Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-02-07 to 2019-04-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22 July 2010

Deviations:

yes

Remarks:

Deviations were considered not to adversely affect the results or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene

EC Number:

933-779-9

Molecular formula:

C10H16

IUPAC Name:

Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene

Test material form:

liquid

Details on test material:

Name: Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene
Public name: Ocimene PQ
EC numbers: 933-779-9 (pre-registration 222-081-3)
CAS numbers: n/a (pre-registration 3338-55-4)
Batch/Lot number: A170524D
Description: Colorless Liquid
Expiry date: 06 June 2019
Purity: Considered as 100%

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
Emerald Kalama Chemical Ltd. (United Kingdom); Lot No. A170524D
- Expiration date of the lot/batch: 2019-06-06
- Purity test date: 2018-02-07

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (15-25oC, ≤70 RH%), under inert gas, protected from humidity (tight closed container)
- Stability under test conditions:Not specified
- Solubility and stability of the test substance in the solvent/vehicle: Not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was weighed and formulations prepared daily on a weight:volume basis (as % (w/v)) in the Pharmacy of Citoxlab Hungary Ltd. Analytical determination of the test item concentration, stability and homogeneity was not performed because of the character and the short period of study.


FORM AS APPLIED IN THE TEST (if different from that of starting material): Colorless liquid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

CaOlaHsd mice

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo (San Pietro al Natisone (UD), Zona Industriale Azzida, 57, 33049 Italy)
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: Only healthy animals were used for the study. Health status was certified by the veterinarian.
- Age at study initiation: 12 weeks old (age-matched, within one week)
- Weight at study initiation: 21.3 – 23.9 grams (The weight variation in animals in the study did not exceed ± 20% of the mean weight.)
- Housing: Group caging / mice were provided with glass tunnel-tubes (Type II polypropylene / polycarbonate cages)
- Diet (e.g. ad libitum): ssniff® SM Rat/Mouse – Breeding and Maintenance, 15 mm, autoclavable “Complete feed for Rats and Mice – Breeding and Maintenance” (Batch number: 639 38520, Expiry date: April 2019 produced by ssniff Spezialdiäten GmbH (Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany), and Gel diet Transport (Batch Number: 60182980010101, Expiry date: 25 October 2019, Scientific Animal Food & Engineering, Route de Saint Bris, 89290 Augy, France) ad libitum.
- Water (e.g. ad libitum): tap water from the municipal supply from 500 mL bottles, ad libitum.
- Acclimation period: 50 days
- Indication of any skin lesions: Not specified but only healthy animals were used for the study. Health status was certified by the veterinarian.

Note: In the Preliminary Experiment, mice of 10 weeks of age (18.2-18.9 grams) were used.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.6 – 25.9°C
- Humidity (%): 22 - 74 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

- IN-LIFE DATES: Preliminary Experiment (From: 2019-01-23 To: 2019-01-28); Experiment (From: 2019-02-07 to 2019-02-13)

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Test material formulations at 50%, 25% and 10% (w/v) in AOO (acetone/olive oil (4:1 v/v))

No. of animals per dose:

4 per dose

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The solubility of the test material was examined in a short Preliminary Compatibility Test. The following standard OECD vehicles was assessed: AOO (acetone:olive oil 4:1 (v:v) mixture). The best vehicle taking into account the test material characteristics and the requirements of the relevant OECD guideline was considered to be AOO. The highest achievable concentration based on the regulatory requirements of OECD 429 and the physical characteristics of the test material was 100% (undiluted). The formulations at 50%, 25% and 10% (w/v) in AOO were also suitable for treatment. The formulations appeared to be solutions by visual examination.

The test material was weighed and formulations prepared daily on a weight:volume basis (as % (w/v)) in the Pharmacy of Citoxlab Hungary Ltd.

Analytical determination of the test item concentration, stability and homogeneity was not performed because of the character and the short period of study.

- Irritation: In the Preliminary Irritation / Toxicity Test, all mice were observed daily for any local irritation at the application site.

- Systemic toxicity: In the Preliminary Irritation / Toxicity Test, all mice were observed daily for any clinical signs of systemic toxicity.

- Ear thickness measurements: Ear thickness was 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 the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals.

- Erythema scores: Both ears of each mouse were observed for erythema and scored using the criteria detailed below:

Erythema Scoring
Observation Score
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beef redness) to eschar formation preventing grading of erythema 4

Note: Excessive local skin irritation is indicated by an erythema score ≥ 3 and/or an increase in ear thickness of ≥ 25 % on any day of measurement.


MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
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. The cages were marked with identity cards with information including study code, cage number, and dose group, sex and individual animal number. The animals were randomised and allocated to the experimental groups. The randomisation was checked by computer software using the body weight to verify the homogeneity and variability between the groups.

- Name of test method: Local Lymph Node Assay (OECD 429)
- Criteria used to consider a positive response: The test material is regarded as a sensitizer if both of the following criteria are fulfilled:

- That exposure to at least one concentration of the test material resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.

- The data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
Test material formulations at 50%, 25% and 10% (w/v) in AOO (acetone:olive oil 4:1 (v:v) mixture) were observed to be suitable for treatment. The formulations appeared to be solutions by visual examination.

The test material was weighed and formulations prepared daily on a weight:volume basis (as % (w/v)) in the Pharmacy of Citoxlab Hungary Ltd.

The Preliminary Irritation/Toxicity Test, was started according to the Study Plan on CBA/CaOlaHsd mice using two doses (2 animals/dose) at test material concentrations of 100% (undiluted)and 50% (w/v) in AOO. The preliminary experiments were conducted in a similar experimental manner to the main study, but it was terminated on Day 6 and the radioactive proliferation assay was not performed.

The maximum concentration of test material in an acceptable vehicle was established according to OECD guideline 429. Based on the observation of the solubility test, the maximum available concentration was 100% (undiluted).

Data from the preliminary study data indicated that the 100% (undiluted) dose group results were above the acceptable limit, and therefore, the 50% (w/v) dose level should be the High dose in the main study.

The experimental groups and dose levels for the main experiment are summarized in Table 1.

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.

Positive control substance(s):

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

Statistics:

Number of radioactive disintegrations per minute (DPM) were measured for each pooled group of nodes. The measured DPM values were corrected with the background DPM value (“DPM”). The average of the two measured DPM values of 5 % (w/v) TCA solutions was used as background DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes) following the industry standard for data presentation.

Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated.

Results and discussion

Positive control results:

The result of the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. The positive control substance was examined at a concentration of 25% in the relevant vehicle (AOO) using CBA/CaOlaHsd mice.

No mortality cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A lymphoproliferative response in line with historical positive control data (stimulation index value of 4.1 was noted for HCA in the main experiment). This value was considered to confirm the appropriate performance of the assay.

Furthermore, the DPN values observed for the vehicle and positive control substance in this experiment were within the historical control range. Each treated and control group included 4 animals.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1

Variability:

DPN = 530.9

Test group / Remarks:

Negative Control (AOO)

Results 2

Key result

Parameter:

SI

Value:

9

Variability:

DPN = 4791.8

Test group / Remarks:

Ocimene PQ 50% (w/v) in AOO

Results 3

Key result

Parameter:

SI

Value:

9.1

Variability:

DPN = 4851.1

Test group / Remarks:

Ocimene PQ 25% (w/v) in AOO

Results 4

Key result

Parameter:

SI

Value:

4.9

Variability:

DPN = 2599.1

Test group / Remarks:

Ocimene PQ 10 (w/v)% in AOO

Results 5

Key result

Parameter:

SI

Value:

4.1

Variability:

DPN = 2151.9

Test group / Remarks:

Positive control (25% (w/v) HCA in AOO

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
The appearance of the lymph nodes was normal in the negative control group. Larger than normal lymph nodes were observed in the 50% and 25% (w/v) dose groups. Normal lymph nodes were observed for 3 animals and slightly enlarged than normal was observed for one animal in the 10% (w/v) dose group. Larger than normal lymph nodes were observed in the positive control group. The results of the proliferation assay are summarized in Table 3.

DETAILS ON STIMULATION INDEX CALCULATION
Number of radioactive disintegrations per minute (DPM) were measured for each pooled group of nodes. The measured DPM values were corrected with the background DPM value (“DPM”). The average of the two measured DPM values of 5 % (w/v) TCA solutions was used as background DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes) following the industry standard for data presentation.

Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated.

The stimulation index values were 9.0, 9.1 and 4.9 at concentrations of 50% (w/v), 25% (w/v) and 10% (w/v), respectively. A lymphoproliferative response in line with historical positive control data (stimulation index value of 4.1 was noted for HCA in the main experiment).

EC3 CALCULATION
The extrapolated EC3 value of Ocimene PQ [Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene] was determined to be 6.6% (w/v).

CLINICAL OBSERVATIONS:
There was no evidence of mortality or systemic toxicity observed during the main study. No evidence of test material residue on the ears of animals was observed and there were no indications of any irritancy at the site of application.

BODY WEIGHTS
Marked body weight loss (≥5%) was observed for one animal in the negative control group, for two animals in the 50% (w/v) and 10% (w/v) dose groups, and in one animal of the 25% (w/v) dose group. However, the mean body weight changes of these groups were within the acceptable range and therefore considered as individual variability.

No treatment-related effects were observed on the mean body weight changes in the main study. Individual and mean body weights are summarised in Table 2.

Ear Thickness Measurement
The ear thickness values and the biopsy weights were within the acceptable range for all the animals in the 25% and 10% (w/v) test item treated groups and in the negative control group. The detected ear thickness values indicated local irritation (≥ 25 %) for one animal (only right ear) of the 50% (w/v) dose group on Day 6 and for one animal (only right ear) of the 25% (w/v) dose group on Day 3. Furthermore, the detected ear thickness values indicated local irritation (≥ 25 %) for one animal (only left ear) of the positive control group on Day 3 and Day 6 and for another animal (only right ear) of the positive control group on Day 3. However, the revealing ear punch weights were within the acceptable range in each case. These facts were considered not to adversely affect the results or integrity of the study.

Any other information on results incl. tables

Table 2. Individual Body Weights for all Animals with Group Means
Animal Number	Identity Number	Test Group Name	Initial Body Weight (g)	Terminal Body Weight* (g)	Change^# (%)
7496	1	Negative (vehicle) control	23.7	21.4	-9.7
7504	2	AOO	22.1	23.3	5.4
7500	3		22.4	22.8	1.8
7499	4		21.3	20.3	-4.7
		Mean	22.4	22.0	-1.8
7508	5	Ocimene PQ	23.6	21.8	-7.6
7505	6	50 (w/v)% in AOO	22.5	22.8	1.3
7514	7		21.6	20.4	-5.6
7495	8		21.5	21.7	0.9
		Mean	22.3	21.7	-2.7
7512	9	Ocimene PQ	23.4	21.6	-7.7
7509	10	25 (w/v)% in AOO	22.5	23.5	4.4
7503	11		21.8	22.7	4.1
7513	12		21.3	21.6	1.4
		Mean	22.3	22.4	0.6
7498	13	Ocimene PQ	23.1	22.1	-4.3
7502	14	10 (w/v)% in AOO	23.1	20.6	-10.8
7510	15		22.4	21.1	-5.8
7507	16		21.3	21.9	2.8
		Mean	22.5	21.4	-4.5
7501	17	Positive control	23.9	23.5	-1.7
7511	18	25 (w/v) % HCA	22.2	22.2	0.0
7497	19	in AOO	21.3	20.8	-2.3
7506	20		21.6	22.0	1.9
		Mean	22.3	22.1	-0.5

Notes:

*: Terminal body weights were measured on Day 6.

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

Table 3. 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)	34 32	-	-	-	-
Negative control (AOO)	4280	4247.0	8	530.9	1.0
Ocimene PQ 50% (w/v) in AOO	38367	38334.0	8	4791.8	9.0
Ocimene PQ 25% (w/v) in AOO	38842	38809.0	8	4851.1	9.1
Ocimene PQ 10 (w/v)% in AOO	20826	20793.0	8	2599.1	4.9
Positive control (25% (w/v) HCA in AOO	17248	17215.0	8	2151.9	4.1

Table 4. Historical Control Data of the Positive and Negative Controls for CBA/CaOlaHsdmice(2014-2018)

	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	472.7	3851.3	9.0	198.7	1988.1	11.2
Range:min	35.8	890.3	3.3	23.0	154.0	3.0
max	1990.1	10336.0	20.2	680.8	6755.8	33.6
Number of cases	92	88	86	234	226	218
	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	256.1	2738.9	11.3	466.0	3014.4	7.2
Range:min	62.0	1201.3	4.9	218.3	1461.3	3.1
max	649.6	5817.9	21.3	934.6	4877.5	14.5
Number of cases	68	68	68	22	22	21
	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	245.1	2278.6	9.4	264.5	4129.5	16.9
Range:min	63.3	817.3	5.8	80.5	1562.5	8.8
max	506.0	4978.0	14.4	516.2	8682.5	36.3
Number of cases	18	18	18	18	19	19

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 values of the group.

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

Under the conditions of the present assay, the test material [Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene], tested in a suitable vehicle, was shown to have a sensitisation potential (sensitizer) in the Local Lymph Node Assay. The extrapolated EC3 value of the test material is 6.6% (w/v). Based on the results observed, Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene meets the criteria to be classified as a skin sensitiser Category 1B (H317: May cause an allergic skin reaction) under EU Regulation (EC) No 1272/2008 (CLP) and GHS (rev. 7) 2017.

Executive summary:

A key OECD Guideline 429 study was conducted to determine the skin sensitisation potential of the test material (Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene) following dermal exposure in CBA/CaOlaHsd mice.

 

A Preliminary Compatibility Test was undertaken and the results of the same along with the test material characteristics, its usage, and on the recommendations of the OECD Guideline, the best vehicle was determined to be AOO (acetone:olive oil 4:1 (v:v) mixture). The highest achievable concentration was 100% (undiluted) with formulations at 50% (w/v), 25% (w/v) and 10% (w/v) in AOO also suitable for treatment.

 

A Preliminary Irritation/Toxicity Test was performed in CBA/CaOlaHsd mice using two doses (2 animals/dose), 100% (undiluted) and 50% (w/v) in AOO. Based on the observations recorded in the preliminary test, the 50% (w/v) dose was selected as top dose for the main test.

 

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

-          three groups received the test material (formulated in AOO) at 50% (w/v), 25% (w/v) and 10% (w/v) concentrations,

-          the negative control group received the vehicle (AOO) only,

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

 

Test material formulations 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 assessed by measuring the incorporation of tritiated methyl thymidine (³HTdR) and the values obtained were used to calculate stimulation indices (SI) in comparison with the control group.

 

There was no evidence of mortality or systemic toxicity observed during the main study. No evidence of test material residue on the ears of animals was observed and there were no indications of any irritancy at the site of application. No treatment-related effects were observed on the mean body weight changes in the main study. The stimulation index values were 9.0, 9.1 and 4.9 at concentrations of 50% (w/v), 25% (w/v) and 10% (w/v), respectively.

 

The result of the positive control substance (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. A lymphoproliferative response (SI = 4.1) in harmony with historical control data was noted for the positive control chemical, this result confirmed the validity of the assay.

 

In conclusion, under the conditions of the present assay, the test material [Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene], tested in a suitable vehicle, was shown to have a sensitisation potential (sensitizer) in the Local Lymph Node Assay. The extrapolated EC3 value of the test material is 6.6% (w/v). Based on the results observed, Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene meets the criteria to be classified as a skin sensitiser Category 1B (H317: May cause an allergic skin reaction) under EU Regulation (EC) No 1272/2008 (CLP) and GHS (rev. 7) 2017.