3-(4,4-dimethylcyclohex-1-en-1-yl)propanal

Endpoint summary

Description of key information

Skin sensitisation: sensitising, EC3 = 54.9 %, female mice, OECD TG 429, 2010

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:

08-12-2009 to 12-02-2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: September 2009 ; signature: November 2009

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Recognised Supplier
- Age at study initiation: 8 to 12 weeks
- Weight at study initiation: 15 to 23g
- Housing: individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet (ad libitum): 2014C Teklad Global Rodent diet (Recognised Supplier); ad libitum
- Water (ad libitum): mains tap water; ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 30 to 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark

IN-LIFE DATES: Fro: 06-02-2012 To: 28-02-2012

Vehicle:

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

Concentration:

- Preliminary test: 100% w/w
- Main test:
EXP 1: 100%, 50%, 25% w/w

No. of animals per dose:

Preliminary test: 1
Main test: 5 per dose group

Details on study design:

RANGE FINDING TESTS:
Using available information regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse per test item concentration. The mouse was treated by daily application of 25 µL to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3) of the undiluted test item (100% v/v). Local skin irritation was scored daily according to the scale included in the full study report. Any clinical signs of toxicity, if present, were also recorded. The body weight of each mouse was recorded on Day 1 (prior to dosing) and on Day 6. No signs of systemic toxicity were noted. Based on this information, the undiluted test item (100% v/v) and the test item at concentrations of 50% and 25% v/v in acetone/olive oil 4:1 were selected for use in the main test.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The test item will be regarded as a sensitiser if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitiser". Applicant assessment indicates: in accordance with the relevant guidelines, the data must additionally be 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:
Groups of five mice were treated with the undiluted test item or the test item at concentrations in acetone/olive oil 4:1. The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3).

3H-Methyl Thymidine Administration:
Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 μl of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 μCi/ml, specific activity 2.0 Ci/mmol) giving a total of 20 μCi to each mouse..

Observations:
- Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
- Bodyweights: The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and prior to 3HTdR treatment and Day 6 (prior to termination).
- Ear thickness measurements: Not recorded.

Preparation of Single Cell Suspension:
Draining lymph nodes were rapidly excised and pooled per female (2 nodes per female). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 μm mesh size). The lymph node cells were rinsed through the gauze with 4 mL of
PBS into a petri dish labelled with the project number and dose concentration. The lymph node cell suspension was transferred to a centrifuge tube. The petri dish was
washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The pooled lymph node cells were pelleted at
1400 rpm (approximately 190 g) for ten minutes. The pellet was resuspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was
resuspended in 3 ml of 5% Trichloroacetic acid (TCA), and then incubated at approximately +4 °C for approximately 18 hours for precipitation of macromolecules.

Determination of 3HTdR Incorporation:
After approximately 18 hours incubation at approximately 4 degrees Celsius, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, resuspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by beta-scintillation counting. The vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left to stand in darkness for approximately 20 minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately 20 minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using a recognised scintillation system.

Positive control substance(s):

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

Statistics:

Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships.

Positive control results:

In a non-concurrent 'positive control study' performed according to OECD TG 429, the sensitivity of the strain of mouse used in this study was assessed using the known sensitiser, α-hexylcinnamaldehyde (85%) at 15% v/v in acetone/olive oil 4:1. The highest concentration tested showed a Stimulation Index (SI) of 3.12 and met the criteria for a 'positive' result. The details on the sensitivity check are documented in the full study report.
The results of routine positive control testing (Historic Control Data) performed according to OECD TG 429, using α-hexylcinnamaldehyde (85%) and Phenylacetaldehyde (90%) indicated positive results using a variety of different vehicles. Information is documented in the full study report.

Parameter:

SI

Value:

2.66

Test group / Remarks:

25% v/v in acetone/olive oil 4:1

Parameter:

SI

Value:

2.67

Test group / Remarks:

50% v/v in acetone/olive oil 4:1

Parameter:

other: disintegrations per minute (DPM)

Value:

36 626.91

Test group / Remarks:

50% v/v in acetone/olive oil 4:1

Remarks on result:

other: See table.

Parameter:

SI

Value:

6.22

Test group / Remarks:

100% v/v (undiluted)

Parameter:

other: disintegrations per minute (DPM)

Value:

85 411.79

Test group / Remarks:

100% v/v (undiluted)

Remarks on result:

other: See table.

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA: See tables.

DETAILS ON STIMULATION INDEX CALCULATION: See tables.

EC3 CALCULATION: The EC3 was determined based on the equation: EC3 = c + [[(3−d)/(b−d)] x (a−c)]
where: a= 100 ; b = 6.22 ; c= 50 ; d = 2.67 : therefore: EC3 = 50 + [[(3-2.67)/(6.22-2.67)] x (100-50)] = 54.6 %

CLINICAL OBSERVATIONS: No mortality occurred and no clinical signs of systemic toxicity were observed in the main study.

BODY WEIGHTS: Body weights and body weight gain of experimental animals remained in the same range as negative (vehicle) controls over the study period.

Table 1.0 - Stimulation Index during the test

Treatment Group	Concentration	dpm	Dpm/node #1	Stimulation Index #2	Result
Vehicle	0% v/v (test item)	13732.74	1716.59	N/A	-
Test Item	25%v/vinacetone/olive oil 4:1	36597.65	4574.71	2.66	Negative
	50%v/vin acetone/olive oil 4:1	36626.91	4578.36	2.67	Negative
	100%	85411.79	10676.47	6.22	Positive

dpm = Disintegrations per minute

#1 = Disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 (total number of lymph nodes)

#2 = Stimulation Index of 3.0 or greater indicates a positive result

N/A = Not applicable

Interpretation of results:

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

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the condition of this study, the test item is considered to be sensitising to skin. The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 54.6%.

Executive summary:

The study was performed to OECD TG 429 and EU Method B.42, Local Lymph Node Assay under GLP to assess the skin sensitisation potential of the test item in the CBA/CaOlaHsd mouse following topical application to the dorsal surface of the ear. In a preliminary screening test single mice were treated by daily application of 25 μl of the solid test item diluted at 100% to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily. Any clinical signs of toxicity, if present, were also recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6. No signs of systemic toxicity, visual local skin irritation or irritation were indicated. Based on the preliminary test, the concentrations selected for the main test were 0% (control), 25%, 50% within Acetone/Olive Oil (4:1) and 100% v/v (undiluted). The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local irritation at the highest suitable concentration (100% v/v). The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded. The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination). At test termination, five hours after administration of 3HTdR, the test organisms were humanly euthanized. For each individual animal of each group the draining auricular lymph nodes were excised and processed. For each individual animal 1 mL of PBS was added to the lymph nodes. Following appropriate preparation, 3HTdR incorporation was measured by β-scintillation counting and the number of radioactive disintegrations per minute (dpm) was measured. The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes. The substance was regarded as sensitising if at least one concentration of the substance resulted in a 3-fold or greater increase in 3HTdR incorporation compared to control values. Any substance failing to produce a 3-fold or greater increase in 3HTdR incorporation was classified as non-sensitising. In the main test, there were no deaths or signs of systemic toxicity, and body weights in treatment groups were comparable to controls. A stimulation index (SI) was recorded for each concentration as follows: 25% v/v: SI = 2.66, 50% v/v: SI = 2.67 and 100% v/v: SI = 6.22. The EC3 was calculated to be 54.6% by linear interpolation. Accordingly, the test item was considered to be sensitising under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Skin Sensitisation:

Key study : in vivo: OECD TG 429, 2010 : The study was performed to OECD TG 429 and EU Method B.42, Local Lymph Node Assay under GLP to assess the skin sensitisation potential of the test item in the CBA/CaOlaHsd mouse following topical application to the dorsal surface of the ear. In a preliminary screening test single mice were treated by daily application of 25 μl of the solid test item diluted at 100% to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily. Any clinical signs of toxicity, if present, were also recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6. No signs of systemic toxicity, visual local skin irritation or irritation were indicated. Based on the preliminary test, the concentrations selected for the main test were 0% (control), 25%, 50% within Acetone/Olive Oil (4:1) and 100% v/v (undiluted). The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local irritation at the highest suitable concentration (100% v/v). The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded. The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination). At test termination, five hours after administration of 3HTdR, the test organisms were humanly euthanized. For each individual animal of each group the draining auricular lymph nodes were excised and processed. For each individual animal 1 mL of PBS was added to the lymph nodes. Following appropriate preparation, 3HTdR incorporation was measured by β-scintillation counting and the number of radioactive disintegrations per minute (dpm) was measured. The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes. The substance was regarded as sensitising if at least one concentration of the substance resulted in a 3-fold or greater increase in 3HTdR incorporation compared to control values. Any substance failing to produce a 3-fold or greater increase in 3HTdR incorporation was classified as non-sensitising. In the main test, there were no deaths or signs of systemic toxicity, and body weights in treatment groups were comparable to controls. A stimulation index (SI) was recorded for each concentration as follows: 25% v/v: SI = 2.66, 50% v/v: SI = 2.67 and 100% v/v: SI = 6.22. The EC3 was calculated to be 54.6% by linear interpolation. Accordingly, the test item was considered to be sensitising under the conditions of the test.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance meets classification criteria under Regulation (EC) No 1272/2008 for skin sensitisation category 1B: H317

 

The weight of evidence indicates that the substance has a low frequency of occurrence in humans and/or low to moderate potency in animals (EC3 >2%) and can be presumed to have the potential to produce sensitisation in humans via the dermal route.