 

CONCLUSION

The test item was considered to be a non-sensitiser under the conditions of the study. The positive control 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.

Reference 3

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Justification for type of information:

See read-across justification attached in Section 13.

Reason / purpose for cross-reference:

read-across source

Parameter:

SI

Value:

1.17

Test group / Remarks:

1% in acetone/olive oil 4:1

Remarks on result:

other: negative

Parameter:

SI

Value:

2.39

Test group / Remarks:

10% in acetone/olive oil 4:1

Remarks on result:

other: negative

Parameter:

SI

Value:

2.99

Test group / Remarks:

100% in acetone/olive oil 4:1

Remarks on result:

other: negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

In vivo

A key investigation was performed to assess the skin sensitisation potential of an analogue test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The study was conducted in compliance with OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted 22 July 2010) and Method B.42 Skin Sensitisation (Local Lymph Node Assay) of Commission Regulation (EC) No. 440/2008.

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 sensitiser, α-Hexylcinnamaldehyde tech, 85 %, at a concentration of 25% v/v in acetone/olive oil 4:1.

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group was reported as 1.17 (1 % test item v/v in acetone/olive oil 4:1; negative), 2.39 (10 % test item v/v in acetone/olive oil 4:1; negative) and 2.99 (100 % test item v/v in acetone/olive oil 4:1; negative). The Stimulation Index expressed as the mean radioactive incorporation for the positive control was reported as 5.13 (25 % v/v positive control item in acetone/olive oil 4:1; positive).

 

The test item was considered to be a non-sensitiser under the conditions of the study. The positive control 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.

Justification for classification or non-classification

Available human data indicates that the registered substance is non-sensitising and these data are supported byLLNA results showing an analogue test item to be non-sensitising (Stimulation Index≤3). In accordance with ECHA Guidance on the Application of the CLP Criteria (Version 4.1; June 2015), classification of the registered substance as a skin sensitiser is not required under the terms of Regulation (EC) No 1272/2008.