T-A06

Endpoint summary

Description of key information

Skin sensitisation

In Vivo: Key study. Method according to OECD 442 B, GLP study. The test substance does not have to be classified as a skin sensitizer.

In Chemico: First key event (molecular initiating event): Data Waiving. Study not technically feasible. Test substance is not solubile in solvents for OECD 442 C. 

In Vitro: Second key event (activation of keratinocytes): Data waiving. The substances is not solubile in solvents for OECD 442 D.

In Vitro: Thrird key event (activation of dendritic cells): Data waiving. According to OECD 442 E, substances with a Log Kow > 3.5 can produce false negative results, as is the case for the test item in question.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
Since the substance has a log Kow greater than 3.5 the study cannot be performed since substance with a log kow greater than 3.5 tend to produce false negative results with this method.

Reason / purpose for cross-reference:

data waiving: supporting information

Reference 2

Endpoint:

skin sensitisation: in vitro

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
Prior to performing OECD 442D a solubility study was performed. The test item was not soluble at the conditions: 200mM in DMOS, 200mM in water, 8mM in 4% DMSO nor 2mM in 1%DMSO. Therefore the study is technically not feasible.

Reference 3

Endpoint:

skin sensitisation: in chemico

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
Prior to performing OECD 442 a solubility study was performed. The test substance was not solulibile in Water, Water/acetonitrile, Isopropanol or Aceton.

Reference 4

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 January 2021 to 26 January 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin sensitisation: Local Lymph Node Assay: BrdU-ELISA or –FCM)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Deviation - please see 'Any other information on materials and methods incl. tables'

Type of study:

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

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Elevage Janvier Labs (F-53941 Le
Genest Saint Isle)
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known:
- Age at study initiation: 8 weeks
- Weight at study initiation: see table 3
- Housing: individually housed in suspended solid-floor polypropylene cages furnished with
softwood woodflakes.
- Diet (e.g. ad libitum): ad libitum ENVIGO 2016
- Water (e.g. ad libitum): ad libitum tap water from public distribution system
- Acclimation period: at least 5 days
- Indication of any skin lesions: see table 1

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25°C
- Humidity (%): 30 to 70 %
- Air changes (per hr): 10x/hr
- Photoperiod (hrs dark / hrs light): 12hrs dark/ 12 hrs light
- IN-LIFE DATES: From: 06 January 2021 To: 26 January 2021

Vehicle:

propylene glycol

Concentration:

Preliminary: highest technically possible concentration: 25µL diluted at 60% in vehicle
Main test: 25µL diluted at 60%, 40% and 20% in vehicle

No. of animals per dose:

4 females

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: highest technically feasable concentration
- Irritation: Non observerd
- Systemic toxicity: recorded on day 1 to day 6
- Ear thickness measurements: recorded on day 1 and 6
- Erythema scores: n/a

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:
* The test item should also be considered as an excessive irritant if the score of erythema is higher or equal to 3.
* Cell proliferation: The test item will be regarded as a sensitiser if at least one concentration of the test item results is greater than 1.6 compared to control values.
* % increase in ear thickness between day 1 and day 3 and/or between day 1 and day 6
Interpretation:
< 10 % Non irritant
10 – 25 % Slightly irritant*
> 25 % Irritan

TREATMENT PREPARATION AND ADMINISTRATION:
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 and 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 four mice received the vehicle alone in the same manner.

Positive control substance(s):

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

Statistics:

The BrdU labelling index was defined as:
BrdU labelling index = (ABSem – ABS blankem) – (ABSref – ABS blankref)

Stimulation Index (SI):
Mean BrdU labelling index/mouse Treated Group/ Mean BrdU labelling index/mouse Control Group

EC1.6
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

Positive control results:

The lab performed a positive control study between 02 September 2020 to 08 September 2020 using the known sensitiser α-Hexylcinnamaldehyde. Three groups, each of four animals, were treated with 50 µL (25 µL per ear) of αHexylcinnamaldehyde, as a solution in acetone/olive oil (4:1, v/v) at concentrations of 5%, 10% and 25% (v/v). A further control group of four animals was treated with acetone/olive oil (4:1, v/v) alone. The results showed an SI of 2.14 (+/- 0.36), which is higher than 1.6 for the 25% concentration (v/v). In conclusion, in view of these results, under these experimental conditions, the substance α-Hexylcinnamaldehyde in accordance with the Regulation (EC) No. 1272/2008 has to be classified in category 1 “Skin sensitisation”.
The signal word “Warning” and hazard statement H317 “May cause an allergic skin reaction” are required.

Key result

Parameter:

SI

Value:

1.24

Test group / Remarks:

20%

Key result

Parameter:

SI

Value:

0.99

Test group / Remarks:

40%

Key result

Parameter:

SI

Value:

0.97

Test group / Remarks:

60%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA

DETAILS ON STIMULATION INDEX CALCULATION
SI = Mean BrdU labelling index/mouse Treated Group/Mean BrdU labelling index/mouse Control Group


EC3 CALCULATION
EC1.6 cannot be determined due to the absence of SI value higher than 1.6.

CLINICAL OBSERVATIONS:

BODY WEIGHTS:
No change in body weight was noted for all treated groups versus control group.


SIGNS OF TOXICITY (including dermal irritation at the site of administration, if any, e.g. increased ear thickness). not observed

Clinical Observations

Groups	Test item	AnimalsNo.		Day 1	Day 2	Day 3	Day 4	Day 5	Day 6
1	PG	Sf	3348	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R
		Sf	3349	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R
		Sf	3350	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R
		Sf	3351	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R
2	20%	Sf	3369	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R
		Sf	3370	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R
		Sf	3371	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R
		Sf	3372	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R
3	40%	Sf	3373	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R
		Sf	3374	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R
		Sf	3375	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R
		Sf	3376	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R
4	60%	Sf	3378	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R
		Sf	3379	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R
		Sf	3380	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R
		Sf	3381	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R	N.t.R

Body weights

Groups	Test item	Animals No.		Bodyweight (g)		Body weight gain (g)
				Day 1	Day 6
1	PG	Sf	3348	20.0	21.0	1.0
		Sf	3349	22.3	22.8	0.5
		Sf	3350	21.6	22.5	0.9
		Sf	3351	21.5	22.4	0.9
	MEAN			21.4	22.2	0.8
	Standard-deviation			1.0	0.8	0.2
2	20%	Sf	3369	19.7	19.9	0.2
		Sf	3370	23.4	24.1	0.7
		Sf	3371	20.5	20.7	0.2
		Sf	3372	22.9	23.4	0.5
	MEAN			21.6	22.0	0.4
	Standard-deviation			1.8	2.0	0.2
3	40%	Sf	3373	22.5	23.7	1.2
		Sf	3374	20.8	21.0	0.2
		Sf	3375	22.3	23.2	0.9
		Sf	3376	21.3	22.8	1.5
	MEAN			21.7	22.7	0.9
	Standard-deviation			0.8	1.2	0.6
4	60%	Sf	3378	21.2	22.8	1.6
		Sf	3379	21.7	22.9	1.2
		Sf	3380	20.5	21.2	0.7
		Sf	3381	21.1	22.8	1.7
	MEAN			21.1	22.4	1.3
	Standard-deviation			0.5	0.8	0.5

Table

Groups	Test item	BrdU-index (mean*)	Stimulation Index SI (mean + standard			Result	EC1.6 value
1	PG	0.252
			n.a			n.a	n.a.
2	20%	0.312	1.24	±	0.03	negative	n.a.
3	40%	0.251	0.99	±	0.21	negative
4	60%	0.244	0.97	±	0.23	negative

Table 

Groups	Test item	Animal No.		BrdU-index (DO Indiv)	BrdU-index (DO mean)	BrdU-index mean*	Stimulation Index S.I. (indiv  ± Standard deviation)
1	PG	Sf	3348	0.279		0.252
				0.283 0.267	0.277		n.a
		Sf	3349	0.248 0.213 0.232	0.231
							n.a
		Sf	3350	0.292 0.222 0.180	0.232
							n.a
		Sf	3351	0.276 0.317 0.214	0.269
							n.a
2	20%	Sf	3369	0.326 0.314 0.303	0.315	0.312	1.25	±	0.05
		Sf	3370	0.356 0.292 0.266	0.305		1.21	±	0.18
		Sf	3371	0.372 0.290 0.260	0.308		1.22	±	0.23
		Sf	3372	0.403 0.346 0.211	0.320		1.27	±	0.39
3	40%	Sf	3373	0.219 0.277 0.252	0.250	0.251	0.99	±	0.12
		Sf	3374	0.472 0.286 0.218	0.326		1.29	±	0.52
		Sf	3375	0.253 0.216 0.142	0.204		0.81	±	0.22
		Sf	3376	0.254 0.202 0.212	0.223		0.88	±	0.11
4	60%	Sf	3378	0.280 0.332 0.313	0.309	0.244	1.22	±	0.10
		Sf	3379	0.240 0.288 0.245	0.258		1.02	±	0.10
		Sf	3380	0.147 0.136 0.221	0.168		0.67	±	0.18
		Sf	3381	0.296 0.307 0.116	0.240		0.95	±	0.43

Table Ear

Groups	Test item	Ear thickness increase D6/D1 (%)	Biopsy ear weight Increase (%)	Excessive irritation #
1	PG	-3.5
			n.a	No
2	20%	-1.2	-0.6	No
3	40%	-2.4	0.9	No
4	60%	-4.8	-8.7	No

Interpretation of results:

GHS criteria not met

Remarks:

EU criteria

Conclusions:

The test substance does not have to be classified as a skin sensitizer.

Executive summary:

The skin sensisitzing properties of the test item were determined according to OECD 442b under GLP conditions. The test substance was dissolved in Propylene glycol and a preliminary study conducted with a single mouse. The test item (25µL in the vehicle (60%) was applied daily for three consecutive days to the dorsal surface of the ear. The daily observations, ear thickness and body weight measurments showed no signs of toxicity after 6 days. Therefore, 3 groups of 4 mice and a control group of four mice recieved 60%, 40% and 20% concentration of the test item in the vehicle applied in the same manner as in the preliminary study. The Clinical observations, body weight, ear thickness and the BrdU measurments by ELISA showed no signs of sensitizing properties. The Stimulation Index was 0.97, 0.99 and 1.24 for 60%,40% and 20% respectively and thus lower than 1.6 and no EC1.6 could be estabilsed. The test substance does not have to be classified as a skin sensitizer.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The skin sensisitzing properties of the test item were determined according to OECD 442b under GLP conditions. The test substance was dissolved in Propylene glycol and a preliminary study conducted with a single mouse. The test item (25µL in the vehicle (60%) was applied daily for three consecutive days to the dorsal surface of the ear. The daily observations, ear thickness and body weight measurments showed no signs of toxicity after 6 days. Therefore, 3 groups of 4 mice and a control group of four mice recieved 60%, 40% and 20% concentration of the test item in the vehicle applied in the same manner as in the preliminary study. The Clinical observations, body weight, ear thickness and the BrdU measurments by ELISA showed no signs of sensitizing properties. The Stimulation Index was 0.97, 0.99 and 1.24 for 60%,40% and 20% respectively and thus lower than 1.6 and no EC1.6 could be estabilsed. The test substance does not have to be classified as a skin sensitizer.

Justification for classification or non-classification

Based on the available data, the substance is not classified for skin sensitization according to CLP Regulation no. 1272/2008.