(1'R,2R)-2-Methyl-4-(2',2',3'-tri-nal and methylcyclopent-3'-en-1'-yl)-4-pent-enal

Endpoint summary

Description of key information

Weight of evidence: Skin sensitising, 2019

1. Skin sensitisation: sensitising, Guinea Pig (female), GPMT, OECD TG 406, 1994

2. Skin sensitisation: non-sensitising, Guinea Pig (female), modified Buehler Assay, OECD TG 406, 1996

3. Skin sensitisation: non-sensitising under conditions tested (EC3 = > 20%), female mice, eq. or similar to OECD TG 429, 2001

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13-07-1994 to 25-08-1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

Method employed in this study for the detection of delayed contact hypersensitivity was the guinea-pig maximization test described by B. Magnusson and A.M. Kligman - "The identification of contact allergens by animal assay, the guinea pig maximisation method"; Invest. Dermatol. 1969. 52, 268-276

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

methods of testing for skin sensitisation described in Document L 383 A (29 December 1992), Annex to Commission Directive 92/69/EEC (31 July 1992)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: October 1992 ; signature: December 1992

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Study conducted prior to Regulation (EC) 1907/2006 and/or Regulation (EC) 1272/2008 publication and implementation.

Species:

guinea pig

Strain:

Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Recognised supplier
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: Young adult (approximately 3 months old).
- Weight at study initiation: 405 – 509 g
- Housing: group housed (5) in stainless steel cages
- Diet (e.g. ad libitum): Complete maintenance diet for guinea pigs (details in the full study report).
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: The acclimatization period was at least 5 days before the start of treatment under laboratory conditions; identical to the test (19 days for main study).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ±2 °C
- Humidity (%): 30 – 70% (actual 51 – 91% - deviations were not considered to impact the integrity of the study)
- Air changes (per hr): at least 12 per hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark

IN-LIFE DATES: From: To: 13-07-1994 to 25-07-1994

Route:

intradermal

Vehicle:

paraffin oil

Concentration / amount:

- Intradermal: 10% test material in light liquid paraffin

Day(s)/duration:

Day 1

Adequacy of induction:

non-irritant substance, but skin pre-treated with 10% SDS

Route:

epicutaneous, occlusive

Concentration / amount:

- Topical: 100% test material (undiluted)

Day(s)/duration:

Day 8

Adequacy of induction:

non-irritant substance, but skin pre-treated with 10% SDS

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

- Challenge: 100%

Day(s)/duration:

Day 22

Adequacy of challenge:

highest non-irritant concentration

No.:

#2

Route:

epicutaneous, occlusive

Vehicle:

other: acetone

Concentration / amount:

- Challenge: 2% and/or 0.5% in acetone

Day(s)/duration:

Day 32

No. of animals per dose:

Test group: 20 ; Control group: 10

Details on study design:

RANGE FINDING TESTS:
A preliminary irritation study was conducted in order to select test substance concentrations to be used in the main study. By intradermal route: tested concentrations: 50%, 25%, 10%, 5%, 1% and 0.5% (w/w). Cutaneous reactions were evaluated approximately 24, 48 hours and daily up to 6 days after the injections. By cutaneous route (occlusive dressing, 24 hours) tested concentrations: 100%, 50%, 25% and 12.5% (w/w). Reactions were evaluated approximately 24 and 48 hours after removal.
At 24 and 48 hours: intradermal and cutaneous/topical route 100%v/v was selected due to absence of irritation. Final concentrations for definitive testing based on preliminary irritation study:
- Intradermal: 10% test material
- Topical: 100% test material (undiluted)
- Challenge: 1st challenge: 100% (undiluted) ; 2nd challenge: 0.5 and 2% in acetone

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 1 intradermal induction; 1 epidermal induction (topical booster)
- Exposure period: Day 1 intradermal induction and Day 8 topical induction (topical booster)
- Test groups: duplicate injections as follows: 2 ID: Freund's Complete Adjuvant diluted at 50% in water; 2 ID: test item at 10% in liquid paraffin ; 2 ID: a test mixture of the test item at 50% in FCA at 50% and in water to give final concentration of 10.0%.
- Control group: Vehicle and 50:50 FCA in water, and vehicle at 50% in 50:50 FCA/water, only.
- Site: intradermal induction – three pairs of injections in clipped interscapular region;
- Frequency of applications:
- Duration: 0-7 days. On day 7, clipped and SLS at 10% application. On day 8 - 48 hours for epidermal induction. The dressing was removed after 48 hours exposure
- Concentrations: Intradermal induction: A) ID: Freund's Complete Adjuvant diluted at 50% in water ; B) test item at 10% in liquid paraffin ; C a test mixture of the test item at 50% in FCA at 50% and in water to give final concentration of 10.0%.. Topical induction: The scapular area between the injection sites was clipped and brushed with a solution of SLS at 10%. Then the following day subsequently treated with 0.5 mL of a 100% test item concentration using occlusive dressing.
The control group were treated as described for the experimental group except that, instead of the test item, the vehicle was administered along with injections of (A) and (C) in three sites.

B. CHALLENGE EXPOSURE
- No. of exposures: 1 initial challenge ; 1 further challenge
- Day(s) of challenge: 24 hours (topical challenge)
- Exposure period: Day 22 the dressing was removed after 24 hours exposure. The treated sites were assessed for challenge reactions 24 and 48 hours after removal of the dressing. The second challenge was performed. The rest period was approximately 5 days.
- Test groups: 1 challenge; test item 100% and 2 challenge: 0.5% and 2%v/v in acetone.
- Control group: 1; vehicle only
- Site: One flank (clipped)
- Concentrations: 100% and 2 challenge: 0.5% and 2%v/v in acetone using occlusive dressing.
- Evaluation (hr after challenge): 24 and 48 hours after dressing removal (at Day 23 and 24) and/or following re-challenge on day 27.
The control group were treated as described for the experimental group except that, instead of the test item, the vehicle was administered.

OTHER: Mortality, toxicity and body weights along with irritation were examined as part of the study

Challenge controls:

(Naive) negative control groups consisting of 10 females were exposed to the vehicle in the induction and challenge, consistent the main study with the difference that instead of test item only the vehicle was administered during induction.

Positive control substance(s):

yes

Remarks:

Mercaptobenzothiazole (10%w/w intradermal and 50%w/w topical)

Positive control results:

A reliability check was performed (presented in the full study report) to check the sensitivity of the test system and the reliability of the experimental techniques used. The study used the same conditions as the main study using Mercaptobenzothiazole (at 10%w/w intradermal induction and 50%w/w topical concentrations) as positive control.
The skin reactions observed in ten experimental animals in response to the > 20 % PC item concentration in the challenge phase were considered indicative of sensitisation, based on the absence of any response in the control animals. These results lead to a sensitisation rate of 100% to the 10%w/w induction and 50%w/w challenge concentrations.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100%

No. with + reactions:

20

Total no. in group:

20

Clinical observations:

Total no. in groups: 20.0 ; Clinical observations: None reported; maximum score = 3 (n=5) to 2 (n=13) (discrete or moderate erythema).

Remarks on result:

positive indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100%

No. with + reactions:

20

Total no. in group:

20

Clinical observations:

Total no. in groups: 20.0 ; Clinical observations: None reported; maximum score = 3 (n=13) to 2 (n=6) (discrete or moderate erythema).

Remarks on result:

positive indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

Total no. in groups: 10.0. ; Clinical observations: None reported; maximum score = 0 (n=10)

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

Total no. in groups: 10.0. ; Clinical observations: None reported; maximum score = 0 (n=10)

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

10% intradermal induction and 50% cutaneous

No. with + reactions:

10

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Remarks:

non-concurrent PC conducted within 6 months of the definitive test; % sensitised was based on comparison of challenge sites with test and controls.

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 conditions of this study, the test item is considered to be a contact sensitizer.

Executive summary:

The study was performed using a method equivalent or similar to guideline OECD TG 406 and EU Method B.6 under GLP. The method was consistent with the Magnusson-Kligman Guinea Pig Maximisation test to assess the skin sensitisation potential of the test item. The concentrations selected for the main study were based on the results of a preliminary study. In the main study, after induction (intradermic injection at 10% in light paraffin oil vehicle and topical application at 100%, on days 1 and 8 respectively) with 10% SLS application on day 7. A treatment group of twenty and a control group of ten, respectively were on day 22, challenged with 100% (undiluted) test item along with parallel control challenged at 100% (undiluted) test item. The left flank was treated with vehicle only. Skin reactions were evaluated 24 to 48 hours after removal of the occlusive dressing. A second challenge was conducted on day 27 with 0.5% and 2% test item in acetone. No clinical signs were reported. Bodyweight gain in the treated group was comparable to controls. In the first challenge, an intense or moderate or discrete erythema (grade 3 or 2 or 1) was noted in 20/20 of the treated group and 0/10 of the control group. The maximum score in control was zero. In the second challenge utilising 2% test item in acetone, a discrete erythema (grade 1) was noted in 6/20 of the treated group and 0/10 of the control group. In the 0.5% test item in acetone and in control, the maximum score was zero. Under the conditions of this study, the test item is considered to be a contact skin sensitizer.

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10-09-2001 to 30-11-2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Method equivalent to guideline, well documented and performed under GLP. All relevant validity criteria were met.

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

Qualifier:

equivalent or similar 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

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/J Hsd strain

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Recognised supplier
- Age at study initiation: approximately 6 to 7 weeks old (prior to acclimatisation)
- Weight at study initiation: 16.3 – 22.1 grams
- Housing: Individually housed, in labelled shoebox style cages
- Diet: Free access to rodent diet (certified, recognised supplier)
- Water: mains tap water ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0 (actual: 17.1 to 25.0°C)
- Humidity (%): 40 to 80 (actual: 39 – 76%)
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 h light / 12 h dark

IN-LIFE DATES: From: 26-09-2001 To: 02-10-2001

Vehicle:

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

Concentration:

0% (vehicle control), 0.5, 1%, 5%, 10%, and 20%
ISOEUGENOL (positive control), 0.5%, 1.0% and 5.0%

No. of animals per dose:

Main test: 8 mice 0% (vehicle control) and 5 mice per test item dose group 0.5, 1%, 5%, 10%, and 20%

Details on study design:

RANGE FINDING TESTS:
Not applicable. The rationale for the test item dose was based on study sponsor selection, which typically would incorporate all available knowledge on the test item potential local/systemic toxicity prior to testing.

MAIN STUDY
- Compound solubility: Fully soluble in vehicle. The choice of AOO (4:1) as a vehicle was based on NIH Publication No. 99-449, "The Murine Local Lymph Node Assay: A Test Method for Assessing the Allergic Contact Dermatitis Potential of Chemicals / Compounds," in which AOO was the vehicle of choice.
- Irritation: None observed.
- Systemic toxicity: None observed.
- Ear thickness measurements: Not conducted.
- Erythema scores: No irritation was reported.

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: Following excision of the nodes. The individual SI is the ratio of the DPM/animal compared to DPM/vehicle control group. If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer.

TREATMENT PREPARATION AND ADMINISTRATION:
Five groups of five animals were treated with one test item concentration per group. One group of five animals was treated with vehicle.
- Induction: The dorsal surface of both ears was topically treated (25 μL/ear) with the test item concentration, at approximately the same time on each day with monitoring for local and systemic toxicity during dosing. A rest period was allowed on days 4 and 5. The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item.
- Node excision: Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) containing 2 μCi of 125-I labelled IuDR and 1x10^-5 M FuDR. After approximately five hours, all animals were terminated. Nodes were dissociated using the frosted ends of glass slides. The cell suspension was washed with Hanks' balanced salt solution (HBSS) and then with PBS prior to being resuspended in 5% trichloroacetic acid (TCA) and refrigerated at approximately 4°C. Approximately 20 hours later, the cells were centrifuged and resuspended in fresh 5% TCA. The radioactivity was measured using a gamma counter.
- Tissue processing and radioactivity measurements: See above. Results from each cell suspension counted on the gamma counter were recorded in counts per minute (CPM). The CPM values were converted to disintegrations per minute (DPM) by dividing by the gamma counter efficiency and multiplying by 100. After the DPM values had been calculated, the mean "blank" DPM was subtracted from each individual DPM to obtain corrected DPM values. The mean corrected DPM and standard error of the mean (SE) were determined for each group. The stimulation index (SI) was then calculated by dividing the treatment-group mean DPM by the control (vehicle)-group mean DPM.

Observations:
- Mortality/Viability: Once daily
- Bodyweights: On Day 1 (pre-dose) and Day 6 (post termination) although post-termination bodyweights were not reported. Applicant assessment: indicates it is feasible that no mention of bodyweight declines within the text of the report is potentially indicative of normal bodyweight gain.
- Clinical Observations: Once daily
- Irritation: Once daily.
- Ear Thickness: Not conducted.

Positive control substance(s):

other: Isoeugenol (CAS No 97-54-1)

Statistics:

A one-sample t test was performed to test whether the individual untransformed SI value for each dose level of each test/reference item was greater than or equal to 3. The natural log transformed DPM values for each compound were compared with vehicle using a Bartlett's Chi-Square test for variance homogeneity. If the Bartlett's Chi-Square results were found to be nonsignificant, a one-way analysis of variance (ANOVA) was performed using dose (concentration). If the ANOVA was found to be significant, then a Dunnett' s t test was performed using an alpha of 0.05. If the Barlett's Chi-Square was found to be significant, nonparametric analyses were conducted, specifically a Kruskal-Wallis (KW) test If the non-parametric analyses-were found to be significant, then a Jonckheere's-Terpera test was performed for dose-dependent trends.

Calculations were performed using Microsoft® Excel and JMP SAS®.

Positive control results:

The concurrent positive control (isoeugenol) had an EC3 = 1.19%. This was considered consistent with previously reported results.

Parameter:

EC3

Remarks:

%

Variability:

C.I. -%

Remarks on result:

other: See table below

Parameter:

SI

Value:

0.7

Variability:

± 0.3

Test group / Remarks:

0.5% in acetone:olive oil (4:1)

Parameter:

SI

Value:

1.3

Variability:

± 0.4

Test group / Remarks:

1% in acetone:olive oil (4:1)

Parameter:

SI

Value:

1

Variability:

± 0.5

Test group / Remarks:

5% in acetone:olive oil (4:1)

Parameter:

SI

Value:

1.2

Variability:

± 0.3

Test group / Remarks:

10% in acetone:olive oil (4:1)

Parameter:

SI

Value:

2.5

Variability:

± 0.6

Test group / Remarks:

20% in acetone:olive oil (4:1)

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA: See tables.

DETAILS ON STIMULATION INDEX CALCULATION: See tables.

EC3 CALCULATION: The data per concentration was fit using a quadratic equation (a linear term and a square term of the concentration). If the quadratic term did not fit, a simple regression model was used. The EC3 was determined from the appropriate regression equation.

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

BODY WEIGHTS: On Day 1 (pre-dose) and Day 6 (post termination) although post-termination bodyweights were not reported. Applicant assessment: indicates it is feasible that no mention of bodyweight declines within the text of the report is potentially indicative of normal bodyweight gain. Since toxicologically significant bodyweight declines would be unusual findings and typically reported alongside systemic toxicity findings.

Table 1. Results from the definitive test

Group	Number	CPM	DPM	Mean DPM	SD	SE	Mean SI	SE	Ears mean % increase	SE
Vehicle Control	1	26.6	30.3
	2	16.4	17.5
	3	68.6	83.2
	4	25.6	29.0
	5	26.0	29.5
	6	13.8	14.2
	7	19.8	21.7
	8	16.6	17.7	26.7	16.5	-	-	-	ND	ND
Test Item 0.5%	9	6.6	5.1
	10	35.8	41.9
	11	34.8	40.6
	12	9.8	9.1
	13	13.8	14.2	22.2	17.7	7.9	0.7	0.3	ND	ND
Test Item 1%	14	17.2	18.5
	15	17.4	18.7
	16	30.6	35.3
	17	32.8	38.1
	18	70.6	85.7	39.3	27.5	1.3	1.3	0.4	ND	ND
Test Item 5%	19	10.4	9.9
	20	24.2	27.3
	21	78.2	95.3
	22	8.4	7.4
	23	16.8	18.0	31.6	36.5	16.3	1.0	0.5	ND	ND
Test Item 10%	24	15.6	16.4
	25	14.2	14.7
	26	44.2	52.5
	27	57.4	69.1
	28	28.0	32.1	37.0	23.5	10.5	1.2	0.3	ND	ND
Test Item 20%	29	108.6	133.6
	30	18.8	20.5
	31	72.0	87.5
	32	71.4	86.8
	33	42.4	50.2	75.7	42.8	19.1	2.5	0.6	ND	ND
Isoeugenol 0.5%	34	15.6	16.4
	35	21.8	24.3
	36	43.4	51.5
	37	31.8	36.9
	38	24.8	28.0	31.4	13.4	6.0	1.0	0.2	ND	ND
Isoeugenol 1.0%	39	50.2	60.0
	40	15.2	15.9
	41	17.6	19.0
	42	31.2	36.1
	43	18.8	20.5	30.3	18.4	8.2	1.0	0.3	ND	ND
Isoeugenol 5.0%	44	272.6	340.3
	45	507.6	636.4
	46	292.4	365.2
	47	362.2	453.2
	48	119.2	147.0	388.4	178.1	79.6	12.8	2.6	ND	ND

Where:

SE = standard error of the mean

SD = standard deviation

ND = not determined

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study the test item is not considered to be sensitising to skin.

Executive summary:

The study was performed to using the local lymph node assay method under GLP to assess the skin sensitisation potential of the test material in the CBA/J strain mouse following topical application to the dorsal surface of the ear. The test item was tested at concentrations of: 0.5%, 1%, 5%, 10% and 20% in acetone/olive oil (4:1) and with a 0% vehicle control group. A concurrent positive control consisting of Isoeugenol at 0.5%, 1.0% and 5.0% concentrations was additionally conducted. Observations were made daily. No irritation or signs of systemic toxicity were observed. Three days after the final auricular application, the animals were injected intravenously with 125-I radiolabelled Iododeoxyuridine to label proliferating cells. 125-I incorporation was quantified using a gamma counter. The SI values calculated for the test item concentrations 0.5%, 1%, 5%, 10% and 20% were 0.7, 1.3, 1.0, 1.2 and 2.5 respectively. The results show that the test item did not elicit an SI ≥ 3. The concurrent positive control Isoeugenol had an EC3 value of 1.19%.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Key study : in vivo, eq. or similar to OECD TG 406, 1994 : The study was performed using a method equivalent or similar to guideline OECD TG 406 and EU Method B.6 under GLP. The method was consistent with the Magnusson-Kligman Guinea Pig Maximisation test to assess the skin sensitisation potential of the test item. The concentrations selected for the main study were based on the results of a preliminary study. In the main study, after induction (intradermic injection at 10% in light paraffin oil vehicle and topical application at 100%, on days 1 and 8 respectively) with 10% SLS application on day 7. A treatment group of twenty and a control group of ten, respectively were on day 22, challenged with 100% (undiluted) test item along with parallel control challenged at 100% (undiluted) test item. The left flank was treated with vehicle only. Skin reactions were evaluated 24 to 48 hours after removal of the occlusive dressing. A second challenge was conducted on day 27 with 0.5% and 2% test item in acetone. No clinical signs were reported. Bodyweight gain in the treated group was comparable to controls. In the first challenge, an intense or moderate or discrete erythema (grade 3 or 2 or 1) was noted in 20/20 of the treated group and 0/10 of the control group. The maximum score in control was zero. In the second challenge utilising 2% test item in acetone, a discrete erythema (grade 1) was noted in 6/20 of the treated group and 0/10 of the control group. In the 0.5% test item in acetone and in control, the maximum score was zero. Under the conditions of this study, the test item is considered to be a contact skin sensitizer.

Key study : in vivo, OECD TG 406, 1996 : The study was performed using a method according to guideline OECD TG 406 using the modified Buehler Method under GLP. The concentrations selected for the main study were based on the results of a preliminary study. The test item was dosed as supplied (100%) at the three inductions and at 50% in ethanol at the challenge phase of the test. There was no erythema noted at the first and second inductions. At twenty-four and forty-eight hours after the third induction, nineteen of twenty had patchy erythema using 100% test item. During the challenge, two of twenty male/females in the test item treated group had patchy erythema at twenty-four hours post dosing. There was no erythema in the naive control group at any scoring period. The positive control substance, dinitrochlorobenzene (DNCB), was dosed at a concentration of 1.0% in ethanol for the three inductions, and at 0.3% in ethanol at the challenge phase of the test. At challenge. twenty-four hours after the challenge, five of ten exhibited patchy erythema, five with confluent erythema. Forty-eight hours after the challenge, six had patchy erythema; two exhibited confluent erythema, and two with no erythema. At seventy-two hours after the challenge, five exhibited patchy erythema, five had no erythema. The positive control DNCB would be considered a sensitizer with a response of 80 to 100% during challenge. In the naive positive control group, none had erythema at any scoring point. Under the conditions of this study, the test item is not considered to be a contact skin sensitizer.

Key study : in vivo, eq. or similar to OECD TG 429, 2001 : The study was performed to using the local lymph node assay method under GLP to assess the skin sensitisation potential of the test material in the CBA/J strain mouse following topical application to the dorsal surface of the ear. The test item was tested at concentrations of: 0.5%, 1%, 5%, 10% and 20% in acetone/olive oil (4:1) and with a 0% vehicle control group. A concurrent positive control consisting of Isoeugenol at 0.5%, 1.0% and 5.0% concentrations was additionally conducted. Observations were made daily. No irritation or signs of systemic toxicity were observed. Three days after the final auricular application, the animals were injected intravenously with 125-I radiolabelled Iododeoxyuridine to label proliferating cells. 125-I incorporation was quantified using a gamma counter. The SI values calculated for the test item concentrations 0.5%, 1%, 5%, 10% and 20% were 0.7, 1.3, 1.0, 1.2 and 2.5 respectively. The results show that the test item did not elicit an SI ≥ 3. The concurrent positive control Isoeugenol had an EC3 value of 1.19%.

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.

 

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.