3-methyl-1-benzofuran-5-ol

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 16 June 2014 and 22 July 2014.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 429 using a Local Lymph Node Assay method and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (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

Sex:

female

Details on test animals and environmental conditions:

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.

Vehicle:

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

Concentration:

Preliminary Screening Test: 50% w/w
Main test: 50%, 25% or 10% w/w

No. of animals per dose:

Groups of five mice / dose.

Details on study design:

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 test item at a concentration of 50% w/w in acetone/olive oil 4:1, 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 and Positive Control Administration
Groups of five mice were treated with the test item at concentrations of 50%, 25% or 10% w/w 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, α 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.

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 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 eighteen hours incubation at approximately 4 C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, resuspended 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 twenty 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 by analysis of homogeneity of variance followed by one way analysis of variance (ANOVA). In the event of a significant result from the ANOVA, pairwise comparisons were performed between control and treated groups. For homogenous datasets Dunnett’s Multiple Comparison test was used and for non homogenous datasets Dunnett’s T3 Multiple Comparison Method was used.

Probability values (p) are presented as follows:
P < 0.001 = ***
P < 0.01 = **
P < 0.05 =*
P ≥ 0.05 = Not significant

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

At 10 % w/w in acetone/olive oil (4:1) the stimulation index was 5.03 (positive). At 25 % w/w in acetone/olive oil (4:1) the stimulation index was 7.85 (positive). At 50 % w/w in acetone/olive oil (4:1) the stimulation index was 10.56 (positive). The positive control at 25 % w/w in acetone/olive oil (4:1) gave a stimulation index was 8.48.

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. A small amount of pale white residual test item was noted on Days 1, 2 and 3 (post dose).

 

Based on this information the dose levels selected for the main test were50%,25% and10% w/winacetone/olive oil 4:1.

Main test - Clinical observations and mortality data

There were no deaths. No signs of systemic toxicity were noted in the test or control animalsduring the test. A small amount of pale white residual test item was noted on Days 1, 2 and 3 (post dose) on mice treated at a concentration of 50% w/w in acetone/olive oil 4:1.

Main test - Body weight

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

Clinical observations, body weight and mortality data - Preliminary screening test

Concentration (%w/w) in acetone/olive oil 4:1	Animal Number	Body Weight (g)		Clinical Observations
				Days
				1		2		3		4	5	6
		Day 1	Day 6	Pre-Dose	Post Dose	Pre-Dose	Post Dose	Pre-Dose	Post Dose
50	S-1	19	20	0	0Rt	0	0Rt	0	0Rt	0	0	0

0=          No signs of systemic toxicity

Rt =       Small amount of pale white residual test item on the ears

Local skin irritation - Preliminary screening test

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

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

50

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 (%w/w) in acetone/olive oil 4:1	Animal Number	Ear Thickness Measurement (mm)
		Day 1		Day 3		Day 6
		pre‑dose		post dose
		left	right	left	right	left	right
50	S-1	0.20	0.20	0.22	0.20	0.21	0.23
overall mean (mm)		0.20		0.21		0.22
overall mean ear thickness change (%)		na		5.00		10.00

na=  Not applicable

Individual disintegrations per minute and stimulation indices

Treatment Group	Animal Number	dpm/ Animala	Mean dpm/Animal (Standard Deviation)	Stimulation Indexb	Result
Vehicle acetone/olive oil 4:1	1-1	4080.51	4237.84 (±1114.21)	na	na
	1-2	3555.95
	1-3	6190.58
	1-4	3827.63
	1-5	3534.53
Test Item 10% w/win acetone/olive oil 4:1	2-1	15783.39	21334.34* (±5429.23)	5.03	Positive
	2-2	25489.21
	2-3	23020.08
	2-4	26975.13
	2-5	15403.91
Test Item 25% w/win acetone/olive oil 4:1	3-1	36216.17	33272.72*** (±5890.12)	7.85	Positive
	3-2	29343.42
	3-3	34057.71
	3-4	25822.17
	3-5	40924.11
Test Item 50% w/win acetone/olive oil 4:1	4-1	37226.68	44736.97*** (±11878.78)	10.56	Positive
	4-2	34549.69
	4-3	41699.16
	4-4	45617.02
	4-5	64592.30
Positive Control Item 25% v/v in acetone/olive oil 4:1	5-1	27527.85	35924.10*** (±14452.18)	8.48	Positive
	5-2	36994.07
	5-3	56408.79
	5-4	40694.49
	5-5	17995.32

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

***=Significantly different from control group p<0.001

The results of the statistical analysis of the data indicated there was a significant difference between the control group and the test and positive groups ***

Individual clinical observations and mortality data

Treatment Group	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 acetone/olive oil 4:1	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
Test Item 10% w/win acetone/olive oil 4:1	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
Test Item 25% w/win acetone/olive oil 4:1	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
Test Item 50% w/win acetone/olive oil 4:1	4-1	0	0Rt	0	0Rt	0	0Rt	0	0	0
	4-2	0	0Rt	0	0Rt	0	0Rt	0	0	0
	4-3	0	0Rt	0	0Rt	0	0Rt	0	0	0
	4-4	0	0Rt	0	0Rt	0	0Rt	0	0	0
	4-5	0	0Rt	0	0Rt	0	0Rt	0	0	0
Positive Control Item 25% v/v in acetone/olive oil 4:1	5-1	0	0	0	0	0	0	0	0	0
	5-2	0	0	0	0	0	0	0	0	0
	5-3	0	0	0	0	0	0	0	0	0
	5-4	0	0	0	0	0	0	0	0	0
	5-5	0	0	0	0	0	0	0	0	0

0=          No signs of systemic toxicity

Rt=        Small amount of pale white residual test item on the ears

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

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

Executive summary:

The skin sensitisation potential of the test substance, TM 11-0230, was assessed as positive according to OECD Test Guideline 429 using a Local Lymph Node Assay method.

At 10, 25 and 50 % the substance showed SI values of 5.03, 7.85 and 10.56, respectively. EC3 value is anticipated to be <10%.

The test item was considered to be a sensitiser and should be classified as skin sensitizer (Category 1) and labeled as H317: May cause an allergic skin reaction.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Migrated from Short description of key information:
The skin sensitisation potential of the test substance, TM 11-0230, was assessed as positive according to OECD Test Guideline 429 using a Local Lymph Node Assay method. EC3 value was predicted to be <10%.

The test substance tested at 0.25% did not cause sensitizing reaction in a repeated insulted patch test with 105 human subjects with NESIL of 138 ug/cm2.

Justification for selection of skin sensitisation endpoint:
The study was conducted on the target substance in vivo in an appropriate test species and according to internationally recognised guidelines.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the EC3 value <10% in the LLNA study and NESIL = 138 ug/cm2 from a human repeat insulted patch test, the substance has to be classified for skin sensitisation.According to EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, it has to be classified for skin sensitisation,Category 1, H317: May cause an allergic skin reaction.