Methyl 5-mesyl-o-anisate

Endpoint summary

Description of key information

Skin sensitization:

Key Study: Method according to OECD 442-B, GLP study. The test item did not show skin sensitisation potential under the tested conditions in the LLNA assay. The Stimulation Indexes were 0.86, 1.06, and 1.05 at concentrations of 10%, 25% and 50% test item in dimethylformamide, respectively.

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:

October 12, 2016 - October 25,2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Test method B.51 Skin Sensitisation (Local Lymph Node Assay) - Council regulation No 640/2012 of 30 May 2012 (E.U. Journal L193)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA): BrdU-ELISA

Specific details on test material used for the study:

Storage conditions:
- Storage condition of test material: room temperature

Species:

mouse

Strain:

CBA:J

Remarks:

CBA/J (CBA/JRj) strain mice (SPF caw)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: levage Janvier Labs (F-53941 Le Genest Saint Isle)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation:8 weeks old
- Weight at study initiation:
GROUP 1: 21.0g
GROUP 2: 20.5g
GROUP 3: 21.0g
GROUP 4: 20.4g
- Housing: The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet: Tkelad Global 16% Protein Rodent Diet (ENVIGO 2016) ad libitum.
- Water. ad libitum: tap water from public distribution system. Microbiological and chemical analyses of the water were carried out once every 6 monts by BUREAU VERITAS-eurofins(FRANCE)
- Acclimation period: five days under stabling and nutritional conditions
- Indication of any skin lesions:NO


ENVIRONMENTAL CONDITIONS
- Temperature (°C):19°C to 25°C
- Humidity (%):30-70%
- Air changes (per hr):10 changes per hour
- Photoperiod (hrs dark / hrs light):12 hours light, 12 hours dark.
- IN-LIFE DATES: From: To:29 June 2016 to 05 July 2016

Vehicle:

dimethylformamide

Concentration:

50%, 25% and 10%

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS:
As no information was available regarding irritant potential or systemic toxicity of the test item in the mouse, a preliminary screening test was performed using one mouse. Twenty-five μL of the test item diluted at 50% in dimethylformamide (DMF) to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). Test was performed with one mouse.
- Compound solubility: As the test item is a powder, it was not possible to test the undiluted test item. The maximum concentration of test item in a acceptable solvent was established according to the OECD guideline. Based on the observation of the solubility test, the maximum available concentration was 50% in dimethylformamide.
- Irritation: NO See 'Any other information on materials and methods incl. tables'.
- Systemic toxicity: NO, See 'Any other information on materials and methods incl. tables'.
- Erythmea: NO See 'Any other information on materials and methods incl. tables'.

The concentration of 50% was chosen as the highest concentration for the main study.

MAIN STUDY
According the result of preliminary test, the Main study was conducted.

- Clinical observations and mortality: All animals were observed daily on Days 1, 2, 3, 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded. Any irritation reaction (erythema and oedema) was recorded in parallel. Any other observation (dryness, presence of residual test item…) was noted.
- Body weights: The body weight of each mouse wa srepocrded on day 1 prior to dosing and on day 6 (prior to termination).
- Ear thickness measurements: On day 1 and on day 3 (before application) as well as on day 6 (after sacrifice) of each experiment, the thickness of the right ear of each animal of the vehicle control and treated groups was measured by a micrometer. Furthermore, on day 6, punch biopsies of 8 mm in diameter of the apical area of both ears were prepared and weighed in order to assess the irritation potential of the test item and the two lymp nodes per mouse were weighed.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Skin sensititation , LLNA:BrdU ELISA
- Number of animals:16
- Criteria used to consider a positive response: The SI value was derived as specified in the guidelines. If at least one concentration of the test item results is greater than 1.6 compared to control values, that is considered a positive response.The strength of the dose-response relationship, the statistical significance and the consistency of the solvent/vehicle and positive control responses may also be used when determining whether a borderline result (i.e. SI value between 1.6 and 1.9) is declared positive.

- the positive test item will be classified in subcategory 1A or 1B in accordance with the following :
Sub-category 1A EC value ≤ 2 %
Sub-category 1B EC value > 2 %

TREATMENT PREPARATION AND ADMINISTRATION:
Groups of four mice were treated with the test item diluted at 50%, 25% and 10% in dimethylformamide. 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.
The concentrations of the test and reference items are indicated in the following:

Groups Number of animals Treatment Concentration (%)
1 4 females Vehicle 0
2 4 females test item 10
3 4 females test item 25
4 4 females test item 50

An additional mouse was treated in each group in case of problem which may occur during the study, in particular during the excision of lymph nodes. As a problem occurred in group 2 (Sf8764) during the collecting of lymph nodes. The lymph nodes of the additional mouse (Sf8767) group 2 were collected and the data concerning this animal was used in the following study.

On day 5, 0.5 mL (5 mg/mouse) of BrdU (10 mg/mL) solution was injected by intra-peritoneal route. On day 6 (end of the test), the animals were anaesthetised with sodium pentobarbital and
administration continued to fatal levels. The draining auricular lymph nodes from the four mice were excised.
From each mouse, a single-cell suspension of lymph node cells (LNC) excised bilaterally was prepared by gentle mechanical disaggregation through a disposable plastic pestle to crush the lymph
nodes followed by passage through a #70 nylon mesh in 15 mL of PBS (Ca2+ / Mg2+ - free) into a well of a multi-well 6. The optimised volume was based on achieving a mean absorbance of the negative control group within 0.1- 0.2.

BrdU was measured by ELISA using a commercial kit (Roche Applied Science, Mannheim, Germany, Catalogue Number 11 647 229 001 – Batch No.11866200). Briefly, 100 μL of the LNC suspension was added to the wells of a flat-bottom microplate at leastin triplicate. After fixation and denaturation of the LNC, anti-BrdU antibody was added to each well and allowed to react. Subsequently the anti-BrdU antibody was removed by washing and the substrate solution was then added and allowed to produce chromogen. After 5 to 30 min, 30μL of 1 M H2SO4 was added in each well, then shaken for one minute. Absorbance at 450 nm with a reference wavelength of 690nm was then measured.

Positive control substance(s):

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

Positive control results:

The results for the last positive control (29/06/2016) were within the acceptable ranges. 5%, 10% and 25% solutions of hexyl cinnamic aldehyde in MEK generated a SI = 1.34, 1.63 and 1.95, respectively.The EC1.6 value was 9.48%. Under the experimental conditions the substance has to be classified in Category 1 " Skin sensitization".

Key result

Parameter:

SI

Value:

ca. 0.86

Variability:

± 0.14

Test group / Remarks:

2 (10% test item)

Remarks on result:

other: No indication of skin irritation

Key result

Parameter:

SI

Value:

ca. 1.06

Variability:

± 0.51

Test group / Remarks:

3 (25% test item)

Remarks on result:

other: No indication of skin irritation

Key result

Parameter:

SI

Value:

ca. 1.05

Variability:

± 0.19

Test group / Remarks:

4 (50% test item)

Remarks on result:

other: No indication of skin irritation

Parameter:

other: EC1.6

Remarks on result:

not determinable

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA: DATA: see 'Any other information on results' below.
DETAILS ON STIMULATION INDEX CALCULATION: as recommended by the guideline. No stimulation index of more than 1.6 was recorded whatever the tested concentration.
The BrdU labelling index was defined as:
BrdU labelling index = (ABSem – ABS blankem) – (ABSref – ABS blankref)
em = emission wavelength; and ref = reference wavelength.

EC1.6 CALCULATION:
The EC1.6 value (theoretical concentration resulting in a SI value of 1.6) was determined by linear interpolation of points on the dose-response curve, immediately above and below the 1.6-fold threshold.
The equation used for calculation of EC1.6 was:
EC1.6 = c + [(1.6 – d) / (b – d)] x (a – c)
a = the lowest concentration giving stimulation index > 1.6
b = the actual stimulation index caused by a
c = the highest concentration failing to produce a stimulation index of 1.6
d = the actual stimulation index caused by c
As no concentration giving stimulation index higher than 1.6, the EC1.6 cannot be determined.

CLINICAL OBSERVATIONS: No mortality and no signs of systemic toxicity were noted in the test and control animals during the test.

LOCAL IRRITATION: No cutaneous reaction was noted whatever the tested concentration. No increase in ear thickness and in ear weight was noted in animals treated at 10%, 25% and 50%.

BODY WEIGHTS: Changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Table No.3 Individual clinical observation and mortality data:

Groups	Test item	Animals	Day 1	Day 2		Day 3		Day 4		Day 5		Day 6
1	DMF	N° Sf 8743	0		0		0		0		0		0
		N° Sf 8744	0		0		0		0		0		0
		N° Sf 8745	0		0		0		0		0		0
		N° Sf 8746	0		0		0		0		0		0
2	10%	N° Sf 8763	0		0		0		0		0		0
		N° Sf 8767	0		0		0		0		0		0
		N° Sf 8765	0		0		0		0		0		0
		N° Sf 8766	0		0		0		0		0		0
3	25%	N° Sf 8768	0		0		0		0		0		0
		N° Sf 8769	0		0		0		0		0		0
		N° Sf 8770	0		0		0		0		0		0
		N° Sf 8771	0		0		0		0		0		0
4	50%	N° Sf 8773	0		0		0		0		0		0
		N° Sf 8774	0		0		0		0		0		0
		N° Sf 8775	0		0		0		0		0		0
		N° Sf 8776	0		0		0		0		0		0

0: no sign of systemic toxicity

Table No.4 :Body weight and body weight gain:

Groups	Test item	Animals	Bodyweight (g))		Body weight gain(g)
1	DMF	N° Sf 8743	20.8	22.2	1.4
		N° Sf 8744	21.3	23.7	2.4
		N° Sf 8745	20.6	21.4	0.8
		N° Sf 8746	21.3	23.1	1.8
		MEAN	21.0	22.6	1.6
		Standart deviation	0.4	1.0	0.7
2	10%	N° Sf 8763	20.4	21.2	0.8
		N° Sf 8767	19.4	20.1	0.7
		N° Sf 8765	21.1	21.6	0.5
		N° Sf 8766	21.2	22.6	1.4
		MEAN	20.5	21.4	0.9
		Standart deviation	0.8	1.0	0.4
3	25%	N° Sf 8768	20.5	21.7	1.2
		N° Sf 8769	21.0	21.2	0.2
		N° Sf 8770	21.1	22.2	1.1
		N° Sf 8771	21.2	21.6	0.4
		MEAN	21.0	21.7	0.7
		Standart deviation	0.3	0.4	0.5
4	50%	N° Sf 8773	22.3	22.4	0.1
		N° Sf 8774	20.2	20.8	0.6
		N° Sf 8775	19.5	21.3	1.8
		N° Sf 8776	19.6	21.4	1.8
		MEAN	20.4	21.5	1.1
		Standart deviation	1.3	0.7	0.9

Table No.5: BrdU index & Stimulation index per group and calculation of EC1.6

Groups	Test Item	BrdU-index (mean*)	Stimulation Index SI (mean + standard deviation)	Result	EC1.6value
1	DMF	0.655	n.a	n.a	n.a
2	10%	0.564	0.86± 0.14	Negative	n.a
3	25%	0.694	1.06± 0.51	Negative
4	50%	0.688	1.05± 0.19	Negative

n.a.: non applicable

*: mean: Σindividual value / 4

No stimulation index of more than 1.6 was recorded whatever the tested concentration.The Stimulation Index (SI) calculated by individual approach was 0.86, 1.06 and 1.05 for the treated groups at 10%, 25% and 50%, respectively. The EC1.6 cannot be determined in this study.

Table 6: BrdU index & Stimulation index (individual data)

Groups	Test item	Animals	BrdU-index (DO Indiv)	BrdU-in(DO mean)dex	BrdU-index mean*	Stimulation Index S.I. (indiv ± Standard deviation)
1	DMF	N° Sf 8743	0.988 0.937 0.844	0.923	0.655	n.a
		N° Sf 8744	0.626 0.569 0.611	0.602		n.a
		N° Sf 8745	0.537 0.506 0.571	0.538		n.a
		N° Sf 8746	0.604 0.613 0.446	0.555		n.a
2	10%	N° Sf 8763	0.638 0.669 0.646	0.651	0.564	0.99±0.02
		N° Sf 8767	0.610 0.642 0.573	0.609		0.93±0.05
		N° Sf 8765	0.617 0.548 0.513	0.560		0.86±0.08
		N° Sf 8766	0.514 0.401 0.390	0.435		0.66±0.10
3	25%	N° Sf 8768	1.176 1.222 1.090	1.163	0.694	1.78±0.10
		N° Sf 8769	0.786 0.658 0.630	0.692		1.06±0.13
		N° Sf 8770	0.489 0.548 0.323	0.424		0.65±0.18
		N° Sf 8771	0.445 0.548 0.495	0.496		0.76±0.08
4	50%	N° Sf 8773	0.768 0.928 0.786	0.828	0.688	1.27±0.13
		N° Sf 8774	0.520 0.722 0.607	0.617		0.94±0.15
		N° Sf 8775	0.699 0.791 0.751	0.747		1.14±0.07
		N° Sf 8776	0.646 0.475 0.561	0.561		0.86±0.13

Table 7: Individual Ear thickness and irritation level

Groups	Test item	Animals	Day1  ear thickness (mm)	Day3 ear thickness (mm)	Day6 ear thickness (mm)	Ear thickness increase D3/D1(%)	Ear thickness increase D6/D1(%)
1	DMF	N° Sf 8743	0.19	0.21	0.21	10.5	10.5
		N° Sf 8744	0.21	0.21	0.21	0.0	0.0
		N° Sf 8745	0.19	0.19	0.20	0.0	5.3
		N° Sf 8746	0.19	0.19	0.21	0.0	10.5
		MEAN	0.20	0.20	0.21	2.6	6.6
		Standart deviation	0.01	0.01	0.01	5.3	5.0
2	10%	N° Sf 8763	0.21	0.21	0.21	0.0	0.0
		N° Sf 8767	0.22	0.22	0.22	0.0	0.0
		N° Sf 8765	0.21	0.21	0.21	0.0	0.0
		N° Sf 8766	0.21	0.21	0.21	0.0	0.0
		MEAN	0.21	0.21	0.21	0.0	0.0
		Standart deviation	0.01	0.01	0.01	0.0	0.0
3	25%	N° Sf 8768	0.21	0.20	0.21	-4.8	0.0
		N° Sf 8769	0.19	0.20	0.21	5.3	10.5
		N° Sf 8770	0.22	0.22	0.22	0.0	0.0
		N° Sf 8771	0.21	0.22	0.22	4.8	4.8
		MEAN	0.21	0.21	0.22	1.3	3.8
		Standart deviation	0.01	0.01	0.01	4.7	5.0
4	50%	N° Sf 8773	0.21	0.21	0.22	0.0	4.8
		N° Sf 8774	0.21	0.21	0.22	0.0	4.8
		N° Sf 8775	0.22	0.22	0.22	0.0	0.0
		N° Sf 8776	0.19	0.21	0.21	10.5	10.5
		MEAN	0.21	0.21	0.22	2.6	5.0
		Standart deviation	0.01	0.01	0.01	5.3	4.3

Table 8: Individual Ear biopsy weight and lymph node weight

Groups	Test item	Animals	ear weight Day 6 (mg)	% of ear weight increased/group1	lymph nodes (mg)
1	DMF	N° Sf 8743	25.3		6.7
		N° Sf 8744	24.5		4.4
		N° Sf 8745	23.9		5.2
		N° Sf 8746	25.9		4.1
		MEAN	24.9		5.1
		Standart deviation	0.9		1.2
2	10%	N° Sf 8763	24.1	-1.4	3.8
		N° Sf 8767	24.4		5.2
		N° Sf 8765	23.8		6.3
		N° Sf 8766	25.9		8.4
		MEAN	24.6		5.9
		Standart deviation	0.9		1.9
3	25%	N° Sf 8768	25.9	1.5	8.1
		N° Sf 8769	26.4		6.1
		N° Sf 8770	24.8		8.3
		N° Sf 8771	24.0		6.6
		MEAN	25.3		7.3
		Standart deviation	1.1		1.1
4	50%	N° Sf 8773	26.2	-0.6	6.9
		N° Sf 8774	24.5		6.6
		N° Sf 8775	24.1		8.2
		N° Sf 8776	24.2		5.5
		MEAN	24.8		6.8
		Standart deviation	1.0		1.1

Table 9: Summary of result – skin irritation

Groups	Test Item	Ear thickness increase D6/D1 (%)	Biopsy ear weight Increase (%)	Excessive irritation #
1	DMF	6.6	n.a	NO
2	10%	0.0	-1.4	NO
3	25%	3.8	1.5	NO
4	50%	5.0	-0.6	NO

#: O.E.C.D. criteria: (% increase in ear thickness lower than 25%, score of erythema lower than 3

DMF: dimethylformamide

Interpretation of results:

GHS criteria not met

Conclusions:

The test item did not show skin sensitisation potential under the tested conditions in the LLNA assay. The Stimulation Indexes were 0.86, 1.06, and 1.05 at concentrations of 10%, 25% and 50% test item in dimethylformamide, respectively.

Executive summary:

The skin sensitisation potential of the test item was studied according to OECD 422B, under GLP conditions. Three groups of four female CBA/J mice received 25μL/ear of 50, 25, and 10% (w/v) test item in dimethylformamide; and one negative control group (4 females) received the vehicle (dimethylformamide). The last positive control results were used for reference. The animals were treated for 3 consecutive days (D1, D2, D3), and injected with BrdU solution on Day 5. On D6, the proliferation of lymphocytes in the draining auricular lymph nodes was determined by measurement of BrdU content in DNA of lymphocyte using an ELISA kit. The values obtained were used to calculate stimulation indices (SI). No mortality and no signs of systemic toxicity were noted in the test and control animals during the test. No increase in ear thickness and in ear weight was noted in animals treated at 50%, 25% and 10%. Therefore, the test item has to be considered as not excessively irritant at these concentrations. The Stimulation Index (SI) was 0.86, 1.06 and 1.05 for the treated groups at 10%, 25% and 50%, respectively. In conclusion, under the conditions of the present assay, the test item, had no sensitisation potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Key study: The skin sensitisation potential of the test item was studied according to OECD 422B, under GLP conditions. Three groups of four female CBA/J mice received 25μL/ear of 50, 25, and 10% (w/v) test item; and one negative control group (4 females) received the vehicle . The animals were treated for 3 consecutive days. On D6, the proliferation of lymphocytes in the draining auricular lymph nodes was determined. No mortality and no signs of systemic toxicity were noted, neither increase of ear thickness and or ear weight. The Stimulation Index (SI) was 0.86, 1.06 and 1.05 (10%, 25% and 50% respectively). In conclusion, the test item, had no sensitisation potential.

Data waiving (study scientifically not necessary / other information available): data from a LLNA study according to OECD 422B was available. Therefore, an in vitro study was not deemed necessary.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available information (SI<1.6 in the OECD 442B test), the substance is not classified as skin sensitizer according to CLP Regulation (EC) no.1272/2008.