5-(3-Phenylpropioloyl)-1,3-isobenzofurandione

Endpoint summary

Description of key information

NEXAMITE® A56 was shown to have sensitisation potential (sensitiser) in the Local Lymph Node Assay. The calculated EC3 value of NEXAMITE® A56 is 0.09 % (w/v).

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:

10 June 2015 to 16 June 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD Guidelines for Testing of Chemicals No.429, Skin Sensitisation: Local Lymph Node Assay (Adopted 22 July 2010)

Deviations:

yes

Remarks:

See "Any other information" for details

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

Commission Regulation (EC) No. 440/2008 of 30 May 2008, B.42. Skin Sensitisation: Local Lymph Node Assay (Official Journal L 142, 31/05/2008) as amended by Commission Regulation (EU) No 640/2012 of 6 July 2012

Deviations:

yes

Remarks:

See "Any other information" for details

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

No further details specified in the study report.

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Species and strain: CBA/CaOlaHsd mice
Source: Envigo (formerly: Harlan Laboratories S.r.l.), San Pietro al Natisone (UD), Zona Industriale Azzida, 57, 33049 Italy
Hygienic level: Specific Pathogen Free (SPF) at arrival; standard housing conditions during the study
Justification of strain: On the basis of OECD Guideline, mice of CBA/Ca or CBA/J strain can be used. Females are used because the existing database is predominantly based on females.
Number of animals: 4 animals / group
Sex: Female, nulliparous, non pregnant
Age of animals at starting: 9 weeks old (age-matched, within one week)
Body weight range at starting: 19.2-19.9 grams (The weight variation in animals in the study did not exceed ± 20 % of the mean weight.)
Acclimatization time: 7 days
Note: In the Preliminary Experiment I, mice of 9 weeks of age (21.9-23.3 grams); in the Preliminary Experiment II, mice of 10 weeks of age (19.1-19.3 grams); in the Preliminary Experiment III, mice of 13 weeks of age (21.9-22.6 grams) were used.

Husbandry
Animal health: Only healthy animals were used for the study. Health status was veterinarian certified.
Housing / Enrichment: Group caging / mice were provided with glass tunnel-tubes
Cage type: Type II. polypropylene / polycarbonate
Bedding: Bedding was available to animals during the study
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 18.0 - 25.9 °C (see Section 7, Deviations to the Study Plan)
Relative humidity: 31 - 79 % (see Section 7, Deviations to the Study Plan)
Ventilation: 15-20 air exchanges/hour
Temperature and relative humidity were recorded twice every day during the acclimatisation and experimental phases.
Room 244 Cabinet 7 (non-radioactive phase, Preliminary Experiment I)
Room 244 Cabinet 5 (non-radioactive phase, Preliminary Experiment II)
Room 244 Cabinet 2 (non-radioactive phase, Preliminary Experiment III)
Room 244 Cabinet 3 (non-radioactive phase, Main Test)
Room 139-140 (radioactive phase)

Food and feeding
Animals received ssniff® SM Rat/Mouse – “Breeding & Maintenance, 15 mm, autoclavable Complete diet for rats/mice” (Batch number: 814 3108, Expiry date: 31 August 2015) produced by ssniff Spezialdiäten GmbH (Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany), ad libitum. The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The contents of the standard diet are detailed in Appendix 3.

Water supply
Animals received tap water from the municipal supply from 500 mL bottles, ad libitum. Water quality control analysis was performed once every three months and microbiological assessment was performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József Attila u. 36.,Hungary). Copies of the relevant Certificates of Analysis are retained in the Archive at CiToxLAB Hungary Ltd.

Bedding
Bedding of certified wood chips especially designed to keep animals in the best natural environment was provided for animals during the study. Lignocel 3/4-S Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH & Co.KG (Holzmühle 1, 73494 Rosenberg, Germany) was available to animals during the study. Nest building material was also provided (Enviro-Dri produced by LBS).

Identification and randomisation
A unique number written on the tail with a permanent marker identified each animal. The animal number was assigned on the basis of CiToxLAB Hungary Ltd.’s master file. Cages were marked with identity cards with information including study code, cage number, dose group, sex and individual animal number. All animals were randomised and allocated to experimental groups. The randomisation was checked by computer software using the body weight to verify the homogeneity and variability between groups.

Vehicle:

dimethyl sulphoxide

Concentration:

0.1, 0.25, 0.5, 1 % (w/v)

No. of animals per dose:

4 animals per dose group

Details on study design:

ADMINISTRATION OF THE TEST ITEM
Dose Selection and Justification of Dose Selection
The maximum possible concentration of test item in an acceptable solvent was established according to OECD guideline 429. Based on the observation of the solubility test, the maximum possible concentration was 50 % (w/v).
A Preliminary Irritation/Toxicity Test was performed on CBA/CaOlaHsd mice using two concentrations (2 animals/concentration), at test item concentrations of 50 and 25 % (w/v) in DMSO. Based on the observed increased ear thickness values, indicating local skin irritation, and the observed body weight loss, an additional test was performed (Preliminary Irritation/Toxicity Test II) using two concentrations (2 animals/concentration) at test item concentrations of 10 and 5 % (w/v) in DMSO. Based on the observed increased ear thickness values, indicating local skin irritation, and the observed body weight loss, an additional test was performed (Preliminary Irritation/Toxicity Test III) using two concentrations (2 animals/concentration) at test item concentrations of 2.5 and 1 % (w/v) in DMSO. The preliminary experiments were conducted in a similar experimental manner to the main study, but were terminated on Day 6 (inclusive of body weight measurement). No radioactive proliferation assay was performed.
In the Preliminary Irritation / Toxicity Tests, all mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Both ears of each mouse were observed for erythema and scored according to OECD Guidelines for Testing of Chemicals No. 404, Table 2. Ear thickness was also measured using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Additional quantification of ear thickness was performed by ear punch weight determination after euthanasia of experimental animals.
During the Preliminary Irritation / Toxicity Tests no mortality or signs of systemic toxicity were observed. Test item precipitate or minimal amount of test item precipitate was observed on the ears of all animals on Days 1-6 in the 50 and 25 % (w/v) dose groups, on Days 2-5 in the 10 % (w/v) group and on Day 3 in the 5 % (w/v) group. Due to the presence of test item precipitate on the ears, erythema could not be scored at some time points by examining only the outer surface. In these instances, erythema was scored inside the ears of the animals, however no erythema was observed. Marked body weight loss (≥5%) was detected for both animals in the 50 and 2.5 % (w/v) dose groups and for one-one animal in the 25 and 10 % (w/v) dose groups.
Ear thickness of the animals was measured using a thickness gauge on Days 1, 3 and 6. Increased ear thickness values were detected in the 50, 25, 10, 5 and 2.5 % (w/v) dose groups indicating excessive local skin irritation. The ear thickness values were slightly increased in the 1 % (w/v) dose group. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of experimental animals. Ear punch weights were out of the historical control range for one animal in the 50 % (w/v) group, for all animals in the 10 and 5 % (w/v) group and for one animal in the 2.5 % (w/v) group.
The draining auricular lymph nodes of animals were visually examined and were considered larger than normal for all animals in the preliminary experiments. (This was a subjective judgement by comparison with observations of former experiments).
Based on these results, concentrations of 50, 25, 10, 5 and 2.5 % (w/v) may have exceeded the maximum local skin irritation criteria and/or body weight loss in the main experiment. Therefore, an additional dose group (a total of four) was used in the main experiment to ensure that there were three acceptable dose groups for evaluation. Therefore, 1, 0.5, 0.25 and 0.1 % (w/v) concentrations were examined in the main test. Ear thickness of the experimental animals in the main test was measured using a thickness gauge on Days 1 and 6 for additional confirmation that skin irritation limits were not exceeded.

Topical application
During the study, animals were topically dosed with 25 μL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

PROLIFERATION ASSAY
Injection of Tritiated Thymidine (3HTdR)
On Day 6, each mouse was intravenously injected via the tail vein with 250 μL of sterile PBS (phosphate buffered saline) containing approximately 20 μCi of 3HTdR using a gauge 25G x 1" hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).

Removal and Preparation of Draining Auricular Lymph Nodes
Five hours (± 30 minutes) after intravenous injection the mice were euthanized by asphyxiation with ascending doses of carbon dioxide (deep anaesthesia was confirmed before making incision, death was confirmed before discarding carcasses).
The draining auricular lymph nodes were excised by making a small incision on the skin between the jaw and sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. The nodes were then removed using forceps. The carcasses were discarded after cervical dislocation or after cutting through major cervical blood vessels. Once removed, the nodes of mice from each test group were pooled and collected in separate Petri dishes containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing.

Preparation of Single Cell Suspension of Lymph Node Cells
A single cell suspension (SCS) of pooled lymph node cells (LNCs) was prepared and collected in disposable tubes by gentle mechanical disaggregating of lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). Pooled LNCs were pelleted with a relative centrifugal force (RCF) of 190 x g (approximately) for 10 minutes at 4 °C.
After centrifugation supernatants were discarded. Pellets were gently resuspended and 10 mL of PBS added to the tubes. The washing step was repeated twice. This procedure was repeated for each group of pooled lymph nodes.

Determination of Incorporated 3HTdR
After the final washing step, supernatants were removed. Pellets were then gently agitated and resuspended in 3 mL of 5 % (w/v) TCA solution for precipitation of macromolecules.
After overnight (approximately 18 hours) incubation at 2-8 °C, precipitates were centrifuged (approximately 190 x g for 10 minutes at 4oC), and supernatants removed. Pellets were resuspended in 1 mL of 5 % (w/v) TCA solution and dispersed using an ultrasonic bath. Samples were transferred into a suitable sized scintillation vial filled with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded into a β-scintillation counter and 3HTdR incorporation measured (10-minute measurement).
The β-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM). Background level was also measured in duplicates by adding 1 mL of 5 % (w/v) TCA solution into a scintillation vial filled with 10 mL of scintillation liquid.

OBSERVATIONS
Clinical Observations
During the study (Day 1 to Day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Individual records were maintained.

Measurement of Ear Thickness
Ear thickness of the experimental animals in the main test was determined using a thickness gauge on Days 1 and 6.

Measurement of Body Weight
Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.

Positive control substance(s):

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

Positive control results:

The result of the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in DMSO was used to demonstrate the appropriate performance of the assay. The positive control substance was examined at 25 % in the same vehicle as used for the test item (DMSO) using CBA/CaOlaHsd mice.
No mortality or signs of toxicity were observed for the positive control substance. However increased ear thickness values were observed for some animals in the positive control group which is not unusual with the selected vehicle according to the historical control data. A significant lymphoproliferative response (stimulation index value of 7.0) was noted for HCA in the main experiment. This value confirmed the appropriate performance of the assay.

Key result

Parameter:

EC3

Value:

0.09

Key result

Parameter:

SI

Value:

9.7

Test group / Remarks:

1% (w/v)

Key result

Parameter:

SI

Value:

13.2

Test group / Remarks:

0.5% (w/v)

Key result

Parameter:

SI

Value:

10.7

Test group / Remarks:

0.25% (w/v)

Key result

Parameter:

SI

Value:

3.6

Test group / Remarks:

0.1% (w/v)

Cellular proliferation data / Observations:

CLINICAL OBSERVATION
No mortality or signs of systemic toxicity were observed during the study.

EAR THICKNESS MEASUREMENT
One out of four animals in the 1 % (w/v) dose group had significantly increased ear thickness values. The ear swelling of the three remaining animals in this group did not reach the limit of excessive local skin irritation criteria (>25% increase of the ear thickness). Therefore this group as well as the other three test item treated dose groups are considered acceptable for evaluation. Increased ear thickness values were observed for some animals in the positive control group which is not unusual with the selected vehicle according to the historical control data.

BODY WEIGHT MEASUREMENT
No treatment related effects were observed on the body weight changes of experimental animals.

PROLIFERATION ASSAY
The results of the proliferation assay are summarized in Table 5 and Figure 1. The visual appearance of the lymph nodes was normal in the negative (vehicle) control group. Larger than normal lymph nodes were observed in the 1, 0.5 and 0.25 % (w/v) dose groups as well as in the positive control group. Slightly enlarged lymph nodes were observed in the 0.1 % (w/v) group.
The stimulation index values were 9.7, 13.2, 10.7 and 3.6 at concentrations of 1, 0.5, 0.25 and 0.1 % (w/v), respectively.
Based on these results, the shape of the curve of the sensitisation potential given in Figure 1 and the fact this chemical is quoted in the documentation from the client as a modified trimellitic anhydride, it is considered that the test item in higher concentration is likely to have cytotoxic effects. The profile with increased concentration of increasing sensitisation response, with a plateau, then a decrease in proliferation is typical for strong sensitisers with cytotoxic potential.

Individual Body Weights for all Animals with Group Means

Identity Number	Animal Number	Test Group Name	Initial Body Weight (g)	Terminal Body Weight* (g)	Change# (%)
8846 8882 8858 8870	1 2 3 4	Negative (vehicle) control DMSO     Mean	19.2 19.9 19.3 19.7 19.5	19.6 19.5 20.1 20.5 19.9	2.1 -2.0 4.1 4.1 2.1
8848 8884 8860 8872	5 6 7 8	NEXAMITE® A56 1% (w/v) in DMSO   Mean	19.9 19.3 19.5 19.3 19.5	21.4 18.6 21.4 19.0 20.1	7.5 -3.6 9.7 -1.6 3.0
8850 8886 8862 8874	9 10 11 12	NEXAMITE® A56 0.5% (w/v) in DMSO   Mean	19.8 19.4 19.5 19.3 19.5	20.0 18.8 18.8 20.3 19.5	1.0 -3.1 -3.6 5.2 -0.1
8852 8888 8864 8876	13 14 15 16	NEXAMITE® A56 0.25% (w/v) in DMSO   Mean	19.4 19.7 19.3 19.6 19.5	20.3 20.5 20.2 19.9 20.2	4.6 4.1 4.7 1.5 3.7
8854 8890 8866 8878	17 18 19 20	NEXAMITE® A56 0.1% (w/v) in DMSO   Mean	19.3 19.9 19.3 19.8 19.6	19.8 19.6 19.8 19.7 19.7	2.6 -1.5 2.6 -0.5 0.8
8856 8892 8868 8880	21 22 23 24	Positive control 25% (w/v) HCA in DMSO   Mean	19.7 19.3 19.7 19.2 19.5	19.3 20.7 19.4 19.0 19.6	-2.0 7.3 -1.5 -1.0 0.7

*: Terminal body weights were measured on Day 6.

#: = (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100

 

DPM, DPN and Stimulation Index Values for all Groups

Test Group Name	Measured DPM / group	DPM	Number of lymph nodes	DPN	Stimulation Index
Background (5% (w/v) TCA)	33 32	-	-	-	-
Negative (vehicle) control (DMSO)	3955	3922.5	8	490.3	1.0
NEXAMITE® A56 1% (w/v) in DMSO	38038	38005.5	8	4750.7	9.7
NEXAMITE® A56 0.5% (w/v) in DMSO	51687	51654.5	8	6456.8	13.2
NEXAMITE® A56 0.25% (w/v) in DMSO	42159	42126.5	8	5265.8	10.7
NEXAMITE® A56 0.1% (w/v) in DMSO	14281	14248.5	8	1781.1	3.6
Positive control (25% (w/v) HCA in DMSO)	27620	27587.5	8	3448.4	7.0

 

Results of the Preliminary Irritation / Toxicity Test I

Individual Body Weights for all Animals with group Means (Preliminary Experiment I)

Animal Number	Identity Number	Test Group Name	Initial Body Weight (g)	Terminal Body Weight* (g)	Change# (%)
8453 8457	1 2	50% (w/v) 50% (w/v) Mean	23.3 21.9 22.6	22.1 20.2 21.2	-5.2 -7.8 -6.5
8455 8459	3 4	25% (w/v) 25% (w/v) Mean	22.5 22.1 22.3	21.1 21.4 21.3	-6.2 -3.2 -4.7

Notes:

1. *: Terminal body weight were measure on Day 6

2. #: = (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100

 

Individual Ear Thickness for all Animals (Preliminary Experiment I)

Identity Number	Animal Number	Test Group Name	Ear Thickness on Day 1 (mm)		Ear Thickness on Day 3 (mm)		Ear Thickness on Day 6 (mm)		Biopsy weight* on Day 6 (mg)
			Right	Left	Right	Left	Right	Left
8453 8457 8455 8459	1 2 3 4	50% (w/v) 50% (w/v) 25% (w/v) 25% (w/v)	0.20 0.21 0.22 0.21	0.22 0.21 0.22 0.21	0.23 0.24 0.25 0.24	0.33 0.23 0.24 0.24	0.42 0.33 0.37 0.37	0.35 0.30 0.33 0.35	22.86 21.89 21.47 22.11

Note:

1. *: Historical control range: 11.92-22.53 mg. Positive response is over 28.16 mg (≥25%)

 

Summarized Clinical Observations (Preliminary Experiment I)

Period	Group	Animal No.	Identity No.	Clinical observations
DAY 1	50% (w/v)	1	8453	Before treatment: symptom-free, ES: 0 After treatment: symptom free*, ES: 0
		2	8457	Before treatment: symptom-free, ES: 0 After treatment: symptom free*, ES: 0
	25% (w/v)	3	8455	Before treatment: symptom-free, ES: 0 After treatment: symptom free**, ES: 0
		4	8459	Before treatment: symptom-free, ES: 0 After treatment: symptom free**, ES: 0
DAY 2	50% (w/v)	1	8453	Before treatment: symptom-free**, ES: 0 After treatment: symptom free*, ES: i.s.: 0
		2	8457	Before treatment: symptom-free**, ES: 0 After treatment: symptom free*, ES: i.s.: 0
	25% (w/v)	3	8455	Before treatment: symptom-free**, ES: 0 After treatment: symptom free*, ES: 0
		4	8459	Before treatment: symptom-free**, ES: 0 After treatment: symptom free*, ES: 0
DAY 3	50% (w/v)	1	8453	Before treatment: symptom-free*, ES: 0 After treatment: symptom free*, ES: i.s.: 0
		2	8457	Before treatment: symptom-free*, ES: 0 After treatment: symptom free*, ES: i.s.: 0
	25% (w/v)	3	8455	Before treatment: symptom-free*, ES: 0 After treatment: symptom free*, ES: 0
		4	8459	Before treatment: symptom-free*, ES: 0 After treatment: symptom free*, ES: 0
DAY 4	50% (w/v)	1	8453	Symptom-free*, ES: 0
		2	8457	Symptom-free*, ES: 0
	25% (w/v)	3	8455	Symptom-free*, ES: 0
		4	8459	Symptom-free*, ES: 0
DAY 5	50% (w/v)	1	8453	Symptom-free*, ES: 0
		2	8457	Symptom-free*, ES: 0
	25% (w/v)	3	8455	Symptom-free**, ES: 0
		4	8459	Symptom-free*, ES: 0
DAY 6	50% (w/v)	1	8453	Symptom-free**, ES: 0
		2	8457	Symptom-free**, ES: 0
	25% (w/v)	3	8455	Symptom-free**, ES: 0
		4	8457	Symptom-free**, ES: 0

Notes:

1. The clinical observations of animals on the first day was performed simultaneously with the body weight measurements.

2. ES: Erythema score

3. *: test item precipitate, **: minimal amount of test item precipitate

4. i.s.: Score made on inner surface of ear. (Due to the test item precipitate on the ears of the animals, erythema could not be scored at some points. If the erythema could not be scored on the outer surface, it was scored inside the ears of the animals).

 

Results of the Preliminary Irritation / Toxicity Test II

Individual Body Weights for all Animals with group Means (Preliminary Experiment II)

Animal Number	Identity Number	Test Group Name	Initial Body Weight (g)	Terminal Body Weight* (g)	Change# (%)
8725 8729	1 2	10% (w/v) 10% (w/v) Mean	19.2 19.3 19.3	18.2 20.4 19.3	-5.2 5.7 0.2
8727 8731	3 4	5% (w/v) 5% (w/v) Mean	19.3 19.1 19.2	19.5 18.7 19.1	1.0 -2.1 -0.5

Notes:

1. *: Terminal body weight were measure on Day 6

2. #: = (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100

 

Individual Ear Thickness for all Animals (Preliminary Experiment II)

Identity Number	Animal Number	Test Group Name	Ear Thickness on Day 1 (mm)		Ear Thickness on Day 3 (mm)		Ear Thickness on Day 6 (mm)		Biopsy weight* on Day 6 (mg)
			Right	Left	Right	Left	Right	Left
8725 8729 8727 8731	1 2 3 4	10% (w/v) 10% (w/v) 5% (w/v) 5% (w/v)	0.21 0.21 0.22 0.20	0.20 0.21 0.21 0.21	0.23 0.24 0.22 0.21	0.22 0.24 0.21 0.22	0.35 0.31 0.33 0.35	0.31 0.35 0.35 0.32	25.67 27.35 24.98 25.70

Note:

1. *: Historical control range: 11.92-22.53 mg. Positive response is over 28.16 mg (≥25%)

 

Summarized Clinical Observations (Preliminary Experiment II)

Period	Group	Animal No.	Identity No.	Clinical observations
DAY 1	10% (w/v)	1	8725	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
		2	8729	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
	5% (w/v)	3	8727	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
		4	8731	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
DAY 2	10% (w/v)	1	8725	Before treatment: symptom-free, ES: 0 After treatment: symptom free*, ES: 0
		2	8729	Before treatment: symptom-free, ES: 0 After treatment: symptom free*, ES: 0
	5% (w/v)	3	8727	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
		4	8731	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
DAY 3	10% (w/v)	1	8725	Before treatment: symptom-free**, ES: 0 After treatment: symptom free*, ES: 0
		2	8729	Before treatment: symptom-free**, ES: 0 After treatment: symptom free*, ES: 0
	5% (w/v)	3	8727	Before treatment: symptom-free, ES: 0 After treatment: symptom free**, ES: 0
		4	8731	Before treatment: symptom-free, ES: 0 After treatment: symptom free**, ES: 0
DAY 4	10% (w/v)	1	8725	Symptom-free**, ES: 0
		2	8729	Symptom-free**, ES: 0
	5% (w/v)	3	8727	Symptom-free, ES: 0
		4	8731	Symptom-free, ES: 0
DAY 5	10% (w/v)	1	8725	Symptom-free**, ES: 0
		2	8729	Symptom-free**, ES: 0
	5% (w/v)	3	8727	Symptom-free, ES: 0
		4	8731	Symptom-free, ES: 0
DAY 6	10% (w/v)	1	8725	Symptom-free, ES: 0
		2	8729	Symptom-free, ES: 0
	5% (w/v)	3	8727	Symptom-free, ES: 0
		4	8731	Symptom-free, ES: 0

Notes:

1. The clinical observations of animals on the first day was performed simultaneously with the body weight measurements.

2. ES: Erythema score

3. *: test item precipitate, **: minimal amount of test item precipitate

 

Results of the Preliminary Irritation / Toxicity Test III

Individual Body Weights for all Animals with group Means (Preliminary Experiment III)

Animal Number	Identity Number	Test Group Name	Initial Body Weight (g)	Terminal Body Weight* (g)	Change# (%)
8558 8562	1 2	2.5% (w/v) 2.5% (w/v) Mean	21.9 22.6 22.3	20.7 20.0 20.4	-5.5 -11.5 -8.5
8560 8563	3 4	1% (w/v) 1% (w/v) Mean	22.1 22.5 22.3	21.3 22.4 21.9	-3.6 -0.4 -2.0

Notes:

1. *: Terminal body weight were measure on Day 6

2. #: = (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100

 

Individual Ear Thickness for all Animals (Preliminary Experiment III)

Identity Number	Animal Number	Test Group Name	Ear Thickness on Day 1 (mm)		Ear Thickness on Day 3 (mm)		Ear Thickness on Day 6 (mm)		Biopsy weight* on Day 6 (mg)
			Right	Left	Right	Left	Right	Left
8558 8562 8560 8563	1 2 3 4	2.5% (w/v) 2.5% (w/v) 1% (w/v) 1% (w/v)	0.22 0.22 0.20 0.22	0.22 0.21 0.21 0.23	0.24 0.25 0.24 0.23	0.23 0.26 0.23 0.24	0.33 0.39 0.30 0.27	0.32 0.34 0.25 0.25	22.11 23.60 17.83 17.94

Note:

1. *: Historical control range: 11.92-22.53 mg. Positive response is over 28.16 mg (≥25%)

 

Summarized Clinical Observations (Preliminary Experiment III)

Period	Group	Animal No.	Identity No.	Clinical observations
DAY 1	225% (w/v)	1	8558	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
		2	8562	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
	1% (w/v)	3	8560	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
		4	8563	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
DAY 2	2.5% (w/v)	1	8558	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
		2	8562	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
	1% (w/v)	3	8560	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
		4	8563	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
DAY 3	2.5% (w/v)	1	8558	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
		2	8562	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
	1% (w/v)	3	8560	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
		4	8563	Before treatment: symptom-free, ES: 0 After treatment: symptom free, ES: 0
DAY 4	2.5% (w/v)	1	8558	Symptom-free, ES: 0
		2	8562	Symptom-free, ES: 0
	1% (w/v)	3	8560	Symptom-free, ES: 0
		4	8563	Symptom-free, ES: 0
DAY 5	2.5% (w/v)	1	8558	Symptom-free, ES: 0
		2	8562	Symptom-free, ES: 0
	1% (w/v)	3	8560	Symptom-free, ES: 0
		4	8563	Symptom-free, ES: 0
DAY 6	2.5% (w/v)	1	8558	Symptom-free, ES: 0
		2	8562	Symptom-free, ES: 0
	1% (w/v)	3	8560	Symptom-free, ES: 0
		4	8563	Symptom-free, ES: 0

Notes:

1. The clinical observations of animals on the first day was performed simultaneously with the body weight measurements.

2. ES: Erythema score

 

Summarized Clinical Observations and Individual Ear Thickness Values

Individual Ear Thickness for all Animals

Identity Number	Animal Number	Test Group Name	Ear Thickness on Day 1 (mm)		Ear Thickness on Day 6 (mm)
			Right	Left	Right	Left
8846 8882 8858 8870	1 2 3 4	Negative (vehicle) control DMSO	0.20 0.20 0.20 0.20	0.20 0.20 0.21 0.21	0.23 0.22 0.20 0.20	0.23 0.21 0.21 0.22
8848 8884 8860 8872	5 6 7 8	NEXAMITE® A56 1% (w/v) in DMSO	0.20 0.20 0.20 0.20	0.21 0.21 0.21 0.20	0.21 0.22 0.25 0.24	0.24 0.23 0.31 0.24
8850 8886 8862 8874	9 10 11 12	NEXAMITE® A56 0.5% (w/v) in DMSO	0.20 0.20 0.22 0.20	0.20 0.21 0.20 0.21	0.21 0.24 0.22 0.21	0.21 0.22 0.22 0.23
8852 8888 8864 8876	13 14 15 16	NEXAMITE® A56 0.25% (w/v) in DMSO	0.20 0.20 0.21 0.20	0.20 0.20 0.20 0.21	0.21 0.23 0.24 0.22	0.21 0.23 0.23 0.22
8854 8890 8866 8878	17 18 19 20	NEXAMITE® A56 0.1% (w/v) in DMSO	0.20 0.20 0.21 0.20	0.20 0.20 0.21 0.20	0.23 0.23 0.23 0.22	0.22 0.23 0.22 0.23
8856 8892 8868 8880	21 22 23 24	Positive control 25% (w/v) HCA in DMSO	0.21 0.20 0.20 0.21	0.20 0.20 0.20 0.21	0.33 0.27 0.25 0.25	0.35 0.29 0.24 0.25

 

Summarized Clinical Observations

Group	Identity No.	CLINICAL OBSERVATIONS
		DAY 1	DAY 2	DAY 3	DAY 4	DAY 5	DAY 6
Negative control (DMSO)	8846   8882   8858   8870	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free
NEXAMITE® A56 1% (w/v) in DMSO	8848   8884   8860   8872	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free
NEXAMITE® A56 0.5% (w/v) in DMSO	8850   8886   8862   8874	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free
NEXAMITE® A56 0.25% (w/v) in DMSO	8852   8888   8864   8876	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free
NEXAMITE® A56 0.1% (w/v) in DMSO	8854   8890   8866   8878	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free
Positive control 25% (w/v) HCA in DMSO	8856   8892   8868   8880	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free

Note:

1. BT: before treatment, AT: after treatment

 

Historical Control Data

Historical Control Data of the Positive and Negative Controls for CBA/J Rj mice (2008-2014)

CBA/J Rj mice
	Vehicles
	Acetone: Olive oil 4: 1 (AOO)			1% Pluronic PE9200 in water (1%Plu)
	DPN values		SI value	DPN values		SI value
	Control	HCA 25%	HCA 25%	Control	HCA 25%	HCA 25%
Average	162.3	1627.0	11.7	117.0	931.2	8.6
Range:min            max	36.4 586.9	304.9 4602.1	3.2 52.7	21.4 469.6	146.3 6258.1	1.8 55.1
Number of cases	106	81	81	127	84	84

 

	Vehicles
	N,N-Dimethylformamide (DMF)			Dimethyl sulfoxide (DMSO)
	DPN values		SI value	DPN values		SI value
	Control	HCA 25%	HCA 25%	Control	HCA 25%	HCA 25%
Average	165.6	2484.6	14.7	210.0	2278.2	11.2
Range:min            max	20.8 1843.0	350.9 28287.0	3.8 75.7	80.6 424.3	848.2 5441.2	4.8 24.1
Number of cases	90	56	56	28	20	20

 

	Vehicles
	Propylene glycol (PG)			Methyl ethyl ketone (MEK)
	DPN values		SI value	DPN values		SI value
	Control	HCA 25%	HCA 25%	Control	HCA 25%	HCA 25%
Average	120.7	1488.1	13.4	203.9	2525.5	14.0
Range:min            max	38.4 288.8	510.4 3231.3	3.7 27.9	72.2 572.2	412.6 4056.4	4.4 26.5
Number of cases	22	21	21	11	9	9

HCA 25% = alpha-Hexylcinnamaldehyde 25% (w/v)

SI (Stimulation Index) = DPN of a treated group divided by DPN of the appropriate control group.

DPN (Disintegrations Per Node) = DPM (Disintegrations Per Minute) divided by the number of lymph nodes.

In case of individual approach, SI values were calculated from the mean DPN value of the group.

 

Historical Control Data of the Positive and Negative Controls for CBA/CaOlaHsd mice (2014-2015)

CBA/CaOlaHsd mice
	Vehicles
	Acetone: Olive oil 4: 1 (AOO)			1% Pluronic PE9200 in water (1%Plu)
	DPN values		SI value	DPN values		SI value
	Control	HCA 25%	HCA 25%	Control	HCA 25%	HCA 25%
Average	415.2	2922.6	7.5	197.7	1825.3	10.0
Range:min            max	111.3 847.3	890.3 7674.5	3.3 15.5	23.0 680.8	154.0 6755.8	3.0 33.1
Number of cases	32	32	30	134	134	128

 

	Vehicles
	N,N-Dimethylformamide (DMF)			Dimethyl sulfoxide (DMSO)
	DPN values		SI value	DPN values		SI value
	Control	HCA 25%	HCA 25%	Control	HCA 25%	HCA 25%
Average	244.6	2522.6	10.8	488.7	3212.1	7.8
Range:min            max	140.8 505.8	1201.3 4804.6	6.3 21.3	238.5 934.6	2017.2 4877.5	3.1 14.5
Number of cases	21	21	21	13	13	12

 

	Vehicles
	Propylene glycol (PG)			Methyl ethyl ketone (MEK)
	DPN values		SI value	DPN values		SI value
	Control	HCA 25%	HCA 25%	Control	HCA 25%	HCA 25%
Average	235.4	2371.8	10.0	260.2	4888.8	19.5
Range:min            max	63.3 506.0	817.3 4978.0	6.5 14.4	183.5 383.3	2456.3 8682.5	8.9 36.3
Number of cases	14	14	14	9	10	10

HCA 25% = alpha-Hexylcinnamaldehyde 25% (w/v)

SI (Stimulation Index) = DPN of a treated group divided by DPN of the appropriate control group.

DPN (Disintegrations Per Node) = DPM (Disintegrations Per Minute) divided by the number of lymph nodes.

In case of individual approach, SI values were calculated from the mean DPN value of the group.

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

In conclusion, under the conditions of the present assay NEXAMITE® A56, tested in a suitable vehicle, was shown to have sensitisation potential (sensitiser) in the Local Lymph Node Assay. The calculated EC3 value of NEXAMITE® A56 is 0.09 % (w/v).

Executive summary:

The aim of this study was to determine the skin sensitisation potential of NEXAMITE® A56 following dermal exposure. The study was performed with vertebrate animals as no regulatory in vitro alternative is available. The minimum number of animals was used, corresponding to the regulatory guidelines being followed.

 

Based on the results of the Preliminary Compatibility Test and on the recommendations of the OECD Guideline, the test item was formulated in Dimethyl sulfoxide (abbreviated as DMSO). Due to the physical characteristics of the test item the maximum possible concentration was 50 % (w/v).

 

The Preliminary Irritation / Toxicity Tests were performed in CBA/CaOlaHsd mice using six test concentrations: 50, 25, 10, 5, 2.5 and 1 % (w/v) in DMSO. Based on the observations recorded in the preliminary tests, 1 % (w/v) was selected as highest concentration for the main test as higher concentrations had indicated they may exceed the maximum permitted local skin irritation criteria and/or body weight loss. A total of four dose groups were selected for the main experiment to ensure three acceptable dose groups for evaluation, in case the 1% concentration exceeded permitted irritation limits.

 

In the main assay, twenty-four female CBA/CaOlaHsd mice were allocated to six groups of four animals each:

- four groups received NEXAMITE® A56 (formulated in DMSO) at 1, 0.5, 0.25 and 0.1 % (w/v) concentrations,

- the negative control group received the vehicle (DMSO),

- the positive control group received 25 % (w/v) HCA (dissolved in DMSO).

 

Test item solutions were applied to the dorsal surface of ears of experimental animals (25 μL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. The cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI).

 

No mortality or signs of systemic toxicity were observed during the study. No treatment related effects were observed on the body weight changes of the experimental animals.

 

One out of four animals had significantly increased ear thickness values in the 1 % (w/v) dose group. The swelling of the three remaining animals in this group did not reach the limit of the excessive local skin irritation criteria (>25% increase of the ear thickness). Therefore, this group as well as the other three test item treated dose groups are considered acceptable for evaluation.

 

The stimulation index values were 9.7, 13.2, 10.7 and 3.6 at concentrations of 1, 0.5, 0.25 and 0.1 % (w/v), respectively.

 

The result of the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in DMSO, the same vehicle, was used to demonstrate the appropriate performance of the assay [1]. A significant lymphoproliferative response (stimulation index value of 7.0) was noted for the positive control chemical, this result confirmed the validity of the assay.

 

In conclusion, under the conditions of the present assay NEXAMITE® A56, tested in a suitable vehicle, was shown to have sensitisation potential (sensitiser) in the Local Lymph Node Assay. The calculated EC3 value of NEXAMITE® A56 is 0.09 % (w/v).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Based on the results of the Preliminary Compatibility Test and on the recommendations of the OECD Guideline, the test item was formulated in Dimethyl sulfoxide (abbreviated as DMSO).

In the main assay, twenty-four female CBA/CaOlaHsd mice were allocated to six groups of four animals each:

- four groups received NEXAMITE® A56 (formulated in DMSO) at 1, 0.5, 0.25 and 0.1 % (w/v) concentrations,

- the negative control group received the vehicle (DMSO),

- the positive control group received 25 % (w/v) HCA (dissolved in DMSO).

 

Test item solutions were applied to the dorsal surface of ears of experimental animals (25 μL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. The cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI).

 

No mortality or signs of systemic toxicity were observed during the study. No treatment related effects were observed on the body weight changes of the experimental animals.

One out of four animals had significantly increased ear thickness values in the 1 % (w/v) dose group. The swelling of the three remaining animals in this group did not reach the limit of the excessive local skin irritation criteria (>25% increase of the ear thickness). Therefore, this group as well as the other three test item treated dose groups are considered acceptable for evaluation.

 

The stimulation index values were 9.7, 13.2, 10.7 and 3.6 at concentrations of 1, 0.5, 0.25 and 0.1 % (w/v), respectively.

 

In conclusion, under the conditions of the present assay NEXAMITE® A56, tested in a suitable vehicle, was shown to have sensitisation potential (sensitiser) in the Local Lymph Node Assay. The calculated EC3 value of NEXAMITE® A56 is 0.09 % (w/v).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available