Balmywood

Endpoint summary

Description of key information

Skin sensitiser, category 1 (OECD 442D, KeratinoSens, GLP, K, rel.1)

 

Sub-categorisation as skin sensitiser category 1B based on the following weight-of-evidence approach:

• OECD 429 study (GLP, WoE, rel.1) on read-across substance (Labdanum gum): the source and the target substances are not a skin sensitizer.


• OECD 429 study (GLP, WoE, rel.1) on read-across substance (Cedarwood Texas cedrol oil): the source and the target substances need to be classified as category 1B skin sensitiser.

--> As the first source substance is not considered to be a skin sensitiser and the second source substance is considered to be a low sensitiser (category 1B), the probability of occurrence of a high sensitisation rate in humans based on animal data for the starting materials is not expected and therefore the target substance does not require a category 1A classification for skin sensitisation. Therefore, the registered substance is considered to be classified as a skin sensitiser (category 1B) and labelled as H317 (May cause an allergic skin reaction).

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:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

See the RAAF document.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Remarks:

Read-across justification document

Positive control results:

The SI values calculated for the item concentrations 5, 10 and 25% were 1.4, 2.4 and 4.3 respectively. An EC3 value of 14.7% was calculated using linear interpolation. The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%. The results of the 6 monthly HCA reliability checks of the recent years were 13.4, 14.1, 17.3, 9.8, 17.8% and 18.0%. The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch was found a appropriate model for testing for contact hypersensitivity.

Key result

Parameter:

EC3

Value:

13.4

Parameter:

SI

Value:

1

Variability:

0.3

Test group / Remarks:

0% dose

Parameter:

SI

Value:

2

Variability:

0.6

Test group / Remarks:

5% dose

Parameter:

SI

Value:

2.3

Variability:

0.6

Test group / Remarks:

10% dose

Parameter:

SI

Value:

5.4

Variability:

1.3

Test group / Remarks:

25% dose

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
-All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

DETAILS ON STIMULATION INDEX CALCULATION
-Mean DPM/animal values for the experimental groups treated with test item concentrations 5, 10 and 25% were 561, 647 and 1524 DPM, respectively. The mean DPM/animal value for the vehicle control group was 285 DPM. The SI values calculated for the test item concentrations 5, 10 and 25% were 2.0, 2.3 and 5.4, respectively.

EC3 CALCULATION
-The EC3 value (the estimated item concentration that will give a SI=3) was determined based on the dose response relationship or calculated using linear interpolation. The test item elicits a SI ≥ 3 when tested at 25%. The data showed a dose response and an EC3 value of 13.4% was calculated.

CLINICAL OBSERVATIONS:
-No erythema was noted for any of the animals. Scaliness was noted on the ears of four animals treated at 25% between Days 3 and 6. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study.

BODY WEIGHTS
-Body weights and body weight gain of experimental animals remained in the same range as controls over the main study period.

Interpretation of results:

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

Remarks:

Based on CLP criteria (Annex I of 1272/2008/EC)

Conclusions:

Based on the results of this LLNA according with OECD Guideline 429, the calculated EC3 value of the source and the target substance is 13.4%. Therefore, Cedrol, Cedarwood Texas oil distilled and the target substance need to be classified as category 1B skin sensitiser according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).

Executive summary:

The skin sensitisation potential of Cedrol, Cedarwood Texas oil distilled was tested according to OECD, Section 4, Health Effects, No.429 (2010).

Three experimental groups of five female CBA/J mice were treated with test item concentrations of 5, 10 or 25% w/w on three consecutive days, by open application on the ears. Test item concentrations selected for the main study were based on the results of a pre-screen test. Five vehicle control animals were similarly treated, but with the vehicle alone (AcOO).  

No erythema was noted for any of the animals, Scaliness was noted on the ears of four animals treated at 25% between days 3 and 6. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals. Body weights and body weight gain of experimental animals remained in the same range as controls over the main study period. Mean DPM/animal values for the experimental groups treated with test item concentrations 5, 10 and 25% were 561, 647 and 1524 DPM, respectively. The mean DPM/animal value for the vehicle control group was 285 DPM. The SI values calculated for the test item concentrations 5, 10 and 25% were 2.0, 2.3 and 5.4, respectively.

The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 13.4% was calculated.

Based on the results of this LLNA according with OECD Guideline 429, the calculated EC3 value of the source and the target substances is 13.4%. Therefore, Cedrol, Cedarwood Texas oil distilled and the target substance need to be classified as category 1B skin sensitiser according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

14 Mar 2017 - 30 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Qualifier:

according to guideline

Guideline:

other: EC, No 440/2008, part B "Skin Sensitization: Local Lymph Node Assay"

Version / remarks:

2008

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and Batch No.of test material: Provided by sponsor, B-64530
- Expiration date of the lot/batch: 25 January 2019
- Purity test date: 26 January 2017
- Test Facility test item number: 207804/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
-Test item was equilibrated to 100C for several minutes until completely liquefied to obtain a homogeneous sample. Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily and dosed within 4 hours after adding the vehicle to the test item. The dosing formulations were kept at room temperature until dosing. The dosing formulations were stirred until and during dosing.

Species:

mouse

Strain:

CBA

Remarks:

J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals: SPF-quality
- Age at study initiation: approx. 10 weeks old
- Weight at study initiation: 18.7 to 23.6 g
- Housing: animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon MIII type; height 18 cm.) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles.
- Diet: ad libitum, pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: ad libitum, municipal tap-water (periodically analysed)
- Acclimation period: at least 5 days
- Indication of any skin lesions: before the initiation of dosing, a health inspection was performed and any assigned animal considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24
- Humidity (%): 42 to 66%
- Air changes (per hr): >10 (no recirculation)
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES:
-Experimental study start date 09 March 2017 - 30 May 2017 (completion of In-life)

Vehicle:

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

Concentration:

Pre-screen: 10, 25, 50, 100 % w/w
Main study: 0, 5, 10, 25 % w/w

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Irritation: erythema and eschar formation observations were performed once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing)
- Systemic toxicity: observations were performed once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
- Ear thickness measurements: ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on Days 1 and 3, and on Day 6
- Erythema scores:
0 No erythema
1 Very slight erythema (barely perceptible)
2 Well-defined erythema
3 Moderate to severe erythema (beet redness) to slight eschar formation (injuries in depth)
4 Severe erythema (beet redness) to eschar formation preventing grading of erythema

MAIN STUDY
In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Aceton/Olive Oil (4:1 v/v)). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA

EVALUATION CRITERIA
DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean. If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer. Consideration was given to the EC3 value (the estimated test item concentration that will give a SI =3), EC3 value ≤ 2%: sub-category 1A, EC3 value > 2%: sub-category 1B.

TREATMENT PREPARATION AND ADMINISTRATION:
Test item dosing formulations (w/w) were homogenized in the vehicle (acetone/olive oil (4:1 v/v)) to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily and dosed within 4 hours after adding the vehicle to the test item. The dosing formulations were kept at room temperature until dosing. The dorsal surface of both ears was topically treated (25 μL/ear) with the test item, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing. The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item.

Positive control substance(s):

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

Statistics:

- DPM values are presented for each animal and for each dose group.
- A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean.
- Consideration was given to the EC3 value (the estimated test item concentration that will give a SI =3)

Positive control results:

The SI values calculated for the item concentrations 5, 10 and 25% were 1.4, 2.4 and 4.3 respectively. An EC3 value of 14.7% was calculated using linear interpolation. The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%. The results of the 6 monthly HCA reliability checks of the recent years were 13.4, 14.1, 17.3, 9.8, 17.8% and 18.0%. The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch was found a appropriate model for testing for contact hypersensitivity.

Key result

Parameter:

EC3

Value:

13.4

Parameter:

SI

Value:

1

Variability:

0.3

Test group / Remarks:

0% dose

Parameter:

SI

Value:

2

Variability:

0.6

Test group / Remarks:

5% dose

Parameter:

SI

Value:

2.3

Variability:

0.6

Test group / Remarks:

10% dose

Parameter:

SI

Value:

5.4

Variability:

1.3

Test group / Remarks:

25% dose

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
-All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

DETAILS ON STIMULATION INDEX CALCULATION
-Mean DPM/animal values for the experimental groups treated with test item concentrations 5, 10 and 25% were 561, 647 and 1524 DPM, respectively. The mean DPM/animal value for the vehicle control group was 285 DPM. The SI values calculated for the test item concentrations 5, 10 and 25% were 2.0, 2.3 and 5.4, respectively.

EC3 CALCULATION
-The EC3 value (the estimated item concentration that will give a SI=3) was determined based on the dose response relationship or calculated using linear interpolation. The test item elicits a SI ≥ 3 when tested at 25%. The data showed a dose response and an EC3 value of 13.4% was calculated.

CLINICAL OBSERVATIONS:
-No erythema was noted for any of the animals. Scaliness was noted on the ears of four animals treated at 25% between Days 3 and 6. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study.

BODY WEIGHTS
-Body weights and body weight gain of experimental animals remained in the same range as controls over the main study period.

Interpretation of results:

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

Remarks:

Based on CLP criteria (Annex I of 1272/2008/EC)

Conclusions:

Based on the results of this LLNA, the calculated EC3 value is 13.4%. Therefore, Cedrol, Cedarwood Texas oil distilled needs to be classified as category 1B skin sensitiser according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).

Executive summary:

The skin sensitisation potential of Cedrol, Cedarwood Texas oil distilled was tested according to OECD, Section 4, Health Effects, No.429 (2010).

Three experimental groups of five female CBA/J mice were treated with test item concentrations of 5, 10 or 25% w/w on three consecutive days, by open application on the ears. Test item concentrations selected for the main study were based on the results of a pre-screen test. Five vehicle control animals were similarly treated, but with the vehicle alone (AcOO).  

No erythema was noted for any of the animals, Scaliness was noted on the ears of four animals treated at 25% between days 3 and 6. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals. Body weights and body weight gain of experimental animals remained in the same range as controls over the main study period. Mean DPM/animal values for the experimental groups treated with test item concentrations 5, 10 and 25% were 561, 647 and 1524 DPM, respectively. The mean DPM/animal value for the vehicle control group was 285 DPM. The SI values calculated for the test item concentrations 5, 10 and 25% were 2.0, 2.3 and 5.4, respectively.

The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 13.4% was calculated.

Based on the results of this LLNA according with OECD Guideline 429, the calculated EC3 value is 13.4%. Therefore, Cedrol, Cedarwood Texas oil distilled needs to be classified as category 1B skin sensitiser according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).

Reference 3

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

See the RAAF document.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Remarks:

Read-across justification document

Positive control results:

The reduced LLNA (rLLNA) , Positive control from study: 41403325 (08 October 2014 to 14 October 2014 ). Positive control: α Hexylcinnamaldehyde, tech., 85% in dimethyl formamide at a concentration of 15% v/v. SI- 5.34

Parameter:

SI

Value:

1.22

Test group / Remarks:

test concentration 2.5%

Parameter:

SI

Value:

1.35

Test group / Remarks:

concentration 5%

Key result

Parameter:

SI

Value:

1.76

Test group / Remarks:

at 10% concentration

Clinical Observations and Mortality Data

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

 

Bodyweight

Body weight change of the test animals between Day 1 and Day 6 were comparable to that observed in the corresponding control group animals over the same period.

Interpretation of results:

GHS criteria not met

Conclusions:

The source and the target substances were considered to be a non skin-sensitizer under the conditions of the test.

Executive summary:

A study was performed to assess the skin sensitisation potential of test material in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was conducted according to the OECD test guideline No 429 and in compliance with GLP.

 

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 10% w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of five animals, were treated with 50 µL (25 µL per ear) of the test item as a solution in dimethyl formamide at concentrations of 10%, 5% or 2.5% w/w. A further group of five animals was treated with dimethyl formamide alone.

 

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

 

Concentration (%w/w) in dimethyl formamide	Stimulation Index	Result
2.5	1.22	Negative
5	1.35	Negative
10	1.76	Negative

 

 

The historical positive control, α-Hexylcinnamaldehyde, gave a SI of 5.34, when tested at 15 % v/v. The test system was therefore considered to be valid.

 

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.Body weight change of the test animals between Day 1 and Day 6 were comparable to that observed in the corresponding control group animals over the same period.

 

Under the test conditions, the source and the target substances are not classified as a skin sensitiser according to the annex VI of the Regulation EC No. 1272/2008 (CLP) .

This study is considered as acceptable and satisfies the requirement for sensitisation endpoint.

Reference 4

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

November 2014 - february 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 22 July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

dark brown solid block

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories Uk, Oxon, UK
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15-23 g
- Housing: Animals were housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet: Food (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK), ad libitum
- Water: Mains tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C
- Humidity: 30-70 %
- Air changes: Approximately 15 changes/h
- Photoperiod: 12 h dark / 12 h light

Vehicle:

dimethylformamide

Concentration:

Preliminary screening test:50%, 25% and 10% w/w in DMF
Main test: 10%, 5% and 2.5%w/w in DMF

No. of animals per dose:

Preliminary screening test: One animal/dose
Main test: 5 animals/dose

Details on study design:

RANGE FINDING TESTS:
- Using available information regarding the irritancy potential of the test item, a preliminary screening test was performed using three mice, one mouse per test item concentration. The mice were treated by daily application of 25 μL of the test item at concentrations of 50%, 25% and 10% w/w in dimethyl formamide, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mice were 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 body weight was recorded on Day 1 (prior to dosing) and on Day 6.
- The thickness of each ear was measured using a Mitutoyo 547-300S gauge (Mitutoyo Corporation), pre-dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization.

- Irritation: No signs of systemic toxicity were noted. Very slight erythema on both ears was noted in the animal treated with the test item at a concentration of 50% w/w in dimethyl formamide. Colorless, sticky residual test item on the ears and fur loss were noted in the animals treated with the test item at concentrations of 50% or 25% w/w in dimethyl formamide.(DMF). A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization. Based on this information the dose levels selected for the main test were 10%, 5% and 2.5% w/w in DMF.


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: The test item will be regarded as a sensitizer 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 sensitizer."

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was formulated within two hours of being applied to the test system. It is assumed that the formulation was stable for this duration.
25 µL of control or test material was applied topically on the dorsal surface of both ears using a micropipette daily for three consecutive days (Days 1-3). 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, ARC UK Ltd) giving a total of 20 μCi to each mouse. Five hours later animals were killed by carbon dioxide asphyxiation followed by cervical separation. The lymph nodes from each group were pooled and a single cell suspension was prepared. Cells were washed with phosphate buffered saline (PBS) and precipitated with 5% trichloroacetic acid (TCA) for 18 h at ca. 4 °C. The pellets were recovered by centrifugation and resuspended in 1 mL of TCA and transferred to a vial containing scintillation fluid. 3HTdR incorporation was measured by β-scintillation counting. The "Poly Q™" 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 twenty minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).
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 (Stimulation Index).

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. Data was first assessed for suitability by analysis of normality and homogeneity of variance. If the assumptions that the data are both normally distributed and has homogeneity of variances, then parametric one way analysis of variance (ANOVA) and Dunnett’s multiple comparison procedure were used to determine statistical significance. If the assumptions were not met, non-parametric Kruskal-Wallis Rank Sum and Mann-Whitney U test procedures were used.
Probability values (p) were presented as follows:
P<0.001 ***
P<0.01 **
P<0.05 *
P≥0.05 (not significant)

Positive control results:

The reduced LLNA (rLLNA) , Positive control from study: 41403325 (08 October 2014 to 14 October 2014 ). Positive control: α Hexylcinnamaldehyde, tech., 85% in dimethyl formamide at a concentration of 15% v/v. SI- 5.34

Parameter:

SI

Value:

1.22

Test group / Remarks:

test concentration 2.5%

Parameter:

SI

Value:

1.35

Test group / Remarks:

concentration 5%

Key result

Parameter:

SI

Value:

1.76

Test group / Remarks:

at 10% concentration

Clinical Observations and Mortality Data

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

 

Bodyweight

Body weight change of the test animals between Day 1 and Day 6 were comparable to that observed in the corresponding control group animals over the same period.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to be a non skin-sensitizer under the conditions of the test.

Executive summary:

A study was performed to assess the skin sensitisation potential of test material in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was conducted according to the OECD test guideline No 429 and in compliance with GLP.

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 10% w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of five animals, were treated with 50 µL (25 µL per ear) of the test item as a solution in dimethyl formamide at concentrations of 10%, 5% or 2.5% w/w. A further group of five animals was treated with dimethyl formamide alone.

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

 

Concentration (%w/w) in dimethyl formamide	Stimulation Index	Result
2.5	1.22	Negative
5	1.35	Negative
10	1.76	Negative

 

 

The historical positive control, α-Hexylcinnamaldehyde, gave a SI of 5.34, when tested at 15 % v/v. The test system was therefore considered to be valid.

 

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.Body weight change of the test animals between Day 1 and Day 6 were comparable to that observed in the corresponding control group animals over the same period.

 

Under the test conditions, test material is not classified as a skin sensitiser according to the annex VI of the Regulation EC No. 1272/2008 (CLP) .

This study is considered as acceptable and satisfies the requirement for sensitisation endpoint.

Reference 5

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 07 March 2022 to 18 March 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD Guideline No. 442D and under GLP compliance with the following deviation: in repetition 2, the EC1.5 of positive control reference (21.33 µM) is very slightly above the historical data range of the laboratory (3-20µM) and within the OECD dataset validation range (7-30µM). This result is considered acceptable and allows the study to be validated without impacting on the results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

Dated June, 25th, 2018

Deviations:

yes

Remarks:

See section 'Principles of method if other than guideline' below for more details.

Principles of method if other than guideline:

The study is performed according to OECD Guideline No. 442D and under GLP compliance with the following deviation: in repetition 2, the EC1.5 of positive control reference (21.33 µM) is very slightly above the historical data range of the laboratory (3-20µM) and within the OECD dataset validation range (7-30µM). This result is considered acceptable and allows the study to be validated without impacting on the results.

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Justification for non-LLNA method:

The in vitro ARE-Nrf2 luciferase KeratinoSens™ test method (hereafter called the KeratinoSens™ test method) underwent validation studies followed by an independent peer review conducted by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM). The KeratinoSens™ test method was considered scientifically valid to be used as part of an IATA, to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard
identification.

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

PREPARATION OF TEST ITEM STOCK, SPIKING AND WORKING SOLUTIONS
- The test item was diluted in DMSO. The stock solution was prepared at 40 mg/mL (i.e. 4%). The test item was tested at 12 concentrations according to a geometric progression of ratio 2 from from 0.2 µg/mL to 400 µg/mL.
Then, a 100-fold concentrated dilutions series was prepared in 96-well plate. The test item was placed in one of the rows B to F. 100 µL of DMSO were distributed from columns 1 to 11. 200 µL of the 40 mg/mL stock solution were placed in column 12 then the series dilutions were prepared by transferring 100 µL from column 12 to column 11 and so on until the column 1. Dilutions were mixed by repeated pipetting, at least 3 times, between each concentration.
Then, the 100-fold DMSO plate was diluted 25-fold in a new plate (4-fold) in treatment medium.

PREPARATION OF THE POSITIVE CONTROL
- The positive control stock solution was prepared at 200 mM in DMSO according to the following formula, then diluted to 6.4 mM:
V = 5 x [(p ÷100) x w / MW] - (w/1000)

V is the volume of DMSO in mL to be added
p is the purity of the positive control in %
MW is the molecular weight of the positive control in g/mol
w is the exact weight of the positive control in mg.

- Then, 100 µL of DMSO were distributed in row G from columns 7 to 10. 200 µL of the 6.4 mM stock solution were placed in column 11 then the series dilutions were prepared by transferring 100 µL from column 11 to column 10 and so on until the column 7. Dilutions were mixed by repeated pipetting, at least 3 times, between each concentration.

NEGATIVE CONTROL
- Treatment culture medium with 1% DMSO was used as solvent control/negative control. 6 wells of solvent control (1% DMSO in treatment medium) with cells and 1 well of solvent control without cell by culture plate were included.
- 100 µL of DMSO were distributed in row G columns 1 to 6 and 12 and in the well H12.

CELL LINE
KeratinoSens™cells (Givaudan) were maintained according to the current working instruction IL 09. Cells are cultured in maintenance medium at 37°C, 5% CO2. Cells are exempt of mycoplasma. Assessment of mycoplasma was performed according to the current working instruction IL 07.
Cells were used at passage 25 in repetition 1 and passage 13 in repetition 2.

CELL CULTURE
- Maintenance medium: DMEM 1 g/l glucose, 9.1% non-heat inactivated foetal calf serum, 0.05% geneticin - Stored at 5°C ± 3°C
- Seeding medium: DMEM 1 g/l glucose, 9.1% non-heat inactivated foetal calf serum - Stored at 5°C ± 3°C
- Treatment medium: DMEM 1 g/l glucose, 1% non-heat inactivated foetal calf serum - Stored at 5°C ± 3°C
- Trypsin (0.5 g/L) - EDTA (0.2 g/L) - stored at -20°C ± 5°C upon opening and 5°C ± 3°C after opening.
- Diluent for the test item and the positive control: DMSO - stored at room temperature 20°C ± 5°C upon opening.
- Luciferase substrate: Lyophilized Bright-Glo™ Substrate: - 20°C ± 5°C, Bright-Glo™ Buffer: Room temperature 20°C ± 5°C and Reconstituted reagent: - 80°C ± 10°C
- Cell washing solution: Dulbecco’s PBS Ca2+ and Mg2+ free complemented with 0.05% EDTA - stored at 5°C ± 3°C.
- Staining solution: 5 mg/ml MTT in solution in PBS - extemporaneously prepared and used within the day
- Desorption solution: 10% SDS in water - stored at room temperature 20°C ± 5°C.

FL REAC 01 and FL REAC 06 forms ensure the traceability of media and reagents used in the study.
The expiry after opening of the media and reagents used in the study is defined in the form FL REAC 05.

EXPERIMENTAL DESIGN
The study was composed of two independent repetitions. For each repetition the test item and the reference items were replicated on three independent plates for the measurement of induction and two plates for the measurement of cytotoxicity. Each repetition was performed on a different day with fresh stock solution.

- Cell seeding (first day): The cells culture, 80 - 90% confluent, were trypsinized according to the current working instruction IL 09. After removal of the culture medium from the culture flask, the cell layer was rinsed with PBS 0.05% EDTA, Ca2 + and Mg² + free to remove all traces of serum. The solution of trypsin-EDTA was added and left for a few minutes at 37°C, 5% CO2 until the detachment of cells. The action of trypsin was stopped by addition of maintenance medium.
Cell concentration was determined using a Malassez cell. Cell suspension was adjusted to a density of 8.10^4 cells/ml in seeding medium.
125 µl of the cell suspension at 8.10^4 cells/ml (i.e. 10^4 cells per well) were distributed in three white cell culture plates (96 wells) for the induction measurement and two transparent cell culture plates (96 wells) to assess the cytotoxicity. In order to ensure a good homogeneity of seeding, cells suspension was regularly mixed all along the seeding. The seeded plates were incubated 24 hours ± 1 hour at 37°C, 5% CO2.
The H12 wells were left without cells and will enable the measurement of blanks.

- Contact between the cells and the test and reference items (second day): The test item and positive control dilutions were prepared. In the 5 seeded plates, the medium was aspirated and replaced with 150 µL of treatment medium. Then the 4-fold plate was replicated 5 times: 50 µL from the 4-fold plate was placed in each of the three white plates and in the two transparent plates. The plates (1-fold) were incubated for 48 hours ± 1 hour (37°C, 5% CO2). To limit the cross-contamination, an empty line was left between each tested element.

- Luciferase activity (day 4): After 48 hours, the medium was aspirated and each well was gently washed once with 200 µL of PBS. Then 100 µL of luciferase substrate (constituted by a mix of luciferine + ATP + lysing agent) were added in each well. The formation of foam was avoided by careful pipetting. The plates were incubated at least 15 minutes at room temperature to ensure cells lysis. The plate was placed in the luminometer then the luciferase activity was measured with an integration time of 2 seconds.

- Cell viability assessment with MTT method (day 4): After 48 hours, the medium was aspirated. Each well was gently washed once with 200 µl of PBS. Then, 225 µl of staining solution diluted at 0.6 mg/ml in treatment medium (from the 5 mg/ml staining solution) were distributed in each well. The plates were incubated for 4 hours ± 30 minutes (37°C, 5% CO2). The staining solution was then removed and the cells were treated with 200 µl of 10% SDS one night in the dark (37°C, 5% CO2). After a 10 minutes homogenization, the absorbances were measured at 540 nm.

DATA ANALYSIS
Two parameters are measured in the KeratinoSens™ test method, the luciferase induction and the cytotoxicity:

- Luciferase induction
Imax, maximal fold induction of luciferase activity value observed at any concentration of the test item and positive control. The induction value I is calculated according to the following formula:

I=[Luminescence(Test item) – Luminescence(Blank)]/[Luminescence(Negative control) – Luminescence(Blank)]

The Imax of a test item is the average of the Imax calculated for each of the repetitions.

EC1.5, value representing the concentration for which induction of luciferase activity is above 1.5 threshold, is obtained according to the following equation:

EC1.5= (Cb - Ca) × [(1.5 - Ia)/(Ib - Ia)] + Ca

Where:
Ca = the lowest concentration with more than 1.5 fold the induction
Cb = the highest concentration with less than 1.5 fold the induction
Ia = induction factor for the lowest concentration with more than 1.5 fold the induction.
Ib = induction factor for the highest concentration with less than 1.5 fold the induction

The EC1.5 of a test item is the geometric average of the EC1.5 calculated for each of the repetitions.

- Cytotoxicity
The viability V is calculated according to the following formula:
V (%)= [Absorbance(Test item) – Absorbance(Blank)/(Absorbance(Negative control) – Absorbance(Blank)] x 100

IC30 and IC50, concentration in µM or µg/ml for which we obtained respectively 30% or 50% viability reduction:

ICx = (Cb - Ca) x [(100-x) - Va) / (Vb - Va)] + Ca

where
x is the % viability at the concentration to be calculated (70 for IC70
Ca is the lowest concentration for which the % viability is lower than X%
Cb is the highest concentration for which the % viability is higher than X%
Va is the % viability at the lowest concentration for which the % viability is lower than X%
Vb is the % viability at the highest concentration for which the % viability is higher than X%

For each concentration showing a luciferase activity induction equal or higher than 1.5 fold, statistical significance is determined (using a two-tailed Student’s t-test) by comparing the luminescence values of the three replicate test item with the luminescence values in the solvent control wells to assess whether the luciferase activity is statistically significant (p<0.05). In addition, at least two consecutive concentrations should have more than 70% viability, otherwise the concentration range should be adjusted.

If the dose-response curve obtained is biphasic (crossing the threshold of 1.5 twice), it is recommended to verify whether this is specific to the test item or due to an experimental artefact. In case the biphasic response is reproducible in an independent repetition, the lower concentration, i.e. when the threshold of 1.5 is crossed the first time, should be recorded.

In case where a statistically non-significant luciferase induction equal or above 1.5 fold is observed, followed by a higher concentration with a statistically significant induction, results from this repetition are only considered as valid and positive if the statistically significant induction equal or above the threshold of 1.5 was obtained for a non-cytotoxic concentration.

If the test item induces a 1.5 fold or higher induction already at the lowest concentration tested (i.e. 0.98 µM or 0.20 µg/ml), the EC1.5 is set to < 0.98 µM or 0.20 µg/ml.

ACCEPTABILITY CRITERIA
To validate the test, it is essential to check the validity criteria for the test:

Positive Control:
- the gene induction (luciferase induction) must be statistically significant above the threshold of 1.5 in at least one of the tested concentration,
- the EC1.5 value should be between IDEA Lab historical data (3.0 µM ≤ EC1.5 ≤ 20 µM) and the average induction, in each repetition, for cinnamaldehyde at 64 µM should be between 2 and 8. If the latter criterion is not fulfilled, the dose-response of cinnamaldehyde should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control. The OECD validation dataset is between 7 µM and 30 µM.

Negative Control
- The average coefficient of variation of the luminescence reading for the solvent controls (3 x 6 wells) should be below 20% in each repetition.
If for one repetition the validity criteria are not met, or in case of equivocal result additional repetitions should be considered.
The validation of the results is carried out by the Study Director in accordance with the current working instruction IL 04.

DATA INTERPRETATION
The test item is identified as potential skin sensitizer if the 4 following conditions are met in 2 of 2 or in 2 of 3 repetitions. Otherwise the Keratinosens™ prediction is considered as negative:

- the Imax is equal to or higher than 1.5 times and statistically significantly different as compared to the negative control (as determined by a two-tailed, unpaired Student’s t-test on the raw RLU values),
- the EC1.5 value is strictly below 200 µg/ml as the test item has no defined molecular weight,
- at the lowest concentration with a gene induction equal to or higher than 1.5, the cell viability must be strictly higher than 70%,
- there is an apparent overall dose-response for luciferase induction, which is similar between the repetitions.

If, in a given repetition, all of the three first conditions are met but a clear dose-response for the luciferase induction cannot be observed, then the result of that repetition should be considered inconclusive and further testing may be required. In addition, a negative result obtained at a maximal test concentration < 200 µg/ml and which do not reach cytotoxicity (< 70% viability) at the maximal tested concentration should also be considered as inconclusive.

If the test item induces the gene activity at a concentration very close to the cytotoxic levels, it can be positive in some repetitions at non-cytotoxic levels, and in other repetitions only at cytotoxic levels. The test item must be tested again with a narrower range using a dilution factor of 4/3 instead of 2.

Test items that only induce the gene activity at cytotoxic levels are not rated as positive, as it is the case for some non-sensitizing skin irritants.

Moreover, when testing multi-constituent substances or mixtures, consideration should be given to possible interference of cytotoxic constituents with the observed responses. The presence of a high content of non-sensitizing cytotoxic constituents may mask the response of weakly sensitizing components or sensitizing components present at low concentration. It might be justified to test either single main constituents forming the major fraction or several fractions of the mixture to conclude on the sensitization potential.

Vehicle / solvent control:

DMSO

Negative control:

not applicable

Positive control:

cinnamic aldehyde [442D]

Positive control results:

Results for Cinnamaldehyde

EXPERIMENT 1
- Induction at 64 µM: 2.34
- EC1.5: 19.01 μM

EXPERIMENT 2
- Induction at 64 µM: 2.48
- EC1.5: 21.33 μM

Results for control solvent (DMSO)
- CV(%) of experiment 1: 11.4 %
- CV(%) of experiment 2: 13.4 %

For both repetitions:
- the gene induction for cinnamaldehyde is statistically significant above the threshold of 1.5,
- the EC1.5 value of the repetition 1 is within the range of the IDEA Lab historical data. The EC1.5 value of the repetition 2 is very slightly above the historical data range of the laboratory (3-20µM) and within the OECD dataset validation range (7-30µM). This result is considered acceptable and the criterion is considered to be met.
- the coefficient of variation of the solvent control is less than 20%.

All validity criteria are fulfilled, which allows to consider the study valid.

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Value:

3.63

Cell viability:

Greater than 70%

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC 1.5 [442D]

Value:

0.48 µg/mL

Cell viability:

Greater than 70%

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

Imax [442D]

Value:

4.18

Cell viability:

Greater than 70%

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC 1.5 [442D]

Value:

0.3 µg/mL

Cell viability:

Greater than 70%

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

In the repetition 1, the maximal fold induction of luciferase activity, Imax, is greater than 1.5, with a value of 3.63. The EC1.5 is less than 200 µg/mL, with a value of 0.48 µg/mL and the EC1.5 viability is greater than 70%. The repetition 1 is considered positive.

In the repetition 2, Imax is greater than 1.5, with a value of 4.18. The EC1.5 is less than 200 µg/mL with a value of 0.30 µg/mL, and the EC1.5 viability is greater than 70%. The repetition 2 is considered positive.

Repetitions 1 and 2 showed reproducible results with a consistent conclusion for both experiments.

In both repetitions:
- the Imax is higher than 1.5 times and statistically significantly different as compared to the negative control,
- the EC1.5 value is below 200 µg/ml,
- at the lowest concentration with a gene induction higher than 1.5, the cell viability is higher than 70%,
- As we can see in the graphs attached in the section 'Overall remarks, attachments', there is an apparent overall dose-response for luciferase induction.

All conditions are met to conclude that both repetitions are positive.

Table 1. Results of reference item

Cinnamaldehyde	4 µM	8 µM	16 µM	32 µM	64 µM	EC1.5	Imax
Rep 1	1.04	1.36	1.47	1.61	2.34	19.01	2.34
Rep 2	1.02	1.28	1.40	1.71	2.48	21.33	2.48
Mean	1.03	1.32	1.43	1.66	2.41	20.14*	2.41

Control solvent	CV % control solvent
Rep 1	11.4
Rep 2	13.4

 

Table 2. Results of test item

 

	VIABILITY		INDUCTION
	IC50 µg/mL	IC30 µg/mL	Imax	Linear EC1.5 µg/mL	EC1.5 Lin/Log µg/mL
Rep 1	6.15	5.28	3.63	0.48	0.46
Rep 2	4.62	3.90	4.18	0.30	0.28
Mean	-	-	3.90	-	-
Geometric mean	5.33	4.54	-	0.38	0.36

 

Table 3. Acceptance Criteria for positive and negative controls

Criterion	Range	Experiment 1	pass/fail	Experiment 2	pass/fail
CV Solvent Control PC (1% DMSO)	< 20%	11.4	pass	13.4	pass
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	2	pass	2	pass
EC1.5 positive control with IDEA Lab historical data range	2 < EC1.5 < 20 µM	19.01 µM	pass	21.33 µM	not pass
EC1.5 positive control with OECD dataset validation range	7 < EC1.5 < 30 µM	19.01 µM	pass	21.33 µM	pass
Induction PC at 64 µM	2.00 < x < 8.00	2.34	pass	2.48	pass

In repetition 2, the EC1.5 of positive control reference (21.33 µM) is very slightly above the historical data range of the laboratory (3-20µM) and within the OECD dataset validation range (7-30µM). This result is considered acceptable and allows the study to be validated without impacting on the results.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Under the retained experimental conditions, BALMYWOOD - REF : ZA3155 code ID-22/01120 may be classified as skin sensitizer.

Executive summary:

The sensitizing potential of BALMYWOOD – REF: ZA3155 has been evaluated by an in vitro sensitization assay according to the OECD Guideline 442D: KeratinoSens ™. The KeratinoSens™ is a test method which quantifies luciferase gene induction as a measure of the activation of the Keap1-Nrf2-antioxidant/electrophile response element (ARE)-dependant pathway in an immortalized adherent cell line derived from HaCaT human keratinocytes transfected with a selectable plasmid.

The test item was tested at 12 concentrations according to a geometric progression of ratio 2 from 0.2 µg/mL to 400 µg/mL, to cover a large concentration range. After 48 hours (± 1 hour) of contact at 37°C, 5% CO2, with the test item, the cells KeratinoSens™ were lysed and the induction of luciferase was quantified. In parallel, the cytotoxicity was measured, in order to exclude a false positive generated by a skin irritation. The cinnamaldehyde was used as the positive control. The study was composed of two independent repetitions. For each repetition the test item and the reference items were replicated on three independent white plates for the measurement of induction and two transparent plates for the measurement of cytotoxicity. Each repetition was performed independently of one another.

 

In the repetition 1, the maximal fold induction of luciferase activity, Imax, is greater than 1.5, with a value of 3.63. The EC1.5 is less than 200 µg/mL, with a value of 0.48 µg/mL and the EC1.5 viability is greater than 70%. The repetition 1 is considered positive.

In the repetition 2, Imax is greater than 1.5, with a value of 4.18. The EC1.5 is less than 200 µg/mL with a value of 0.30 µg/mL, and the EC1.5 viability is greater than 70%. The repetition 2 is considered positive.

Repetitions 1 and 2 showed reproducible results with a consistent conclusion for both experiments.

In both repetitions:
- the Imax is higher than 1.5 times and statistically significantly different as compared to the negative control,
- the EC1.5 value is below 200 µg/ml,
- at the lowest concentration with a gene induction higher than 1.5, the cell viability is higher than 70%,
- There is an apparent overall dose-response for luciferase induction in the graph describing dose-response curves for induction of luciferase activity and viability;

All conditions are met to conclude that both repetitions are positive.

 

Based of on results of the study, BALMYWOOD – REF : ZA3155 may be classified as potential skin sensitizer with the in vitro sensitization test: KeratinoSens™.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

• A key study was identified (IDEA Lab, 2022).

In this study, the sensitizing potential of BALMYWOOD – REF: ZA3155 has been evaluated by an in vitro sensitization assay according to the OECD Guideline 442D: KeratinoSens ™. The KeratinoSens™ is a test method which quantifies luciferase gene induction as a measure of the activation of the Keap1-Nrf2-antioxidant/electrophile response element (ARE)-dependant pathway in an immortalized adherent cell line derived from HaCaT human keratinocytes transfected with a selectable plasmid.The test item was tested at 12 concentrations according to a geometric progression of ratio 2 from 0.2 µg/mL to 400 µg/mL, to cover a large concentration range. After 48 hours (± 1 hour) of contact at 37°C, 5% CO2, with the test item, the cells KeratinoSens™ were lysed and the induction of luciferase was quantified. In parallel, the cytotoxicity was measured, in order to exclude a false positive generated by a skin irritation. The cinnamaldehyde was used as the positive control. The study was composed of two independent repetitions. For each repetition the test item and the reference items were replicated on three independent white plates for the measurement of induction and two transparent plates for the measurement of cytotoxicity. Each repetition was performed independently of one another.

In the repetition 1, the maximal fold induction of luciferase activity, Imax, is greater than 1.5, with a value of 3.63. The EC1.5 is less than 200 µg/mL, with a value of 0.48 µg/mL and the EC1.5 viability is greater than 70%. The repetition 1 is considered positive.

In the repetition 2, Imax is greater than 1.5, with a value of 4.18. The EC1.5 is less than 200 µg/mL with a value of 0.30 µg/mL, and the EC1.5 viability is greater than 70%. The repetition 2 is considered positive.

Repetitions 1 and 2 showed reproducible results with a consistent conclusion for both experiments.

In both repetitions:
- the Imax is higher than 1.5 times and statistically significantly different as compared to the negative control,
- the EC1.5 value is below 200 µg/ml,
- at the lowest concentration with a gene induction higher than 1.5, the cell viability is higher than 70%,
- There is an apparent overall dose-response for luciferase induction in the graph describing dose-response curves for induction of luciferase activity and viability;

All conditions are met to conclude that both repetitions are positive.

Based of on results of the study, BALMYWOOD – REF : ZA3155 may be classified as potential skin sensitizer with the in vitro sensitization test: KeratinoSens™.

 

• A weight-of-evidence approach was used to sub-categorise the registered substance as skin sensitiser category 1B based on the following read-across data: 
  
  
  
  1. OECD 429 study (GLP, WoE, rel.1) on read-across substance (Labdanum gum).
  
  
  2. OECD 429 study (GLP, WoE, rel.1) on read-across substance (Cedarwood Texas cedrol oil).
  
  
  
  

 

Description of the first weight-of-evidence data (Harlan, 2015):

A study was performed to assess the skin sensitisation potential of test material in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was conducted according to the OECD test guideline No 429 and in compliance with GLP.

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 10% w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of five animals, were treated with 50 µL (25 µL per ear) of the test item as a solution in dimethyl formamide at concentrations of 10%, 5% or 2.5% w/w. A further group of five animals was treated with dimethyl formamide alone.

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (%w/w) in dimethyl formamide	Stimulation Index	Result
2.5	1.22	Negative
5	1.35	Negative
10	1.76	Negative

The historical positive control, α-Hexylcinnamaldehyde, gave a SI of 5.34, when tested at 15 % v/v. The test system was therefore considered to be valid.

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.Body weight change of the test animals between Day 1 and Day 6 were comparable to that observed in the corresponding control group animals over the same period.

Under the test conditions, the source and the target substances are not classified as a skin sensitiser according to the annex VI of the Regulation EC No. 1272/2008 (CLP) .

 

Description of the second weight-of-evidence data (CRL, 2017):

The skin sensitisation potential of Cedrol, Cedarwood Texas oil distilled was tested according to OECD, Section 4, Health Effects, No.429 (2010).

Three experimental groups of five female CBA/J mice were treated with test item concentrations of 5, 10 or 25% w/w on three consecutive days, by open application on the ears. Test item concentrations selected for the main study were based on the results of a pre-screen test. Five vehicle control animals were similarly treated, but with the vehicle alone (AcOO).  

No erythema was noted for any of the animals, Scaliness was noted on the ears of four animals treated at 25% between days 3 and 6. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals. Body weights and body weight gain of experimental animals remained in the same range as controls over the main study period. Mean DPM/animal values for the experimental groups treated with test item concentrations 5, 10 and 25% were 561, 647 and 1524 DPM, respectively. The mean DPM/animal value for the vehicle control group was 285 DPM. The SI values calculated for the test item concentrations 5, 10 and 25% were 2.0, 2.3 and 5.4, respectively.

The data showed a dose-response and an EC3 value (the estimated test item concentration that will give a SI =3) of 13.4% was calculated.

Based on the results of this LLNA according with OECD Guideline 429, the calculated EC3 value of the source and the target substances is 13.4%. Therefore, Cedrol, Cedarwood Texas oil distilled and the target substance need to be classified as category 1B skin sensitiser according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Harmonised classification:

The substance has no harmonised classification according to the Regulation (EC) No. 1272/2008 (CLP).

Self classification:

Based on an in vitro skin sensitisation study (OECD 442D) and on read-across data on source substances, the registered substance is classified as skin sensitiser: Skin sensitiser Category 1B (H317: May cause an allergic skin reaction) according to the criteria of the Regulation (EC) No. 1272/2008 (CLP).

No data was available for respiratory sensitisation.