Benzeneacetaldehyde, cyclic acetal with glycerol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Remarks:

LLNA was performed before October 2016

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 8 August 2016 and 7 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

LLNA was performed before October 2016 when the REACH regulation came into force requesting in vitro testing first.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 22 July 2010

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

1

Reference substance name:

Benzeneacetaldehyde, cyclic acetal with glycerol

EC Number:

249-934-2

EC Name:

Benzeneacetaldehyde, cyclic acetal with glycerol

Cas Number:

29895-73-6

Molecular formula:

C11H14O3

IUPAC Name:

Reaction mass of cis-2-benzyl-1,3-dioxan-5-ol and rel-[(2R,4R)-2-benzyl-1,3-dioxolan-4-yl]methanol and rel-[(2R,4S)-2-benzyl-1,3-dioxolan-4-yl]methanol and trans-2-benzyl-1,3-dioxan-5-ol

Test material form:

liquid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Animal Information:
Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Envigo RMS B.V., Inc., Horst, The Netherlands. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non-pregnant. After an acclimatization 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.

Animal Care and Husbandry:
The 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 Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study.
The temperature and relative humidity were set to achieve limits of 19 to 25 oC and 30 to 70%, respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light 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.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

Undiluted test item or the test item at concentrations of 30% or 10% v/v in dimethyl formamide.

No. of animals per dose:

Groups of five mice were treated

Details on study design:

Preliminary Screening Test
As no toxicological information was available 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 for erythema. 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 five mice were treated with the undiluted test item or the test item at concentrations of 30% or 10% v/v in dimethyl formamide. 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 five mice received the vehicle alone in the same manner.

Routine positive control study (reduced LLNA) was performed to assess the sensitivity of the strain of mouse used at these laboratories to a known sensitizer. A group of five animals was treated with 50 µL (25 µL per ear) of α-Hexylcinnamaldehyde, tech., 85% as a solution in dimethyl formamide at a concentration of 25% v/v. A further control group of five animals was treated with dimethyl formamide alone.

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 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 labeled 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 oC, 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 beta-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 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:

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)

Results and discussion

Positive control results:

The positive control item, α-Hexylcinnamaldehyde, tech., 85%, gave a Stimulation Index of >3 (6.08) when tested at a concentration of 25 % v/v in dimethyl formamide and was considered to be a sensitizer under the conditions of the test.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

EC3

Remarks on result:

other: The test item, tested up to 100%, did not produce a threefold or greater increase in 3HTdR incorporation compared to control values.

Results 2

Parameter:

SI

Remarks on result:

other: The following SI values were derived at 10, 30 and 100%: 0.82, 0.62 and 0.79, respectively.

Cellular proliferation data / Observations:

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.

Any other information on results incl. tables

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 undiluted test item (100%) and the test item at concentrations of 30% and 10% v/v in dimethyl formamide were selected for the 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 (% v/v) in dimethyl formamide	Stimulation Index	Result
10	0.82	Negative
30	0.62	Negative
100	0.79	Negative

The test item, tested up to 100%, did not produce a threefold or greater increase in³HTdR incorporation compared to control values.

Clinical Observations, Body Weight and Mortality Data – Preliminary Screening Test:

Concentration	Animal Number	Body Weight (g)		Day
				1		2		3		4	5	6
		Day 1	Day 6	Pre-Dose	Post Dose	Pre-Dose	Post Dose	Pre-Dose	Post Dose
100%	S-1	20.3	20.6	0	0	0	0	0	0	0	0	0

0=    No signs of systemic toxicity

Local Skin Irritation – Preliminary Screening Test:

Concentration

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

100%

S-1

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	Animal Number	Ear Thickness Measurement (mm)
		Day 1		Day 3		Day 6
		pre‑dose		post dose
		left	right	left	right	left	right
100%	S-1	0.22	0.21	0.22	0.22	0.22	0.22
overall mean (mm)		0.215		0.220		0.220
overall mean ear thickness change (%)		na		2.326		2.326

na=  Not applicable

Individual Disintegrations per Minute and Stimulation Index:

Concentration (% v/v) in dimethyl formamide	Animal Number	dpm/ Animal (a)	Mean dpm/Animal (Standard Deviation)	Stimulation Index (b)	Result
Vehicle	1-1	2533.24	2578.18 (±501.14)	na	na
	1-2	3182.37
	1-3	1798.34
	1-4	2738.61
	1-5	2638.35
10	2-1	2816.29	2107.34 (±537.78)	0.82	Negative
	2-2	2166.11
	2-3	2370.71
	2-4	1749.61
	2-5	1433.98
30	3-1	1742.74	1588.82* (±337.53)	0.62	Negative
	3-2	1583.59
	3-3	1164.23
	3-4	2053.62
	3-5	1399.92
100	4-1	1837.89	2031.54 (±434.99)	0.79	Negative
	4-2	2056.53
	4-3	2211.82
	4-4	2611.47
	4-5	1440.01

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 control group p<0.05

Individual Clinical Observations and Mortality Data:

Concentration (% v/v) in dimethyl formamide	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
	1-5	0	0	0	0	0	0	0	0	0
10	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
	2-5	0	0	0	0	0	0	0	0	0
30	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
	3-5	0	0	0	0	0	0	0	0	0
100	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
	4-5	0	0	0	0	0	0	0	0	0

0=    No signs of systemic toxicity

Individual Body Weights and Body Weight Change:

Concentration (% v/v) in dimethyl formamide	Animal Number	Body Weight (g)		Body Weight Change (g)
		Day 1	Day 6
Vehicle	1-1	22.9	23.4	0.5
	1-2	20.3	22.0	1.7
	1-3	20.8	21.0	0.2
	1-4	21.2	22.1	0.9
	1-5	19.8	20.3	0.5
10	2-1	19.0	20.1	1.1
	2-2	19.2	19.9	0.7
	2-3	19.0	20.1	1.1
	2-4	19.2	20.4	1.2
	2-5	17.8	17.8	0.0
30	3-1	20.2	20.0	-0.2
	3-2	19.4	19.1	-0.3
	3-3	21.1	21.3	0.2
	3-4	19.8	20.1	0.3
	3-5	20.3	20.7	0.4
100	4-1	20.2	20.2	0.0
	4-2	22.2	22.1	-0.1
	4-3	20.4	20.4	0.0
	4-4	19.0	18.8	-0.2
	4-5	18.1	18.3	0.2

Applicant's summary and conclusion

Interpretation of results:

other: not a skin sensitizer

Remarks:

in accordance with EU CLP 1272/2008 and its amendments

Conclusions:

The test item, tested up to 100%, did not produce a threefold or greater increase in 3HTdR incorporation compared to control values, therefore the test item was considered not to be a skin sensitiser under the conditions of the test.

Executive summary:

The skin sensitization potential of the test substance has been tested according to OECD TG 429: Local Lymph Node Assay and GLP. 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 (100%) or the test item as a solution in dimethyl formamide at concentrations of 30% or 10% v/v. A further group of five animals was treated with dimethyl formamide alone. At 10, 30 and 100% the substance showed SI values of 0.82, 0.62 and 0.79, respectively. The test item, tested up to 100%, did not produce a threefold or greater increase in 3HTdR incorporation compared to control values. The test item was considered not to be a skin sensitizer under the conditions of the test.