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Toxicological information

Skin sensitisation

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
ethyl (2E)-3-(cyclopropylamino)-2-[(2,4-dichloro-3-methylphenyl)carbonyl]prop-2-enoate
Molecular formula:
C16H17Cl2N1O3
IUPAC Name:
ethyl (2E)-3-(cyclopropylamino)-2-[(2,4-dichloro-3-methylphenyl)carbonyl]prop-2-enoate
Test material form:
solid: particulate/powder

In vivo test system

Test animals

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

Study design: in vivo (LLNA)

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



Results and discussion

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.

In vivo (LLNA)

Resultsopen allclose all
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

Any other information on results incl. tables

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


Applicant's summary and conclusion

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.

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