2-Propenenitrile, reaction products with 3-amino-1,5,5-trimethylcyclohexanemethanamine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 16, 2014 to September 16, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted according to OECD Guideline 429 and EU Method B.42, in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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:

2-Propenenitrile, reaction products with 3-amino-1,5,5-trimethylcyclohexanemethanamine

EC Number:

292-053-3

EC Name:

2-Propenenitrile, reaction products with 3-amino-1,5,5-trimethylcyclohexanemethanamine

Cas Number:

90530-15-7

Molecular formula:

C10 H22 N2 . C3 H3 N1

IUPAC Name:

3-(aminomethyl)-3,5,5-trimethylcyclohexan-1-amine; 3-[(3-{[(2-cyanoethyl)amino]methyl}-3,5,5-trimethylcyclohexyl)amino]propanenitrile; 3-{[(5-amino-1,3,3-trimethylcyclohexyl)methyl]amino}propanenitrile; 3-{[3-(aminomethyl)-3,5,5-trimethylcyclohexyl]amino}propanenitrile

Test material form:

other: liquid

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J Rj

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: ELEVAGE JANVIER, Route des Chènes Secs B.P. 4105, 53940 LE GENEST-ST-ISLE, France
- Age at study initiation: 8 weeks old (age-matched, within one week)
- Weight at study initiation: 20.0-21.9 g
- Housing: Group caging / mice were provided with glass tunnel-tubes
- Diet: Animals received ssniff® SM Rat/Mouse – “Breeding & Maintenance, 15 mm, autoclavable Complete diet for rats/mice” (Batch number: 672 1467 / 190 1786, Expiry date: November 2014 / January 2015) produced by ssniff Spezialdiäten GmbH (Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany), ad libitum
- Water: tap water from the municipal supply from 500 mL bottle, ad libitum
- Acclimation period: At least 5 d

ENVIRONMENTAL CONDITIONS
- Temperature: 20.3-26.2°C
- Humidity: 31-70%
- Air changes: 15-20 air exchanges/h
- Photoperiod: 12 h dark/12 h light

IN-LIFE DATES: From July 16, 2014 to September 16, 2014

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

10, 5, 2.5 and 1% (w/v)

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
Preliminary Irritation/Toxicity Test I
Preliminary Irritation/Toxicity Test I was performed on CBA/J Rj mice using two doses (2 animals/dose), at test substance concentrations of 100% (undiluted) and 50% (w/v) in Acetone- Olive oil (AOO). The preliminary experiment was conducted in a similar experimental manner to the main study, but they were terminated on Day 6 with a body weight measurement and the radioactive proliferation assay was not performed.
The maximum concentration of test substance in an acceptable vehicle was established according to OECD guideline 429. Due to the physical characteristics of the test substance, the maximum available concentration was 100% (undiluted).

Preliminary Irritation/Toxicity Test II
Based on the observed results an additional experiment (Preliminary Irritation/Toxicity Test II.) was performed for dose selection. The Preliminary Irritation/Toxicity Test II was started on CBA/J Rj mice using three doses (2 animals/dose) at test substance concentrations of 25, 10 and 5% (w/v) in AOO. This experiment was conducted similarly to the Preliminary Irritation/Toxicity Test I.

MAIN STUDY
Based on the results of preliminary irritation tests, 10, 5, 2.5 and 1% (w/v) doses were selected for the main test. In addition, a negative (vehicle) control (AOO) and a positive control (25% HCA in AOO) were also included. 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.

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 at least 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 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.

Ear Thickness Measurements
In the preliminary experiments, both ears of each mouse were observed for erythema and scored according to OECD Guideline. 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. Ear thickness measurements were performed in the main test in a similar way.

PROLIFERATION ASSAY
Injection of Tritiated Thymidine (3HTdR)
On Day 6, animals were taken to the radioactive suite and 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 h (± 30 minutes).

Removal and Preparation of Draining Auricular Lymph Nodes
5 h (± 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 was 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 the 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 was 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 gently agitated resuspended and 3 mL of 5% (w/v) TCA solution was added to the tubes for precipitation of macromolecules. After overnight (approximately 18 h) incubation at 2-8°C, precipitates were centrifuged (approximately 190 x g for 10 minutes at 4°C), and supernatants were removed. Pellets were resuspended in 1 mL of 5% (w/v) TCA solution and dispersed by 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 was 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.

Positive control substance(s):

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

Results and discussion

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

44.8

Test group / Remarks:

4 animals, 10%

Results 2

Key result

Parameter:

SI

Value:

24.6

Test group / Remarks:

4 animals, 5%

Results 3

Key result

Parameter:

SI

Value:

6

Test group / Remarks:

4 animals, 2.5%

Results 4

Key result

Parameter:

SI

Value:

1.9

Test group / Remarks:

4 animals, 1%

Results 5

Key result

Parameter:

EC3

Value:

1.4

Any other information on results incl. tables

Preliminary Irritation/Toxicity Test I

In the Preliminary Irritation / Toxicity Test I., no mortality or signs of systemic toxicity were observed. No marked body weight loss (>5%) was observed. Crust at the parotic was observed in the animals in the 100% (undiluted) dose group for one animal on Days 3-6. Alopecia was observed in the 100% (undiluted) dose group on Day 6. Rigid ears were observed in the 100% (undiluted) dose group on Days 3-6 and in the 50 % (w/v) dose group on Days 4-6. Epithelisation was observed in the 50% (w/v) dose group on Day 6. Erythema (scored 1 and 2) was observed in the 100% (undiluted) dose group on Days 2-6 and in the 50% (w/v) dose group on Days 3-5. Increased ear thickness data (over the limit of positivity) were observed in both dose groups. The revealing ear punch weights were over the historical control range for all animals, and the obtained values

exceeded the criteria (≥25% increase in ear thickness). Therefore, due to the strong local irritation the examined concentrations were considered as not acceptable for a valid main test. The draining auricular lymph nodes of the animals were visually examined: the appearance of the lymph nodes was larger than normal in all animals (subjective judgement by analogy with observations of former experiments).

Preliminary Irritation/Toxicity Test II

In the Preliminary Irritation/Toxicity Test II., no mortality or signs of systemic toxicity were observed. No marked body weight loss was observed in the examined dose groups. Rigid ears and erythema (score 1) were detected in the 25% (w/v) dose group on Days 3-6, slightly rigid ears were detected for one animal in the 10% (w/v) dose group on Day 6. Scales and alopecia were detected in the 25% (w/v) dose group on Days 5-6. Increased ear thickness values (>25% increase) were detected for both animals in the 25% (w/v) dose group and for one animal in the 10% (w/v) dose group indicating excessive local skin irritation. Borderline effect was observed for the other animal in the 10% (w/v) dose group. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals. The revealing ear punch weight was out of the historical control range in the 25% (w/v) dose group. However, ear punch weight did not exceed the limit of positive response (upper limit of historical control range + 25%). The draining auricular lymph nodes of the animals were visually examined in this experiment: the appearance of the lymph nodes was larger than normal in all animals (subjective judgement by analogy with observations of former experiments). Based on the observed effects and local irritation, 100% (undiluted), 50 and 25% (w/v) concentrations are clearly above the maximum acceptable concentration for a main study.

CLINICAL OBSERVATIONS

No mortality or signs of systemic toxicity were observed during the study. There were no indications of any irritancy at the site of application.

BODY WEIGHT MEASUREMENT

No treatment related effects were observed on body weights. No marked body weight loss (>5%) was detected for any experimental animals.

EAR THICKNESS MEASUREMENT

Increased ear thickness values were recorded for all animals in the 10% (w/v) dose group on Day 3 and Day 6, and in some sporadic cases in other test substance treated groups, but none of these values were above the regulatory threshold of 25% (as limit of positivity). The biopsy weights were within the historical control range for all test substance treated animals.

PROLIFERATION ASSAY

The appearance of the lymph nodes was normal in the negative (vehicle) control group and in the 1% (w/v) test substance treated group. Larger than normal lymph nodes were observed in the positive control group and in the 10 and 5% (w/v) test substance treated groups, slightly enlarged lymph nodes were observed in the 2.5% (w/v) test substance treated group (subjective judgement by analogy with observations of former experiments).

The stimulation index values were 44.8, 24.6, 6.0 and 1.9 at concentrations of 10, 5, 2.5 and 1% (w/v), respectively.

INTERPRETATION OF OBSERVATIONS

The test substance was a yellowish liquid, which was formulated in AOO in the main test. Since there were no confounding effects of irritation at any dose level, or significant systemic toxicity at the other concentrations, the proliferation values obtained are considered to reflect the real potential of the test substance to cause lymphoproliferation in the Local Lymph Node Assay. The resulted stimulation index values observed at the three highest concentrations of 10, 5 and 2.5% (w/v) were clearly above the threshold limit of 3, thus they were considered to be good evidence that the test substance is a sensitiser. Furthermore, the obtained data are compatible with a conventional dose response. The size of lymph nodes was in good correlation with the observed dose-dependent positive effect.

The calculated EC3 value of the test substance is 1.4% (w/v). Based on this value, the test substance can be classified as skin sensitizer Category 1 (sub-category 1A) according to the UN GHS system.

RELIABILITY OF THE TEST

The result of the positive control substance a-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% (w/v) in the relevant vehicle (AOO) using CBA/J Rj mice. No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A significant lymphoproliferative response (stimulation index value of 9.8) 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.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Based on the study results, the test substance is considered to be sensitising to skin.

Executive summary:

A study was conducted to assess the skin sensitizing potential of the test substance in mouse according to OECD Guideline 429 (Local Lymph Node Assay) and EU method B.42, in compliance with GLP. Test substance concentrations selected for the main study were based on the results of preliminary irritation/toxicity tests. In the main study, 4 experimental groups of four female CBA/J Rj mice were treated at concentrations of 10, 5, 2.5 and 1% (w/v) on three consecutive days, by open application on the ears. Four control animals were similarly treated with vehicle alone (acetone-olive oil). Three days after the last exposure, all animals were injected with tritiated thymidine (3HTdR) and after 5 h, the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as a stimulation index (SI) and was subsequently calculated for each group. No mortality or signs of systemic toxicity were observed during the study. There were no indications of any irritancy at the site of application. No treatment related effects were observed on body weights. No marked body weight loss (>5%) was detected for any experimental animals. Increased ear thickness values were recorded for all animals in the 10% (w/v) dose group on Day 3 and Day 6 and in some sporadic cases in other test substance treated groups, but none of these values were above the regulatory threshold of 25% (as limit of positivity). The biopsy weights were within the historical control range for all test substance treated animals. The appearance of the lymph nodes was normal in the negative (vehicle) control group and in the 1% (w/v) test substance treated group. Larger than normal lymph nodes were observed in the positive control group and in the 10 and 5% (w/v) test substance treated groups, slightly enlarged lymph nodes were observed in the 2.5% (w/v) test substance treated group (subjective judgment by analogy with observations of former experiments). The stimulation index values were 44.8, 24.6, 6.0 and 1.9 at concentrations of 10, 5, 2.5 and 1% (w/v), respectively. The resulted stimulation index values observed at the three highest concentrations of 10, 5 and 2.5% (w/v) were clearly above the threshold limit of 3, thus they were considered to be good evidence that the test substance is a sensitiser. The calculated EC3 value of the test substance is 1.4% (w/v). A significant lymphoproliferative response (stimulation index value of 9.8) was noted for positive control (HCA) in the main experiment. This value was considered to confirm the appropriate performance of the assay. Based on the study results, the test substance is considered to be sensitising to skin (Hargitai J, 2014).