Reaction mass of (E)-3-methylcyclotetradec-5-en-1-one and (Z)-3-methylcyclotetradec-5-en-1-one

Endpoint summary

Description of key information

Cosmone LLNA study - 2004 - OECD guideline No. 429 : EC3 = 16.4% which corresponds to a skin sensitizer to mice

Cosmone OET study - 2005 - CFTA safety guidelines : positive indication of skin sensitization on guinea pigs for challenge concentrations of 10, 5, 3 and 1 % in all four test groups previously induced at 10, 20, 40 and 100 %

Cosmone - HRIPT 6%, 10% and 20% - 2004, 2005 and 2006 : no indication of skin sensitisation on humans for Cosmone at concentrations 6%, 10% and 20%

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 28 Jan 2004 to 07 Apr 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

conducted under GLP conditions

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2002

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

Description: Colourless to pale yellow liquid
Batch number: TQT0300497
Purity: 90.9%
Stability of test item: Stable under storage conditions
Expiry date: 28-NOV-2004
Storage conditions: In the original container at room temperature (20 °C ± 3 °C), away from direct sunlight.
Safety precautions: Routine hygienic procedures (gloves and goggles).

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Test system: Mice, CBA/CaOlaHsd
Rationale: Recognized as the recommended test system.
Source: Harlan Netherlands B.V. Postbus 6174 NL - 5960 AD Horst/ The Netherlands
Number of animals for the pre-test (non-GLP): 2 females
Number of animals for the main study: 16 females
Number of animals per group: 4 females (nulliparous and non-pregnant)
Number of test groups: 3
Number of control (vehicle) group: 1
Age: 8 - 12 weeks (beginning of acclimatization)
Body weight: 16 g - 24 g (ordered)
Identification: Each cage by unique cage card.
Randomization: Randomly selected by computer algorithm at time of delivery.
Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

HUSBANDRY
Room no. 129 B / RCC ltingen
Conditions: Standard Laboratory Conditions. Air-conditioned with target ranges for room temperature 22 ± 3 °C, relative
humidity 30 - 70 % and 10 - 15 air changes per hour. Room temperature and humidity were monitored continuously and values outside of these ranges occasionally occurred, usually following room cleaning. These transient variations are considered not to have any influence on the study and, therefore, these data are not reported but are retained at RCC. There was a 12 hour fluorescent light / 12 hour dark cycle with at least 8 hours music during the light period.
Accomodation: Individual in Makrolon type-2 cages with standard softwood bedding ("Ugnocel", Schill AG, CH-4132 Muttenz).
Diet:
Pelleted standard Kliba 3433, batch no. 78/03 mouse maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst) available ad libitum. Results of analyses for contaminants are archived at RCC)
Water: Community tap water from ltingen, available ad libitum. Results of representative bacteriological, chemical and contaminant analyses are archived at RCC.

Vehicle:

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

Concentration:

1 %, 10 % and 25 % (w/v) in acetone:olive oil, 4:1 (v/v)

No. of animals per dose:

4

Details on study design:

Topical application:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 1 %, 10 % and 25 % (w/v) in acetone:olive oil, 4:1 (v/v). The application volume, 25 µI, was spread over the entire dorsal surface (0 - 8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals). A hair dryer was passed briefly over the ear's surface to prevent the loss of any of the test item applied.

Administration of 3H-Methylthymidine:
3H-methyl thymidine (3HTdR) was purchased from Amersham International (Amersham product code no. TRA 310; specific activity, 2 Ci/mmol; concentration, 1 mCi/ml).
Five days after the first topical application, all mice were administered with 250 µI of
79.6 µCi/ml 3HTdR (equal to 19.9 µCi 3HTdR) by intravenous injection via a tail vein.

Determination of incorporated 3HTDR:
Approximately five hours after treatment with 3HTdR all mice were euthanized with dry ice
(CO2).
The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline {approx. 10 ml) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to glass scintillation vials with 10 ml of 'Ultima Gold' scintillation liquid and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a 13-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The 13-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Interpretation of raw data:
The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of 3HTdR incorporated into lymph node cells of test group relative to that recorded for control group (STIMULATION INDEX) (S.I.). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.
A test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled:
- First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the STIMULATION INDEX.
- Second, that 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.

Observations:
In addition to the sensitizing reactions the following observations and data were recorded during the test and observation period:
Mortality / Viability: Twice daily from acclimatization start to the termination of in-life phase.
Body weights: Prior to the 1st application and prior to necropsy.
Clinical signs (local I systemic): Daily from acclimatization start to the termination of in-life phase. Particular attention was paid to the treatment sites.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated in the body weight tables.
A statistical analysis was conducted for assessment of the dose-response relationship, and the EC3 value was calculated according to the equation
EC3 = (a-c) [(3-d)/(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.

Positive control results:

The test item ALPHA-HEXYLCINNAMALDEHYDE was found to be a skin sensitizer and an EC3 value of 11.7 % (w/v) was derived.

Parameter:

EC3

Value:

16.4

Variability:

% (w/v)

Parameter:

SI

Value:

1.4

Test group / Remarks:

Group 2 - 1% in acetone:olive oil, 4:1 (v/v)

Parameter:

SI

Value:

1.8

Test group / Remarks:

Group 3 - 10% in acetone:olive oil, 4:1 (v/v)

Parameter:

SI

Value:

4.6

Test group / Remarks:

Group 4 - 25% in acetone:olive oil, 4:1 (v/v)

Cellular proliferation data / Observations:

Proliferation data: see table "Results from definitive test"
Viability/Mortality: No deaths occurred during the study period.
Clinical signs: No clinical signs were observed in any animals of the control group, Group 2 (1 %) or Group  3 (10 %). On the second application day, a slight to moderate ear erythema was observed at both dosing sites in all mice of Group 4 (25 %), persisting for a total of three  days.
Body weights: The body weight of the animals, recorded prior to the1stapplication and prior to necropsy, was within the range commonly recorded for animals of this strain and age.

Table. Results from definitive test

Test item concentration % (w/v)		Measurement dpm	Calculation			Result
			dpm -BGa>	number of lymph nodes	dpm perlymph nodebl	S.I.
--	BG I	10	--	--	--	--
--	BG11	3	--	--	--	--
--	CG 1	2201	2194	8	274	--
1	TG2	3097	3090	8	386	1.4
10	TG3	3937	3930	8	491	1.8
25	TG4	10007	10000	8	1250	4.6

BG=Background (1 ml 5 % trichloroacetic acid) in duplicate CG = Control Group

TG = Test Group

S.I.= Stimulation Index

a)      = The mean value was taken from the figures BG I and BGII

b)      = Since the lymph nodes of the animals of a dose group were pooled,   DPM/nodewasdetermined by dividing the measured value by the number of lymph nodespooled

The proliferative capacity of pooled lymph node cells was determined by the incorporation  of3H-methyl thymidine measured on a 13-scintillation counter.

	Test item concentration %(w/v)	S.I.
Group 2	1	1.4
Group 3	10*	1.8*
Group 4	25*	4.6*
EC3=16.4 %(w/v)
A dose-response relation was observed. *This value was used in calculation of EC3.

Interpretation of results:

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

Conclusions:

In this study STIMULATION INDICES of 1.4, 1.8 and 4.6 were determined with the test item at concentrations of 1 %, 10 % and 25 % (w/v), respectively, in acetone:olive oil, 4:1 (v/v).
A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the STIMULATION INDEX (S.I.).
The test item KARMALONE was found to be a skin sensitizer and an EC3 value of 16.4 % (w/v) was derived.

Executive summary:

In order to study a possible contact allergenic potential of KARMALONE, three groups each of four female mice were treated daily with the test item at concentrations of 1 %, 10 % and 25%(w/v) in acetone:olive oil, 4:1 (v/v) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. A control group of four mice was treated with the vehicle (acetone:olive oil,4:1(v/v)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine {3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of3H-methyl thymidine measured in a ­ scintillationcounter.

No clinical signs were observed in any animals of the control group, Group 2 (1 %) or Group 3 (10 %). On the second application day, a slight to moderate ear erythema was observed at both dosing sites in all mice of Group 4 (25 %), persisting for a total of threedays.

All treated animals survived the scheduled study period.

The results obtained (STIMULATION INDEX (S.I.)) are reported in the following table. The estimated concentration of test item required to produce a S.I.of 3 is referred to as the EC3 value.

	Test item concentration %(w/v)	S.I.
Group 2	1	1.4
Group 3	10*	1.8*
Group 4	25*	4.6*
EC3=16.4 % (w/v)
A dose-response relation was observed. *This value was used in calculationofEC3.

In this study STIMULATION INDICES of 1.4, 1.8 and 4.6 were determined with the test item at concentrations of 1 %, 10 % and 25 % (w/v), respectively, in acetone:olive oil, 4:1(v/v).

A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the STIMULATION INDEX (S.I.).

The test item KARMALONE was found to be a skin sensitizer and an EC3 value of 16.4 % (w/v) was derived.

Reference 2

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

from 18 aug 2004 to 29 apr 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Remarks:

conducted under GLP conditions

Qualifier:

according to guideline

Guideline:

other: CTFA Safety Testing Guidelines, The Cosmetic, Toiletry and Fragrance Association, Inc. Washington, D.C. 20036; "Guidelines for Evaluating Photodermatitis"

Version / remarks:

1991

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

open epicutaneous test

Justification for non-LLNA method:

LLNA study shows that Cosmone is a skin sensitizer on mice. Here the substance is tested on another animals (guinea pigs).

Specific details on test material used for the study:

Description: Colourless to pale yellow liquid
Batch number: TQT0300497
Purity: 90.9%
Stability of test item: Stable under storage conditions
Expiry date: 28-NOV-2004
Storage conditions: In the original container at room temperature (20 °C ± 3 °C), away from direct sunlight.
Safety precautions: Routine hygienic procedures (gloves and goggles).

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male

Details on test animals and environmental conditions:

Test system: Albino Dunkin Hartley Guinea Pig, CRL:(HA)BR, SPF
Source: Charles River Deutschland GmbH Stolzenseeweg 32-36 D-88353 Kisslegg / Germany
Number of animals: 30 males
Age at beginning of acclimatization period: 4 - 6 weeks
Body weight at beginning of acclimatization period: Control and test groups 332 - 388 g
Identification: By unique cage number and corresponding individual animal number.
Randomization: Randomly selected by hand at time of delivery. No computer randomization.
Acclimatization: Five days for the control group and test groups 1 to 3 under test conditions after health examination. One day for the test group 4. Only animals without any visible signs of illness were used for the study.

HUSBANDRY:
Room no. 112 / RCC Ltd, FOllinsdorf
Conditions: Standard Laboratory Conditions
Air-conditioned with target ranges for room temperature 22
± 3 °C, relative humidity 30-70 % and approximately 10-15 air changes per hour. Room temperature and humidity were monitored continuously and values outside of these ranges occasionally occurred, usually following room cleaning. These transient variations are considered not to have any influence on the study and, therefore, these data are not reported but are retained at RCC. The animals were provided with an automatically controlled light cycle of 12 hours light and 12 hours dark. Music was played during the daytime light period.
Accommodation: Individually in Makrolon type-4 cages with a wire-mesh floor.
Diet: Pelleted standard Provimi Kliba 3418, batch nos. 39/04 and 55/04, guinea pig breeding / maintenance diet, containing Vitamin C (Provimi Kliba AG, CH-4303 Kaiseraugst), ad libitum. Results of analyses for contaminants are archived at RCC Ltd.
Water: Community tap water from FOllinsdorf, ad libitum. Results of bacteriological, chemical and contaminant analyses are archived at RCC Ltd.

Route:

epicutaneous, open

Vehicle:

unchanged (no vehicle)

Concentration / amount:

100%

Day(s)/duration:

4 weeks

Adequacy of induction:

highest technically applicable concentration used

Route:

epicutaneous, open

Vehicle:

corn oil

Concentration / amount:

10% in corn oil

Day(s)/duration:

4 weels

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Route:

epicutaneous, open

Vehicle:

corn oil

Concentration / amount:

20% in corn oil

Day(s)/duration:

4 weeks

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Route:

epicutaneous, open

Vehicle:

corn oil

Concentration / amount:

40% in corn oil

Day(s)/duration:

4 weeks

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

No.:

#1

Route:

epicutaneous, open

Vehicle:

corn oil

Concentration / amount:

10%

Day(s)/duration:

day 29

Adequacy of challenge:

highest non-irritant concentration

No.:

#1

Route:

epicutaneous, open

Vehicle:

corn oil

Concentration / amount:

5%

Day(s)/duration:

day 29

No.:

#1

Route:

epicutaneous, open

Vehicle:

corn oil

Concentration / amount:

3%

Day(s)/duration:

day 29

No.:

#1

Route:

epicutaneous, open

Vehicle:

corn oil

Concentration / amount:

1%

Day(s)/duration:

day 29

No.:

#2

Route:

epicutaneous, open

Vehicle:

corn oil

Concentration / amount:

0.75%

Day(s)/duration:

day 51

No.:

#2

Route:

epicutaneous, open

Vehicle:

corn oil

Concentration / amount:

0.50%

Day(s)/duration:

day 51

No.:

#2

Route:

epicutaneous, open

Vehicle:

corn oil

Concentration / amount:

0.25%

Day(s)/duration:

day 51

No.:

#2

Route:

epicutaneous, open

Vehicle:

corn oil

Concentration / amount:

0.10%

Day(s)/duration:

day 51

No. of animals per dose:

6

Details on study design:

Test item preparation:
The test item and vehicle were placed into a glass beaker on a tared Mettler PM 460 or AE 260 balance and a weight by weight dilution was prepared. A homogenizer was used to ensure homogeneous distribution of the test item in the vehicle. Each concentration was applied on standard areas of the clipped flank of each animal using a 1 ml tuberculin syringe.
The preparations were made prior to each dosing. Dose levels were in terms of material as supplied.

Pretest: Before starting the induction procedure, the threshold irritation concentration of the test item was estimated. The test item was applied epicutaneously, uncovered, undiluted and dissolved at 40 %, 20 % and 10 %. Corn oil was used for the dilutions. A single amount of
0.025 ml of each test item concentration was simultaneously applied to one of the areas, measuring 2 cm2 of the left flank previously clipped and marked with a circular stamp. The animals used for this previous test, were those of group V (test group 4).
The skin reactions were assessed 24 and 48 hours after the application of the test item. Three hours prior to the 24-hour reading the fur of the animals was depilated with an approved depilatory cream (VEET Cream, Reckitt & Colman AG, CH-4123 Allschwil) at the application site. The depilatory cream was placed on the test sites and surrounding areas, and left on for 3-5 minutes. It was then thoroughly washed off with a stream of warm running water. The animals were then dried with a disposable towel and returned to their cages. The minimal irritating and the maximal non-irritating test item concentration were determined by a binary criterion (yes/no). The minimal irritating concentration is defined as the lowest causing skin irritation. The maximal non-irritating concentration is defined as the highest not causing macroscopic skin reactions in any of the animals.
Comparable results were seen in all concentrations tested. Discrete/patchy erythema (grade 1) was recorded at 100, 40, 20 and 10 %.

Induction: Days 1-26:
On day 1, application of 0.1 ml of the test item, undiluted and at the concentrations of 1O %, 20 % and 40 % in com oil was performed to an area measuring 7 cm2 on the clipped right flank skin of the test guinea pigs. Four groups of six test animals each, were treated with
only one test item concentration (the lowest concentration of 10 % in the test group 1 to the highest concentration of 100 % in the test group 4). The applications were repeated daily (5 times per week) for 4 weeks using the same skin site. The application site was left uncovered and read approximately 24 hours after each application. The last application was read twice, at the 24- and 48-hour reading. The maximal non-irritating and the minimal irritating concentration after repeated applications were determined by the same all-or-none criterion as described above. If very strong skin reactions were provoked, the application sites were changed. The animals of the control group were treated in the same way with corn oil only.
On the 15 3rd and 5th day of the induction weeks (except in the third induction week on the
1st and 4th day), the test animals were shaved on the right flank to improve the contact of the skin and to facilitate the evaluation of a possible erythema and edema reaction.

Challenges:
To determine whether or not contact sensitization was induced, all groups of guinea pigs previously treated in the induction as well as the 6 control animals, were treated on day 29 (first challenge) and on day 51 (second challenge) on the clipped left (first challenge) and right (second challenge) flank with the test item at the same procedure as described above for the induction period but at concentrations of 10 %, 5 %, 3 % and 1 % in corn oil for the first challenge and 0.75 %, 0.50 %, 0.25 % and 0.10 % in corn oil for the second challenge. The minimal irritating concentration of test item(= the lowest tested concentration of 10 % in the pretest procedure) was used to confirm the biological activity determined before starting the induction and to exclude false results based on instability of test item. These tests were
performed by applying with syringe 0.025 ml of each concentration to skin areas measuring 2 cm2• Approximately 3 hours prior to the 24-hour reading the fur of the animals was depilated at the application site to avoid false scoring. The reactions were read after 24, 48 and 72 hours.


Reading and scoring: The scoring system was performed by visual scoring of erythema, oedema and other clinical changes of skin conditions. They were assessed as follows:
O = no visible change
1 = discrete or patchy erythema
2 = moderate and confluent erythema
3 = intense erythema and swelling
Grading of all animals was done by positioning the animal under true-light (Philips TLD 36W/84 or Osram 36W/31 830).
This procedure determines the minimal sensitizing concentration necessary for inducing allergic contact hypersensitivity and the minimal eliciting concentration necessary to cause a positive reaction. The test item is considered allergenic at a concentration to which at least one out of 6 animals of the concentration group concerned shows positive reactions when non-irritating concentrations were used for challenge, i.e. its threshold concentration causing skin reactions is shifted into the lower part of the concentration range used for challenge after comparison with control animals.

Observations:
In addition to the sensitizing reactions the following observations and data were recorded during the test and observation period:
Viability/Mortality: Daily from delivery of the animals to the termination of the test
Clinical signs (systemic): Daily from delivery of the animals to the termination of the test
Skin reactions/local symptoms: At the times specified during the pretest, induction and challenge periods
Body weights: At beginning of the acclimatization period, day 1 and termination of the test.

Necropsy: No necropsies were performed in the animals sacrificed at termination of the observation period.
The animals were sacrificed by intraperitoneal injection of Vetanarcol at a dose of at least
2.0 mUkg body weight (equivalent to 324 mg sodium pentobarbitone/kg body weight) and discarded.

Positive control substance(s):

yes

Remarks:

ALPHA-HEXYLCINNAMALDEHYDE

Positive control results:

The highest challenge concentration of 10 % chosen as well as the concentration of 5 % were irritant as revealed by the positive challenge results obtained with the control group. The threshold for induction of skin sensitization was approximately at 5 %.
No sensitization was induced with 1, 0.3 and 0.1 %. The challenge threshold was dependent on the induction concentration used. The declining frequency of positive animals indicated that the threshold was achieved at 3 %.
Therefore, ALPHA-HEXYLCINNAMALDEHYDE is considered to possess skin sensitization (contact allergenic) potential in albino guinea pigs and proves the sensitivity of the test item at low challenge concentrations.

Reading:

1st reading

Hours after challenge:

72

Group:

negative control

Dose level:

0% (control group); challenge concentrations 10%, 5%, 3% and 1%

No. with + reactions:

6

Total no. in group:

6

Clinical observations:

Discrete/patchy erythema

Remarks on result:

positive indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

72

Group:

test chemical

Dose level:

induction concentrations 100%, 40%, 20% and 10% ; challenge concentrations 10%, 5% and 3%

No. with + reactions:

6

Total no. in group:

6

Clinical observations:

Discrete/patchy to moderate/confluent erythema

Remarks on result:

positive indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

72

Group:

test chemical

Dose level:

induction concentration 20% ; challenge concentration 1%

No. with + reactions:

4

Total no. in group:

6

Clinical observations:

Discrete/patchy to moderate/confluent erythema

Remarks on result:

positive indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

72

Group:

test chemical

Dose level:

induction concentrations 100%, 40% and 10% ; challenge concentration 1%

No. with + reactions:

5

Total no. in group:

6

Clinical observations:

Discrete/patchy to moderate/confluent erythema

Remarks on result:

positive indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

72

Group:

negative control

Dose level:

0% (control group) ; challenge concentrations 0.75%, 0.50%, 0.25% and 0.10%

No. with + reactions:

0

Total no. in group:

6

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

induction concentrations 10% and 20% ; challenge concentration 0.75%

No. with + reactions:

4

Total no. in group:

6

Clinical observations:

Discrete/patchy erythema

Remarks on result:

positive indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

induction concentrations 40% and 100% ; challenge concentration 0.75%

No. with + reactions:

2

Total no. in group:

6

Clinical observations:

Discrete/patchy erythema

Remarks on result:

positive indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

induction concentrations 20% and 40% ; challenge concentration 0.50%

No. with + reactions:

2

Total no. in group:

6

Clinical observations:

Discrete/patchy erythema

Remarks on result:

positive indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

induction concentrations 100% and 10% ; challenge concentration 0.50%

No. with + reactions:

0

Total no. in group:

6

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

induction concentrations 100%, 40%, 20%, 10% ; challenge concentration 0.25%

No. with + reactions:

0

Total no. in group:

6

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

induction concentrations 100%, 40%, 20%, 10% ; challenge concentration 0.10%

No. with + reactions:

0

Total no. in group:

6

Remarks on result:

no indication of skin sensitisation

Viability/Mortality: There were no deaths during the course of the study, hence no necropsies were performed.

Clinical signs (systemic): No symptoms of systemic toxicity were observed in the animals.

Body weights: Animal no. 132 of group V (test group 4) showed a loss of body weight (7.2 %) during the acclimatization period. It recovered between the treatment start and the end of the study.

The body weight of the other animals was within the range commonly recorded for animals of this strain and age.

Interpretation of results:

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

Conclusions:

The initial challenge concentrations of 10, 5, 3 and 1 % in all four test groups previously induced at 10, 20, 40 and 100 %, respectively, indicated that all of the test animals (except in one occasion) had been sensitized. However, the nature of the reactions (no concentration dependency between the different test groups) and the presence of erythema at all four concentrations of 10, 5, 3 and 1 %, even slight in the control group, confirm they are in fact be due to skin irritation. Rechallenge in the test groups under identical conditions on the opposite flank by applying lower concentrations at 0.75, 0.50, 0.25 and 0.10 % in corn oil demonstrated that the very few slight erythema observed, were observed only at the 24-hour reading after treatment with the test item at 75 %. The fading of the reactions after the 24-hour reading and the absence of induction concentration dependency let to the conclu­ sion that the reactions in the test animals are not of an allergic nature.
Therefore, KARMALONE is considered to possess no skin sensitization (contact allergenic) potential in albino guinea pigs.

Executive summary:

In this open epicutaneous test (OET), the test item and its dilutions were applied epicutaneously uncovered to the test site with intact skin surface. Constant volumes per square centimeter of each test item concentration were applied to standard areas of the clipped flank skin during the induction and challenge phase. Six male albino guinea pigs were used for each test concentration and vehicle group.

The induction required daily applications, on 5 consecutive days per week during 4 weeks,  of

0.1 ml of the test item concentrations at 10 %, 20 %, 40 % and 100 %, applied in the test groups 1, 2, 3 and 4, respectively using a skin site of 7cm2on the contralateral right flank. Corn oil was used for thedilutions.

The first challenge was performed with all test groups treated in the same way with the test item at the minimal irritating concentration of 10 % and three lower primary non-irritating concentrations of 5 %, 3 % and 1 %. The concentrations were applied at a dose of 0.025 ml using a test site area of 2 cm2on the contralateral clipped left flank. Control animals were treated similarly, except that the use of test item was omitted during the induction.

A second challenge was performed 22 days after the first challenge due to the equivocal results obtained after the first challenge. The second challenge was performed in the same way as in the first challenge but with the concentrations of 0.75 %, 0.50 %, 0.25 % and

0.10 % in corn oil.

The reactions were read and recorded 24 hours after each induction application and 24, 48 and 72 hours after challenging.

Results

Skm'   Reactions after the First ChaIIenge procedure

GROUPS	INDUCTION CONCENTRATION(%)	FIRST CHALLENGE CONCENTRATION(%)POSITIVE/ TOTAL ANIMALS
		10 %	5%	3%	1%
I   (controlgroup)	corn oil	6/6	6/6	6/6	6/6
II  (test group 1)	10 %	6/6	6/6	6/6	5/6
Ill (test group 2)	20%	6/6	6/6	6/6	4/6
IV (test qroup 3)	40%	6/6	6/6	6/6	5/6
V (test group 4)	100 %	6/6	6/6	6/6	5/6

S.km Reactions after the second ChaIIenge procedure

GROUPS	INDUCTION CONCENTRATION(%)	SECOND CHALLENGE CONCENTRATION(%) POSITIVE/ TOTAL ANIMALS
		0.75%	0.50%	0.25%	0.10 %
I  (control qroup)	corn oil	0/6	0/6	0/6	0/6
II  (test qroup 1)	10%	4/6	0/6	0/6	0/6
Ill (test qroup 2)	20%	4/6	2/6	0/6	0/6
IV (test group 3)	40%	2/6	2/6	0/6	0/6
V (test group 4)	100 %	2/6	0/6	0/6	0/6

No toxic symptoms were evident in the guinea pigs of the control or test group. No deaths occurred.

The initial challenge concentrations of 10, 5, 3 and 1%in all four test groups previously induced at 10, 20, 40 and 100%,respectively, indicated that all of the test animals (except in one occasion) had been sensitized. However, the nature of the reactions (no concentration dependency between the different test groups) and the presence of erythema at all four concentrations of 10, 5, 3 and 1%, even slight in the control group, confirm they are in fact be due to skin irritation. Rechallenge in the test groups under identical conditions on the opposite flank by applying lower concentrations at 0.75, 0.50, 0.25 and 0.10%in corn oil demonstrated that the very few slight erythema observed, were observed only at the 24-hour reading after treatment with the test item at 75%.The fading of the reactions after the 24-hour reading and the absence of induction concentration dependency let to the conclu­ sion that the reactions in the test animals are not of an allergic nature.

Therefore, KARMALONE is considered to possess no skin sensitization (contact allergenic) potential in albino guinea pigs.

Reference 3

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

from December 29 2004 to February 4 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Repeated application under occlusive patch test conditions to the skin of human subjects

GLP compliance:

no

Remarks:

conducted under good clinical practice

Type of study:

other: human repeated insult patch test

Justification for non-LLNA method:

LLNA study shows that Cosmone is a skin sensitizer on animals. Here the substance is tested with 10% concentration on humans to see if there are skin reactions on humans too.

Specific details on test material used for the study:

Cosmone concentration: 10%
Description: Clear liquid
Test article ID: 10% C-61597

Species:

other: human

Strain:

not specified

Remarks:

not applicable

Sex:

male/female

Details on test animals and environmental conditions:

A total of 110 subjects, 20 males and 90 females ranging in age from 19 to 69 years, were empaneled for this test.

The subjects chosen were dependable and able to read and understand instructions. The subjects did not exhibit any physical or dermatological condition that would have precluded application of the test article or determination of potential effects of the test article.

Route:

epicutaneous, occlusive

Vehicle:

not specified

Concentration / amount:

10%

Day(s)/duration:

48 hours

Adequacy of induction:

not specified

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

not specified

Concentration / amount:

10%

Day(s)/duration:

72 hours

Adequacy of challenge:

not specified

No. of animals per dose:

110 subjects (20 males and 90 females)

Details on study design:

Induction phase:
A sufficient amount of the test article (approximately 0.2 ml) was placed onto a Parke-Davis Readi-Bandage® occlusive patch and applied to the back of each subject between the scapulae and waist, adjacent to the spinal mid-line. This procedure was performed by a trained technician/examiner and repeated every Monday, Wednesday and Friday until 9 applications of the test article had been made.

The subjects were instructed to remove the patch 24 hours after application. Twenty-four hour rest periods followed the Tuesday and Thursday removals and 48-hour rest periods followed each Saturday removal. Subjects returned to the Testing Facility and the site was scored by a trained examiner just prior to the next patch application.

If a subject developed a positive reaction of a level 2 erythema or greater during the Induction phase or if, at the discretion of the Study Director, the skin r sponse warranted a change in site, the patch was applied to a previously unpatched, adjacent site for the next application. If a level 2 reaction or greater occurred at the new site, no further applications were made. However, any reactive subjects were subsequently Challenge patch tested.

Challenge phase:
After a rest period of approximately 2 weeks (no applications of the test article), the Challenge patch was applied to a previously unpatched (virgin) test site. The site was scored 24 and 72 hours after application. All subjects were instructed to report any delayed skin reactivity that occurred after the final Challenge patch reading. When warranted, selected test subjects were called back to the Clinic for additional examinations and scoring to determine possible increases or decreases in Challenge patch reactivity.

Dermal responses for both the Induction and Challenge phases of the study were scored according to the following 6-point scale:
0 = No evidence of any effect
+ = Barely perceptible (Minimal, faint, uniform or spotty erythema) 1 = Mild (Pink, uniform erythema covering most of the contact site) 2 = Moderate (Pink-red erythema uniform in the entire contact site)
3 = Marked (Bright red erythema with/without petechiae or papules) 4 = Severe (Deep red erythema with/without vesiculation or weeping)
All other observed dermal sequelae (eg, edema, dryness, hypo- or hyperpigmentation) were appropriately recorded on the data sheet and described as mild, moderate or severe.

Positive control substance(s):

no

Reading:

1st reading

Hours after challenge:

72

Group:

test chemical

Dose level:

10%

No. with + reactions:

0

Total no. in group:

110

Remarks on result:

no indication of skin sensitisation

One hundred three (103/110) subjects satisfactorily completed the test procedure on Test Article: 10% C - 61597. Six (6/110) subjects discontinued for personal reasons unrelated to the conduct of the study.One (1/110) test panelist (Subject No.27[PanelNo.04343]) was discontinued due to violation of the Protocol;the subject was inadvertently enrolled prior to completion of the required rest period between studies. Discontinued panelist data are shown up to the point of discontinuation,but are not used in the Conclusions section of this final report.

There was no skin reactivity observed at any time during the course of the study.

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the conditions of a repeated insult (occlusive) patch test procedure in 103 subjects, Test Article: 10% C - 61597 was "Dermatologist-Tested" and did not induce skin irritation nor show any evidence of induced allergic contact dermatitis in human subjects.

Executive summary:

The objective of this study was to determine the irritation and/or sensitization potential of the test article after repeated application under occlusive patch test conditions to the skin of human subjects (non-exclusive panel).

Under the conditions of a repeated insult (occlusive) patch test procedure in 103 subjects, Test Article: 10% C - 61597 was "Dermatologist-Tested" and did not induce skin irritation nor show any evidence of induced allergic contact dermatitis in human subjects.

Reference 4

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

February 15 2006 to March 31 2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Repeated application under occlusive patch test conditions to the skin of human subjects

GLP compliance:

no

Remarks:

conducted under good clinical practices

Type of study:

other: Human Repeated Insult Patch test

Justification for non-LLNA method:

LLNA study shows that Cosmone is a skin sensitizer on animals. Here the substance is tested with 20% concentration on humans to see if there are skin reactions on humans too.

Specific details on test material used for the study:

Cosmone concentration: 20%
Description: Clear liquid
Test article ID: 20% C-615988

Species:

other: human

Strain:

not specified

Remarks:

not applicable

Sex:

male/female

Details on test animals and environmental conditions:

A total of 110 subjects, 20 males and 90 females ranging in age from 18 to 74 years, were empaneled for this test.
The subjects chosen were dependable and able to read and understand instructions. The subjects did not exhibit any physical or dermatological condition that would have precluded application of the test article or determination of potential effects of the test article.

Route:

epicutaneous, occlusive

Vehicle:

not specified

Concentration / amount:

20%

Day(s)/duration:

48 hours

Adequacy of induction:

not specified

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

not specified

Concentration / amount:

20%

Day(s)/duration:

72 hours

Adequacy of challenge:

not specified

No. of animals per dose:

110 humans: 20 males and 90 females

Details on study design:

Induction phase:
A sufficient amount of the test article (approximately 0.2 ml) was placed onto a modified Parke-Davis Readi-Bandage® occlusive patch and applied to the upper arm of each subject. This procedure was performed by a trained technician/examiner and repeated every Monday, Wednesday and Friday until 9 applications of the test article had been made.

The subjects were instructed to remove the patch 24 hours after application. Twenty-four hour rest periods followed the Tuesday and Thursday removals and 48-hour rest periods followed each Saturday removal. Subjects returned to the Testing Facility and the site was scored by a trained examiner just prior to the next patch application.
If a subject developed a positive reaction of a level 2 erythema or greater during the Induction phase or if, at the discretion of the Study Director, the skin response warranted a change in site, the patch was applied to a previously unpatched, adjacent site for the next application. If a level 2 reaction or greater occurred at the new site, no further applications were made. However, any reactive subjects were subsequently Challenge patch tested.

Challenge phase:
After a rest period of approximately 2 weeks (no applications of the test article), the Challenge patch was applied to a previously unpatched (virgin) test site. The site was scored 24 and 72 hours after application. All subjects were instructed to report any delayed skin reactivity that occurred after the final Challenge patch reading. When warranted, selected test subjects were called back to the Clinic for additional examinations and scoring to determine possible increases or decreases in Challenge patch reactivity.

Dermal responses for both the Induction and Challenge phases of the study were scored according to the following 6-point scale:
O = No evidence of any effect
+ = Barely perceptible (Minimal, faint, uniform or spotty erythema) 1 = Mild (Pink, uniform erythema covering most of the contact site) 2 = Moderate (Pink-red erythema uniform in the entire contact site)
3 = Marked (Bright red erythema with/without petechiae or papuies) 4 = Severe (Deep red erythema with/without vesiculation or weeping)

All other observed dermal sequelae (eg, edema, dryness, hypo- or hyperpigmentation) were appropriately recorded on the data sheet and described as mild, moderate or severe.

Positive control substance(s):

no

Reading:

1st reading

Hours after challenge:

72

Group:

test chemical

Dose level:

20%

No. with + reactions:

0

Total no. in group:

97

Remarks on result:

no indication of skin sensitisation

Ninety-seven (97/110) subjects satisfactorily completed the test procedure on Test Article: 20% C-615988. Thirteen (13/110) subjects discontinued for personal reasons unrelated to the conduct of the study. Discontinued panelist data are shown up to the point of discontinuation, but are not used in the Conclusions section of this final report.There was no skin reactivity observed at any time during the course of the study.

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the conditions of repeated insult (occlusive) patch test procedure conducted in 97 subjects, Test Article: 20% C-615988 was "Dermatologist­ Tested" and did not induce skin irritation nor show any evidence of induced allergic contact dermatitis in human subjects.

Executive summary:

The objective of this study was to determine the irritation and/or sensitization potential of the test article after repeated application under occlusive patch test conditions to the skin of human subjects (exclusive panel).

Under the conditions of repeated insult (occlusive) patch test procedure conducted in 97 subjects, Test Article: 20% C-615988 was "Dermatologist­ Tested" and did not induce skin irritation nor show any evidence of inducedallergic contact dermatitis in human subjects.

Reference 5

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

April 21 to May 27 2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Repeated application under occlusive patch test conditions to the skin of human subjects

GLP compliance:

no

Remarks:

Conducted under Good Clinical Practices

Type of study:

other: Human repeated insult patch test

Justification for non-LLNA method:

LLNA study shows that Cosmone is a skin sensitizer on animals. Here the substance is tested with 6% concentration on humans to see if there are skin reactions on humans too.

Specific details on test material used for the study:

Cosmone concentration: 6%
Description: Clear liquid
Test article ID: 6% C-61596F

Species:

other: human

Strain:

not specified

Remarks:

not applicable

Sex:

male/female

Details on test animals and environmental conditions:

A total of 55 subjects, 7 males and 48 females ranging in age from 20 to 69 years, were empaneled for this test.
The subjects chosen were dependable and able to read and understand instructions. The subjects did not exhibit any physical or dermatological condition that would have precluded application of the test article or determination of potential effects of the test article.

Route:

epicutaneous, occlusive

Vehicle:

not specified

Concentration / amount:

6%

Day(s)/duration:

48 hours

Adequacy of induction:

not specified

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

not specified

Concentration / amount:

6%

Day(s)/duration:

72 hours

Adequacy of challenge:

not specified

No. of animals per dose:

55(7 males and 48 females)

Details on study design:

Induction phase:
A sufficient amount of the test article (approximately 0.2 ml) was placed onto a Parke-Davis Readi-Bandage® occlusive patch and applied to the back of each subject between the scapulae and waist, adjacent to the spinal mid-line. This procedure was performed by a trained technician/examiner and repeated every Monday, Wednesday and Friday until 9 applications of the test article had been made.

The subjects were instructed to remove the patch 24 hours after application. Twenty-four hour rest periods followed the Tuesday and Thursday removals and 48-hour rest periods followed each Saturday removal. Subjects returned to the Testing Facility and the site was scored by a trained examiner just prior to the next patch application.

If a subject developed a positive reaction of a level 2 erythema or greater during the Induction phase or if, at the discretion of the Study Director, the skin response warranted a change in site, the patch was applied to a previously unpatched, adjacent site for the next application. If a level 2 reaction or greater occurred at the new site, no further applications were made. However, any reactive subjects were subsequently Challenge patch tested.

Challenge phase:
After a rest period of approximately 2 weeks (no applications of the test article), the Challenge patch was applied to a previously unpatched (virgin) test site. The site was scored 24 and 72 hours after application. All subjects were instructed to report any delayed skin reactivity that occurred after the final Challenge patch reading. When warranted, selected test subjects were called back to the Clinic for additional examinations and scoring to determine possible increases or decreases in Challenge patch reactivity.

Dermal responses for both the Induction and Challenge phases of the study were scored according to the following 6-point scale:
O =No evidence of any effect
+ =Barely perceptible (Minimal, faint, uniform or spotty erythema) 1 =Mild (Pink, uniform erythema covering most of the contact site) 2 =Moderate (Pink-red erythema uniform in the entire contact site) 3 =Marked (Bright red erythema with/without petechiae or papules)
4 =Severe (Deep red erythema with/without vesiculation or weeping)

All other observed dermal sequelae (eg, edema, dryness, hypo- or hyperpigmentation) were appropriately recorded on the data sheet and described as mild, moderate or severe.

Positive control substance(s):

no

Reading:

1st reading

Hours after challenge:

72

Group:

test chemical

Dose level:

6%

No. with + reactions:

0

Total no. in group:

54

Remarks on result:

no indication of skin sensitisation

Fifty-four (54/55) subjects satisfactorily completed the test procedure on Test Article: 6% C-61596F. One (1/55) subject discontinued for personal reasons unrelated to the conduct of the study. Discontinued panelist data are shown up to the point of discontinuation, but are not used in the Conclusions section of this final report.

 

A barely perceptible (+) patch test response was observed on one (1/54) test panelist (Subject No. 19) during the Induction phase of the study. This response is judged to be non-specific in nature and is not indicative of clinically significant irritation.

There were no responses on any subject during the Challenge phase.

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the conditions of a repeated insult (occlusive) patch test procedure, Test Article: 6% C-61596F was "Dermatologist-Tested" and did not induce clinically significant skin irritation nor show any evidence of induced allergic contact dermatitis in human subjects.

Executive summary:

The objective of this study was to determine the irritation and/or sensitization potential of the test article after repeated application under occlusive patch test conditions to the skin of human subjects (exclusive panel).

Under the conditions of a repeated insult (occlusive) patch test procedure, Test Article: 6% C-61596F was "Dermatologist-Tested" and did not induce clinically significant skin irritation nor show any evidence of induced allergic contact dermatitis in human subjects.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Justification for classification or non-classification

According to the EU CLP regulation (No 1272/2008 and its adaption 286/2011), EC3 value from LLNA study for Cosmone is 16.4% so > 2% that is why Cosmone is classified as a skin sensitizer category 1B, H317: May cause an allergic skin reaction.