Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-03-06 to 2019-03-12

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)

Deviations:

yes

Remarks:

Deviations were not considered 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 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile

EC Number:

915-371-2

Molecular formula:

C13H19N

IUPAC Name:

Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile

Test material form:

liquid

Details on test material:

Substance name: Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile
Public name: Azuril
EC numbers: 915-371-2 (268-417-2 / 244-530-2)
CAS numbers: n/a (68084-04-8 / 21690-43-7)
Batch/lot number: A170421E
Description: Clear, pale yellow liquid
Expiry date: 06 June 2019
Purity: 99.35%

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); Batch no: A170421E
- 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: Controlled room temperature (15-25oC, <70% relative humidity)
- Stability under test conditions: Analytical determination of the test item concentration, stability and homogeneity was not performed because of the character and the short period of study.
- Solubility and stability of the test substance in the solvent/vehicle: The solubility of the test item was examined in a short Preliminary Compatibility Test. The following standard OECD 429 vehicle was assessed: Acetone: Olive oil 4:1 (v/v) mixture (AOO). The best vehicle taking into account the test item characteristics, and the requirements of the relevant OECD guideline was considered to be AOO. 100% (undiluted) was the highest concentration which was suitable for the test. The formulations appeared to be solutions by visual examination.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Clear, pale yellow liquid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

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, nulliparous, non-pregnant
- Microbiological status of animals, when known: SPF at arrival
- Age at study initiation: 11 weeks old (age-matched, within one week)
- Weight at study initiation: 20.2 – 21.9 grams (The weight variation in animals in the study did not exceed ± 20% of the mean weight.)
- Housing: Group caging (Type II. polypropylene / polycarbonate) / mice were provided with glass tunnel-tubes
- 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: 30 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 and Batch Number: 60190070110101, Expiry date: 08 January 2020, 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: at least 14 days
- Indication of any skin lesions: Not specified

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: From: 2019-02-06 To: 2019-03-12

Study design: in vivo (LLNA)

Vehicle:

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

Remarks:

Acetone: Olive oil 4:1 (v/v) mixture (AOO)

Concentration:

Preliminary Irritation / Toxicity Test: Test material concentrations of 100% (undiluted), 50, 25, and 10% (w/v) in AOO

Main Test: Test material concentrations of 10, 5, and 2.5% (w/v) in AOO

No. of animals per dose:

4 females per concentration

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The solubility of the test item was examined in a short Preliminary Compatibility Test. The following standard OECD 429 vehicle was assessed: Acetone: Olive oil 4:1 (v/v) mixture (AOO). The best vehicle taking into account the test item characteristics, and the requirements of the relevant OECD guideline was considered to be AOO. 100% (undiluted) was the highest concentration which was suitable for the test. The formulations appeared to be solutions by visual examination.

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.

- Irritation: A Preliminary Irritation/Toxicity Test was started according to the Study Plan on CBA/CaOlaHsd mice using four doses (2 animals/dose) at test material concentrations of 100% (undiluted), 50, 25, and 10% (w/v) in AOO. The preliminary experiment was 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 item in an acceptable solvent was established according to OECD guideline 429. Based on the observation of the solubility test, the maximum available concentration was 100% (undiluted).

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

During the main study 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 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.

- Ear thickness measurements: In the Preliminary Irritation / Toxicity Tests, all mice were observed daily for local irritation at the application site. Both ears of each mouse were observed for erythema and scored using the scoring system presented below. 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 the ear thickness was performed by ear punch weight
determination after the euthanasia of the experimental animals.

- Erythema scores: Erythema Scoring

Observation Score
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet redness) to eschar formation 4
preventing grading of erythema
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
- Name of test method: Local Lymph Node Assay (LLNA)
- 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 item 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:
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.

The Preliminary Irritation/Toxicity Tests were started according to the Study Plan on CBA/CaOlaHsd mice using four doses (2 animals/dose) at test item concentrations of 100% (undiluted), 50, 25, and 10% (w/v) in AOO. The preliminary experiment was 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.

Based on these observations in the preliminary test, 10% (w/v) dose was selected as the top dose for the main test.

Positive control substance(s):

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

Statistics:

The number of radioactive disintegrations per minute (DPM) was 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. A stimulation index of 3 or greater is an indication of a positive result.

Results and discussion

Positive control results:

Larger than normal lymph nodes were observed in the positive control group. 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 4.4) 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 positive control substance in this
experiment were in within the historical control range.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1

Variability:

DPN: 594.3

Test group / Remarks:

Negative control (AOO)

Results 2

Key result

Parameter:

SI

Value:

1.2

Variability:

DPN = 719.8

Test group / Remarks:

Test Material - 10% (w/v) in AOO

Results 3

Key result

Parameter:

SI

Value:

1.1

Variability:

DPN = 639.6

Test group / Remarks:

Test Material - 5% (w/v) in AOO

Results 4

Key result

Parameter:

SI

Value:

0.9

Variability:

DPN = 556.8

Test group / Remarks:

Test Material - 2.5% (w/v) in AOO

Results 5

Key result

Parameter:

SI

Value:

4.4

Variability:

DPN = 2618.8

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 and in the test item treated dose groups. Larger than normal lymph nodes were observed in the positive control group.

DETAILS ON STIMULATION INDEX CALCULATION
The number of radioactive disintegrations per minute (DPM) was 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. A stimulation index of 3 or greater is an indication of a positive result.

The stimulation index values were 1.2, 1.1, and 0.9 at concentrations of 10, 5, and 2.5% (w/v), respectively.

EC3 CALCULATION
Not specified

CLINICAL OBSERVATIONS:
No mortality or signs of systemic toxicity were observed during the study. No test material residue was present on the ears of the animals.

BODY WEIGHTS
Decreases were noted in body weight, however were considered incidental and not related to treatment. Two out of 4 animals in the negative control, 3 out of 4 animals in the 10% (w/v) dose group, and 1 out of 4 animals in the positive control group showed marked body weight loss (>5%). The mean body weight loss in the 10% (w/v) dose group was -6.2% (mean value). Based on the unequivocal result for this dose group in the preliminary assays, the lack of systematic toxicity in any of the dose groups, and marked body weight losses in both the negative and positive control groups, these changes were considered to be due to individual variety, and not a factor to effect the results of the proliferation assay for this dose group.

Any other information on results incl. tables

Table 2. Summary of Preliminary Study Data
Preliminary Concentrations	Physical Formulation	Clinical Observations	Body Weight	Erythema	Ear Thickness	Ear Biopsy Weight
100% (undiluted)	A	U	E	E	A	A
50% (w/v)	A	U	E	E	A	A
25% (w/v)	A	U	E	A	A	A
10%(w/v)	A	A	E	A	A	A

U = Unacceptable; A = Acceptable; E = Equivocal

Table 3. Individual Body Weights for all Animals with Group Means
Animal Number	Test Group Name	Initial Body Weight (g)	Terminal Body Weight* (g)	Change# (%)
7585	Negative (vehicle) control in AOO	21.6	19.9	-7.9
7598		21.2	21.0	-0.9
7595		20.6	20.4	-1.0
7597		20.7	19.6	-5.3
	Mean	21.0	20.2	-3.8
7586	Test material 10% (w/v) in AOO	21.5	19.7	-8.4
7592		21.0	20.6	-1.9
7596		20.8	19.3	-7.2
7593		20.3	18.8	-7.4
	Mean	20.9	19.6	-6.2
7599	Test material 5% (w/v) in AOO	21.9	21.1	-3.7
7584		20.7	20.8	0.5
7588		20.6	21.3	3.4
7600		20.4	21.7	6.4
	Mean	20.9	21.2	1.7
7587	Test material 2.5% (w/v) in AOO	21.5	22.5	4.7
7602		21.7	22.4	3.2
7594		20.3	20.5	1.0
7590		20.2	19.6	-3.0
	Mean	20.9	21.3	1.5
7603	Positive control 25% (w/v) HCA in AOO	21.5	21.2	-1.4
7591		21.9	20.6	-5.9
7601		20.3	19.5	-3.9
7589		20.2	21.5	7.4
	Mean	21.0	20.8	-1.0

Notes:

*: Terminal body weights were measured on Day 6.

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

 

Table 4. 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)	35	-	-	-	-
Negative control (AOO)	4789	4754.0	8	594.3	1.0
Test Material 10% (w/v) in AOO	5793	5758.0	8	719.8	1.2
Test Material 5% (w/v) in AOO	5152	5117.0	8	639.6	1.1
Test Material 2.5% (w/v) in AOO	4489	4454.0	8	556.8	0.9
Positive control (25% (w/v) HCA in AOO)	20985	20950.0	8	2618.8	4.4

Applicant's summary and conclusion

Interpretation of results:

other: Not a skin sensitiser

Remarks:

Based on EU CLP and GHS

Conclusions:

Under the conditions of the present assay, the test material (Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile), tested in AOO, did not show a sensitisation potential (non-sensitiser) in the local lymph node assay.

No classification/labelling is triggered according to Regulation (EC) No 1272/2008 (CLP) / 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 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile) following dermal exposure in mice.

 

The solubility of the test material was examined in a short Preliminary Compatibility Test. Acetone: Olive oil 4:1 (v/v) mixture (AOO) was used as the vehicle and 100% (undiluted) was the highest concentration found to be suitable for the test. A preliminary irritation/toxicity test was performed in CBA/CaOlaHsd mice using four doses (2 animals/dose): 100% (undiluted), 50, 25, and 10% (w/v) in AOO. Based on the results, 10% (w/v) was selected as top dose for the main test.

 

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

- three groups of animals received the test material (formulated in AOO) at either 10, 5 or 2.5% (w/v),

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

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

 

Test material solutions were applied to the dorsal surface of the ears of the experimental animals (25 µL/ear) for three consecutive days (Days 1, 2 and 3) and then maintained on study for an additional 3 days. Cell proliferation in the (local) lymph nodes was assessed by measuring disintegrations per minute after the incorporation of tritiated methyl thymidine (³HTdR) into the lymph nodes and the values obtained were used to calculate stimulation indices (SI) in comparison with the control group.

 

No mortality or signs of systemic toxicity were observed during the study.Decreases were noted in body weight, however were considered incidental and not related to treatment. Two out of 4 animals in the negative control, 3 out of 4 animals in the 10% (w/v) dose group, and 1 out of 4 animals in the positive control group showed marked body weight loss (>5%). The mean body weight loss in the 10% (w/v) dose group was -6.2%.

 

The SI values were 1.2, 1.1, and 0.9 at test material concentrations of 10, 5, and 2.5% (w/v), respectively.

 

The SI value for the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in AOO was 4.4 therefore demonstrating the appropriate performance of the assay. The lymphoproliferative response of the HCA was in line with historical control data for the positive control, therefore confirming the validity of the assay.

 

Under the conditions of this assay, the test material (Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile), tested in AOO, did not show a sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.

 

No classification/labelling is triggered according to EU Regulation (EC) No 1272/2008 (CLP) or GHS (rev. 7) 2017.