Tall oil, reaction products with ethanolamine

Endpoint summary

Description of key information

The skin sensitisation potential of the test item was assessed in accordance with OECD Guideline 429.  The test item was considered to be a non-sensitiser under the conditions of the test. Alpha-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 when tested at a concentration of 25%  v/v in acetone/olive oil, 4:1 thus demonstrating  the sensitivity and reliability of the test system.

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:

16 December 2014 to 03 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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Female CBA/Ca (CBA/CaOlaHsd) strain mice were nulliparous and non-pregnant.
Acclimatization period of at least five days.
Animals were in the weight range of 15 to 23 g, and were eight to twelve weeks old.
Free access to mains tap water and food.
The temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70%, respectively.
The rate of air exchange was approximately fifteen changes per hour.
The lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.

Vehicle:

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

Concentration:

50, 5.0 and 0.5% w/w

No. of animals per dose:

5

Details on study design:

Groups of five mice were treated with the test item at concentrations of 50, 5 or 0.5% w/w in the vehicle. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3).
The test item formulation was administered using an automatic micropipette and spread over the dorsal surface ofthe ear using the tip of the pipette.
A further group of five mice received the vehicle alone in the same manner.
The positive control animals were similarly treated to the test animals except that 25 µL of the positive control item, alpha-Hexylcinnamaldehyde, tech., 85%, at a concentration of 25% v/v in acetone/olive oil 4:1 was applied to the dorsal surface of each ear.
The thickness of each ear was measured and recorded as described in the preliminary screening test.

Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation.
Preparation of Single Cell Suspension: A single cell suspension of the lymph node cells for each individual animal was prepared by gentle mechanical disaggregation. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labeled with the study number and dose concentration. The lymph node cells suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifiige tube. The lymph node cells were pelleted at 1400 rpm (approximately 190 g) for ten minutes. The pellet was re-suspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was re-suspended in 3 mL of 5% Trichloroacetic acid (TCA).
3HTdR incorporation was measured by B-scintillation counting. The number of radioactive disintegrations per minute was then measured.

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 meandisintegrations 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 ariances, then parametric one way analysis of variance (ANOVA) and Durmett’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.

Positive control results:

25% v/v of the positive control in acetone/olive oil, 4:1 gives positive results with a stimulation Index score of 5.17

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

0.5% w/w of the test item in the vehicle gave negative results with a stimulation Index score of 1.08 5.0% w/w of the test item in the vehicle gave negative results with a stimulation Index score of 0.80 50% w/w of the test item in the vehicle gave negative results with a stimulation Index score of 1.40

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

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

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. No observations of erythema were noted on the ears of all animals treated with the test item or positive control item.

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

Interpretation of results:

GHS criteria not met

Conclusions:

The skin sensitisation potential of the test item was assessed in accordance with OECD Guideline 429. The test item was considered to be a non-sensitiser under the conditions of the test. Alpha-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 when tested at a concentration of 25% v/v in acetone/olive oil, 4:1 thus demonstrating the sensitivity and reliability of the test system.

Executive summary:

Introduction

A study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

Methods

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 50 % w/w in acetone/olive oil, 4:1, 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 butanone at concentrations of 50 % or 5 % or 0.5 % w/w. A further group of five animals was treated with butanone alone. A concurrent positive control test, using a group of five animals, was also performed with the known sensitizer, alpha-Hexylcinnamaldehyde tech., 85 %, at a concentration of 25 % v/v in acetone/olive oil, 4:1..

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:

0.5 % w/w of the test item in the vehicle gave negative results with a stimulation Index score of 1.08

5.0 % w/w of the test item in the vehicle gave negative results with a stimulation Index score of 0.80

50 % v/v of the test item in the vehicle gave negative results with a stimulation Index score of 1.4

25 % v/v of the positive control in the vehicle gave positive results with a stimulation Index score of 5.17

Conclusion

The test item was considered to be a non-sensitiser under the conditions of the test. Alpha-Hexylcinnamaldehyde, tech., 85 % gave a Stimulation Index of greater than 3 when tested at a concentration of 25 % v/v in acetone/olive oil, 4:1 thus demonstrating the sensitivity and reliability of the test system.

Reference 2

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Remarks:

Guinea Pigs-Modified Buehler Design

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 2013 - July 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

The design of this study was based on the study objective(s), the overall product development strategy for the test substance, and the following study design guidelines: OECD Guideline 406 and EPA Health Effects Test Guideline OPPTS 870.2600.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

Buehler test

Justification for non-LLNA method:

The Buehler study assesses the entire sensitisation pathway (induction and elicitation) and is most frequently used for regulatory purposes worldwide.

Species:

guinea pig

Strain:

other: Hartley-derived albino guinea pigs

Sex:

male/female

Details on test animals and environmental conditions:

Animal Identification
Each animal was identified by a cage card and plastic ear tag.

Environmental Acclimation
The animals were acclimated to their designated housing for at least 7 days before the first day of dosing.

Selection, Assignment, and Disposition of Animals
- The animals chosen for study were arbitrarily selected from healthy animals. All animals received a detailed pretest observation prior to dosing. Only healthy animals were chosen for study use.
- The male range-finding animals were approximately 5 weeks of age on the day prior to dosing with body weights of 319 grams and 329 grams. The female range-finding animals were approximately 6 weeks of age on the day prior to dosing with body weights of 323 grams and 342 grams.
- The male main phase animals were approximately 6 weeks of age on the day prior to Induction 1 dosing with body weights ranging from 345 grams to 443 grams. The female main phase animals were approximately 6 weeks of age on the day prior to Induction 1 dosing with body weights ranging from 348 grams to 433 grams.
- The disposition of all animals was documented in the Study Records.

Husbandry

Housing
The animals were pair housed throughout the study in polycarbonate cages containing direct bedding material equipped with an automatic watering valve. Housing and care were as specified in the USDA Animal Welfare Act (9 CFR, Parts 1, 2, and 3) and as described in the Guide for the Care and Use of Laboratory Animals from the National Research Council.

Environmental Conditions
Temperatures of 71°F to 72°F with a relative humidity of 53% to 68% were maintained. A 12-hour light/12-hour dark cycle was maintained, except when interrupted for designated procedures. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Food
PMI Nutrition International Certified Guinea Pig Chow No. 5026 was provided ad libitum throughout the study, except during designated procedures. The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the dietary analyses were provided by the supplier for each lot of diet and are on file at the Testing Facility. It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study.

Water
Municipal tap water after treatment by reverse osmosis and ultraviolet irradiation was freely available to each animal via an automatic watering system, except during designated procedures. Water bottles and/or hydrogel supplemental water were provided when required. The water is analyzed semi-annually for microbial contamination and for total dissolved solids, hardness, and various environmental contaminants. Results of these analyses are maintained on file at the Testing Facility. It is considered that there are no known contaminants in the water that could interfere with the outcome of the study.

Animal Enrichment
Beginning at receipt, guinea pigs were pair housed in solid bottom cages containing direct bedding material. In addition, a timothy hay cube was provided to each animal at least weekly.

Veterinary Care
Veterinary care was available throughout the course of the study; however, no examinations or treatments were required.

Route:

epicutaneous, occlusive

Vehicle:

other: Mineral oil

Concentration / amount:

On the day prior to the first induction dose administration (Day -1), all main phase animals were weighed and the hair was removed from the left side of the test and HCA test animals. On the day following clipping (Day 0), chambers were applied @ 100% (neat test substance)

Day(s)/duration:

The induction procedure was repeated on Day 7 and Day 14 so that a total of 3 consecutive induction exposures were made to the test animals.

Adequacy of induction:

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

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: Mineral oil

Remarks:

Challenge

Concentration / amount:

On the day prior to challenge dose administration, the test, HCA test, challenge control, and HCA challenge control animals were weighed and the hair was removed from the right side of the animals. On the day following clipping (Day 28), chambers were applied at 100%

Day(s)/duration:

day 28

Adequacy of challenge:

highest non-irritant concentration

No.:

#2

Route:

epicutaneous, occlusive

Vehicle:

other: Mineral oil

Remarks:

Rechallenge

Concentration / amount:

On the day prior to rechallenge dose administration, the test and rechallenge control animals were weighed and the hair was removed from the right side of the animals. On the day following clipping (Day 35), chambers were applied at 75 % in mineral oil

Day(s)/duration:

Day 35

Adequacy of challenge:

other: Based on the response noted in the challenge control animals, a rechallenge was conducted to attempt to elicit additional responses.

No. of animals per dose:

Range finder
2 males 2 females per dose

Main study 10 males 10 femails
The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test substance. This study was designed such that it did not require an unnecessary number of animals to accomplish its objectives.
At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.

Details on study design:

Test Substance Characterization
The Sponsor provided to the Testing Facility documentation of the identity, strength, purity, composition, and stability for the test substance.

Analysis of Test Substance
The stability of the bulk test substance was not determined during the course of this study. Information to support the stability of each lot of the bulk test substance was provided by the Sponsor.

Reserve Samples
A reserve sample was collected for each batch (lot) of test substance, control substance (1 mL), and positive control substance (1 g) and maintained under the appropriate storage conditions by the Testing Facility.

Test Substance Inventory and Disposition
Records of the receipt, distribution, storage, and disposition of the test substance (including empty containers) were maintained. With the exception of reserve samples, all unused Sponsor-supplied bulk test substance will be returned to the Sponsor (after issuance of the final reports of all studies using this material). All empty containers were maintained for the duration of the study.

Dose Formulation and Analysis
Preparation of Test Substance
The test substance, was administered as received and/or diluted with the control substance on the day of dosing during the range-finding phases, induction, challenge, and rechallenge. Selected doses were achieved by adjustment of test substance concentration in the control substance. The solubility of the test substance in the control substance was determined prior to preparation of the test substance dosing formulations..

HCA Preparation
HCA dosing formulations were prepared at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared, protected from light, and dispensed on the day of dosing. Details of the preparation and dispensing of the positive control substance have been retained in the Study Records.

Sample Collection and Analysis
No samples for analytical analysis were collected by the Testing Facility.

Test System

Receipt
On 02 Jul 2013, 44 male and 44 female Hartley-derived albino guinea pigs were received from Charles River Laboratories, Raleigh, NC. The animals were examined and weighed on the day following receipt.

Justification for Test System and Number of Animals
The Hartley-derived guinea pig was chosen as the animal model for this study as it is an accepted rodent species for preclinical toxicity testing by regulatory agencies.


The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test substance. This study was designed such that it did not require an unnecessary number of animals to accomplish its objectives.
At this time, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.

Experimental Design – Range-Finding Phases

Text Table 1
Experimental Design for the First Range-Finding Phase


Site No. Test Material Dose Level Number of Animals
Males Females
1 Test Substance 100%
2 Test Substance 75%* 2 2
3 Test Substance 50%*
4 Test Substance 25%*
* The vehicle used was mineral oil.

Justification of Route and Dose Levels
The dermal route of exposure was selected because this is the intended potential route of human exposure.The dose concentration for the main phase was based upon the results of the range-finding portion of the study.

Administration of Test Materials
On the day prior to dosing, the guinea pigs selected for the topical range-finding studies were weighed and the hair removed from the right and left side of the animals with a small animal clipper. Care was taken to avoid abrading the skin during the clipping procedures.
On the following day, a 0.3 mL dose of the appropriate test or positive control substance was placed on a 25 mm Hilltop Chamber® backed by adhesive tape (occlusive patch). The chambers were then applied to the clipped surface of the appropriate animals as quickly as possible.
Following chamber application, the trunk of the animal was wrapped with elastic wrap which was secured with adhesive tape to prevent removal of the chamber and the animal was returned to its cage.
Approximately 6 hours after chamber application, the binding materials were removed. The test sites were then wiped 2 times with gauze moistened in mineral oil, followed by dry gauze, followed by gauze moistened in deionized water, followed by dry gauze, to remove test substance residue, and the animals were returned to their cages.

In-life Procedures, Observations, and Measurements – Range-Finding Phases
The in-life procedures, observations, and measurements listed below were performed for all range-finding animals.

Mortality/Moribundity Checks
The animals were observed for general health/mortality and moribundity twice daily, once in the morning and afternoon, throughout the study.

Dermal Observations
The test sites of each topical range-finding animal were graded for irritation at approximately 24 hours and 48 hours after chamber application using the Macroscopic Dermal Grading System in see attached Appendix 3 according to Buehler.2

Body Weights
Each topical range-finding animal was weighed on the day prior to dosing (Day -1).

Scheduled Euthanasia
Following the 48-hour scoring interval, all range-finding animals were euthanized by carbon dioxide inhalation and discarded.

Experimental Design – Main Phase

Table 2 Experimental Design for the Main Phase Phase/Treatment Number of Animals
Group Induction 1 to 3 Challenge Rechallenge a Males Females
Test Test Substance (100%) Test Substance (100%) Test Substance (75%)b 10 10
Challenge Control - Test Substance (100%)b - 5 5
Rechallenge Control - - Test Substance (75%)b 5 5
HCA Test 5.0% HCAc 2.5% and 1.0% HCA d - 5 5
HCA Control - 2.5% and 1.0% HCA d - 5 5

- not applicable.
b The vehicle used was mineral oil.
c The vehicle used was ethanol.
d The vehicle used was acetone.

Justification of Route and Dose Levels
The dermal route of exposure was selected because this is a potential route of human exposure.
The dose concentration for the main induction phase was based upon the results of the range-finding portion of the study. The test substance concentration used for challenge was expected to produce no systemic toxicity and dermal responses generally consisting of Grades 0 to ±. A rechallenge was performed to assess a lower formulation concentration. The test substance concentration used for rechallenge was expected to produce no systemic toxicity and dermal responses consisting of Grades 0 to ±.


Administration of Test Materials
On the day prior to dosing, the guinea pigs selected for the main study had the hair removed with a small animal clipper. Care was taken to avoid abrading the skin during the clipping procedures.
On the following day, a 0.3 mL dose of the appropriate test or positive control substance was placed on a 25 mm Hilltop Chamber® backed by adhesive tape (occlusive patch). The chamber was then applied to the clipped surface of the appropriate animals as quickly as possible.
Following chamber application, the trunk of the animal was wrapped with elastic wrap to prevent removal of the chamber and the animal was returned to its cage.
Approximately 6 hours after chamber application, the binding materials were removed. The test sites were then wiped 2 times with gauze moistened in mineral oil, followed by dry gauze, followed by gauze moistened in RODI water, followed by dry gauze, to remove test substance residue, and the animals were returned to their cages.


Induction
On the day prior to the first induction dose administration (Day -1), all main phase animals were weighed and the hair was removed from the left side of the test and HCA test animals. On the day following clipping (Day 0), chambers were applied as indicated in Text Table 4.

Text Table 3 Induction Dosing

Group Test Substance Induction No. Dose Volume (mL) Concentration (%) Site No. of Animals
Males Females

Test Test Substance 1 0.3 100a 1 10 10
2 0.3 100a 1
3 0.3 100a 1

HCA Test HCA 1 0.3 5.0b 1 5 5
2 0.3 5.0b 1
3 0.3 5.0b 3c
a As received.
b The vehicle used was ethanol.
c Based on irritation, the test site was changed for Induction 3.
The induction procedure was repeated on Day 7 and Day 14 so that a total of 3 consecutive induction exposures were made to the test animals.


Challenge
On the day prior to challenge dose administration, the test, HCA test, challenge control, and HCA challenge control animals were weighed and the hair was removed from the right side of the animals. On the day following clipping (Day 28), chambers were applied as indicated in Text Table 4.

Text Table 4 Challenge Dosing
Group Test Substance Dose Volume (mL) Concentration (%) Site No. of Animals
Males Females
Test Test Substance 0.3 100 2 10 10
Challenge Control Test Substance 0.3 100 2 5 5
HCA Test HCA 0.3 2.5 b 2 5 5
0.3 1.0 b 4
HCA Control HCA 0.3 2.5 b 2 5 5
0.3 1.0 b 4
b The vehicle used was acetone.

Rechallenge
On the day prior to rechallenge dose administration, the test and rechallenge control animals were weighed and the hair was removed from the right side of the animals. On the day following clipping (Day 35), chambers were applied as indicated in Text Table 5.

Table 5 Rechallenge Dosing


Group Test Substance Dose Volume (mL) Concentration (%) Site No. of Animals
Males Females
Test Test Substance 0.3 75 a 4 10 10
Rechallenge Control Test Substance 0.3 75 a 4 5 5
a The vehicle used was mineral oil.

In-life Procedures, Observations, and Measurements
The in-life procedures, observations, and measurements listed below were performed for main study animals.

Mortality/Moribundity Checks
The animals were observed for general health/mortality and moribundity twice daily, once in the morning and afternoon, throughout the study.

Dermal Observations
The test sites of each main study animal were graded for irritation at approximately 24 hours and 48 hours after chamber application (induction) or 24 hours and 48 hours after chamber removal (challenge and rechallenge) using the Macroscopic Dermal Grading System

Body Weights
Each main study animal was weighed on the day prior to the first induction (Day -1) and on the day prior to challenge and rechallenge dosing for the appropriate test, challenge control, and rechallenge control animals.

Scheduled Euthanasia
Following the rechallenge phase 48-hour scoring interval, all main study animals were euthanized by carbon dioxide inhalation and discarded.

COMPUTERIZED SYSTEMS
Critical computerized systems used in the study are listed below. All computerized systems used in the conduct of this study have been validated; when a particular system has not satisfied all requirements, appropriate administrative and procedural controls were implemented to assure the quality and integrity of data.

Text Table 6
Critical Computerized Systems

System Name Version No. Description of Data Collected and/or Analyzed
Systems 600 Apogee Insight System 3.11 Temperature and humidity (animal rooms, refrigerators, freezers, and compound storage, as applicable)
Instem Life Science Systems, DISPENSE 7.03 Test material receipt, accountability

STATISTICAL ANALYSIS
The sensitization potential of the test substance was based on the dermal responses observed on the test and control animals at challenge, rechallenge, and second rechallenge. Generally, dermal scores of ≥ 1 in the test animals with scores of 0 to ± noted in the controls were considered indicative of sensitization. Dermal scores of 1 in both the test and control animals were generally considered equivocal unless a higher dermal response (≥ grade 2) was noted in the test animals. Group mean dermal scores were calculated for challenge and rechallenge. A response of at least 15% in a nonadjuvant test was expected for a mild to moderate sensitizer.

Challenge controls:

Challenge
On the day prior to challenge dose administration, the test, HCA test, challenge control, and HCA challenge control animals were weighed and the hair was removed from the right side of the animals. On the day following clipping (Day 28), chambers were applied as indicated in the following table

Challenge Dosing
Group Test Substance Dose Volume (mL) Concentration (%) Site No. of Animals
Males Females
Test Test Substance 0.3 100 a 2 10 10
Challenge Control Test Substance 0.3 100 a 2 5 5
HCA Test HCA 0.3 2.5 b 2 5 5
HCA Test HCA 0.3 1.0 b 4
HCA Control HCA 0.3 2.5 b 2 5 5
HCA Control HCA 0.3 1.0 b 4

a As received.
b The vehicle used was acetone.

Rechallenge
On the day prior to rechallenge dose administration, the test and rechallenge control animals were weighed and the hair was removed from the right side of the animals. On the day following clipping (Day 35), chambers were applied as follows:

Group Test Substance Dose Volume (mL) Concentration (%) Site No. of Animals
Males Females
Test Test Substance 0.3 75 a 6 10 10
Rechallenge Control Test Substance 0.3 75 a 2 5 5
a The vehicle used was mineral oil.

Positive control substance(s):

yes

Remarks:

HCA Preparation HCA dosing formulations were prepared at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared, protected from light, and dispensed on the day of dosing.

Positive control results:

Challenge Phase
Following challenge with 2.5% w/v HCA in acetone, dermal scores of 1 were noted in 8/10 HCA test animals at the 24-hour scoring interval, and in 9/10 HCA test animals at the 48-hour scoring interval. Dermal reactions in the HCA control animals were limited to scores of 0 or ±. Group mean dermal scores were higher in the HCA test animals compared to the HCA control animals.
Following challenge with 1.0% w/v HCA in acetone, dermal scores of 1 were noted in 3/10 HCA test animals at the 24-hour scoring interval, and in 4/10 test animals at the 48-hour scoring interval. Dermal reactions in the HCA control animals were limited to scores of 0 or ±. Group mean dermal scores were higher in the HCA test animals compared to the HCA control animals.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100%

No. with + reactions:

4

Total no. in group:

20

Clinical observations:

Dermal scores of 1 were noted in 4/20 test animals at the 24-hour scoring interval

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100%

No. with + reactions:

3

Total no. in group:

20

Clinical observations:

At the 48-hour scoring interval, dermal scores of 1 were noted in 3/20 test animals.

Remarks on result:

no indication of skin sensitisation

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

75%

No. with + reactions:

1

Total no. in group:

20

Clinical observations:

dermal scores of 1 were noted in 1/20 test animals at the 24- hour scoring interval

Remarks on result:

no indication of skin sensitisation

Reading:

rechallenge

Hours after challenge:

48

Group:

test chemical

Dose level:

75%

No. with + reactions:

2

Total no. in group:

20

Clinical observations:

Dermal scores of 1 were noted in 2/20 test animals at the 48-hour scoring interval

RESULTS

Mortality

No mortality occurred during the study.

 

Range-Finding Phase

Exposure to the test material at concentrations of 25%, 50%, 75%, and 100% resulted in dermal scores of 0 or ±. Therefore, induction was determined to be acceptable at 100% (as received).

 

Main Phase

Induction Phase

During the induction, dermal scores of 0, ± (slight patchy erythema), and 1 (slight, confluent or moderate patchy erythema) were noted for the test animals. Additional observations included edema scores of 1, and dermal irritation outside the test site.

Challenge Phase

Following challenge with 100% (as received) the test material, dermal scores of 1 were noted in 4/20 test animals at the 24-hour scoring interval. At the 48-hour scoring interval, dermal scores of 1 were noted in 3/20 test animals. Dermal reactions in the remaining test and challenge control animals were limited to scores of 0 and ±. Group mean dermal scores were higher in the test animals as compared with the challenge control animals. Based on the response noted in the challenge control animals, a rechallenge was conducted to attempt to elicit additional responses.

Following challenge with 2.5% w/v HCA in acetone, dermal scores of 1 were noted in 8/10 HCA test animals at the 24-hour scoring interval, and in 9/10 HCA test animals at the 48-hour scoring interval. Dermal reactions in the HCA control animals were limited to scores of 0 or ±. Group mean dermal scores were higher in the HCA test animals compared to the HCA control animals.

Following challenge with 1.0% w/v HCA in acetone, dermal scores of 1 were noted in 3/10 HCA test animals at the 24-hour scoring interval, and in 4/10 test animals at the 48-hour scoring interval. Dermal reactions in the HCA control animals were limited to scores of 0 or ±. Group mean dermal scores were higher in the HCA test animals compared to the HCA control animals.

Rechallenge Phase

Following rechallenge with 75% w/w the test material in mineral oil, dermal scores of 1 were noted in 1/20 test animals at the 24-hour scoring interval and dermal scores of 1 were noted 2/20 test animals at and the 48-hour scoring interval. Dermal reactions in the remaining test and rechallenge control animals were limited to scores of 0 and ±. Group mean dermal scores were higher in the test animals as compared to the rechallenge control animals.

Body Weights

No the test material-related effects on body weight were observed in the test animals during the study. Weight gain in the animals throughout the study interval was indicative of good health in the test and control animals.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of this study, the test substance is not considered to be a contact sensitizer in guinea pigs, as the criterion for sensitization (dermal scores ≥ 1 in at least 15% of the test animals) was not met. The results of the HCA positive control study demonstrated that a valid test was performed and indicated that the test design would detect potential contact sensitizers.

Executive summary:

SUMMARY

The dermal sensitization potential of the test material was evaluated in Hartley-derived albino guinea pigs. Ten male and 10 female guinea pigs were topically treated with 100% (as received)

the test material once per week, for 3 consecutive weeks. Following a 2-week rest period, a  challenge was performed whereby the 20 test and 10 previously untreated (naïve) challenge control guinea pigs were topically treated with 100% the test material as received. Challenge responses in the test animals were slightly higher than those of the challenge control animals. A rechallenge was performed in which the 20 test and 10 previously untreated (naïve) rechallenge control guinea pigs were topically treated with 75% w/w the test material in mineral oil. Rechallenge responses in the test animals were slightly higher than those of the control animals.

 

An α-Hexylcinnamaldehyde (HCA) positive control group consisting of 10 HCA test and 10 HCA control guinea pigs was included in this study. The animals were treated as above with the HCA test animals receiving 5% w/v HCA in ethanol for induction and 2.5% and 1.0% w/v HCA in acetone for challenge.

 

Testing Groups

Following challenge with 100% (as received) the test material, dermal scores of 1 were noted in 4/20 test animals at the 24-hour scoring interval. At the 48-hour scoring interval, dermal scores of 1 were noted in 3/20 test animals. Dermal reactions in the remaining test and challenge control animals were limited to scores of 0 and ±. Group mean dermal scores were higher in the test animals as compared with the challenge control animals. Based on the response noted in the challenge control animals, a rechallenge was conducted in attempt to elicit additional responses.

 

Following rechallenge with 75% w/w the test material in mineral oil, dermal scores of 1 were noted in 1/20 test animals at the 24- hour scoring interval, and in 2/20 test animals at the 48-hour scoring interval. Dermal reactions in the remaining test and rechallenge control animals were limited to scores of 0 and ±. Group mean dermal scores were higher in the test animals as compared to the rechallenge control animals.

 

α-Hexylcinnamaldehyde (HCA)

Following challenge with 2.5% w/v HCA in acetone, dermal scores of 1 were noted in 8/10 HCA test animals at the 24-hour scoring interval, and in 9/10 HCA test animals at the 48-hour scoring interval. Dermal reactions in the HCA control animals were limited to scores of 0 or ±. Group mean dermal scores were higher in the HCA test animals compared to the HCA control animals.

 

Following challenge with 1.0% w/v HCA in acetone, dermal scores of 1 were noted in 3/10 HCA test animals at the 24-hour scoring interval, and in 4/10 test animals at the 48-hour scoring interval. Dermal reactions in the HCA control animals were limited to scores of 0 or ±. Group mean dermal scores were higher in the HCA test animals compared to the HCA control animals.

 

Conclusion

Based on the results of this study, the test material is not considered to be a contact sensitizer in guinea pigs, as the criterion for sensitization (dermal scores ≥ 1 in at least 15% of the test animals) was not met. The results of the HCA positive control study demonstrated that a valid test was performed and indicated that the test design would detect potential contact sensitizers.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Study 1: LLNA

Introduction

A study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

 

Methods

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 50 %, 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 acetone/olive oil 4:1 at concentrations of 50 % or 5 % or 0.5 % w/w. A further group of five animals was treated with acetone/olive oil 4:1 alone. A concurrent positive control test, using a group of five animals, was also performed with the known sensitizer, alpha-Hexylcinnamaldehyde tech., 85 %, at a concentration of 25 % v/v in acetone/olive oil 4: 1

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:

0.5 % w/w of the test item in acetone/olive oil 4:1 gives negative results with a stimulation Index score of 1.08

5 % w/w of the test item in acetone/olive oil 4:1 gives negative results with a stimulation Index score of 0.8

50 % w/w of the test item in acetone/olive oil 4:1 gives negative results with a stimulation index of 1.40

25 % v/v of the positive control in acetone/olive oil 4:1 gives positive results with a stimulation Index score of 5.17

 

Conclusion

The test item was considered to be a non-sensitiser under the conditions of the test. Alpha-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 when tested at a concentration of 25% v/v in acetone/olive oil 4:1 thus demonstrating the sensitivity and reliability of the test system.

 

Study 2: Buehler

The dermal sensitization potential of the test material was evaluated in Hartley-derived albino guinea pigs. Ten male and 10 female guinea pigs were topically treated with 100% (as received) the test material once per week, for 3 consecutive weeks. Following a 2-week rest period, a challenge was performed whereby the 20 test and 10 previously untreated (naïve) challenge control guinea pigs were topically treated with 100% the test material as received. Challenge responses in the test animals were slightly higher than those of the challenge control animals. A rechallenge was performed in which the 20 test and 10 previously untreated (naïve) rechallenge control guinea pigs were topically treated with 75% w/w the test material in mineral oil. Rechallenge responses in the test animals were slightly higher than those of the control animals. An α-Hexylcinnamaldehyde (HCA) positive control group consisting of 10 HCA test and 10 HCA control guinea pigs was included in this study. The animals were treated as above with the HCA test animals receiving 5% w/v HCA in ethanol for induction and 2.5% and 1.0% w/v HCA in acetone for challenge.

 

Testing Groups

Following challenge with 100% (as received) the test material, dermal scores of 1 were noted in 4/20 test animals at the 24-hour scoring interval. At the 48-hour scoring interval, dermal scores of 1 were noted in 3/20 test animals. Dermal reactions in the remaining test and challenge control animals were limited to scores of 0 and ±. Group mean dermal scores were higher in the test animals as compared with the challenge control animals. Based on the response noted in the challenge control animals, a rechallenge was conducted in attempt to elicit additional responses. Following rechallenge with 75% w/w the test material in mineral oil, dermal scores of 1 were noted in 1/20 test animals at the 24- hour scoring interval, and in 2/20 test animals at the 48-hour scoring interval. Dermal reactions in the remaining test and rechallenge control animals were limited to scores of 0 and ±. Group mean dermal scores were higher in the test animals as compared to the rechallenge control animals.

 

α-Hexylcinnamaldehyde (HCA)

Following challenge with 2.5% w/v HCA in acetone, dermal scores of 1 were noted in 8/10 HCA test animals at the 24-hour scoring interval, and in 9/10 HCA test animals at the 48-hour scoring interval. Dermal reactions in the HCA control animals were limited to scores of 0 or ±. Group mean dermal scores were higher in the HCA test animals compared to the HCA control animals.Following challenge with 1.0% w/v HCA in acetone, dermal scores of 1 were noted in 3/10 HCA test animals at the 24-hour scoring interval, and in 4/10 test animals at the 48-hour scoring interval. Dermal reactions in the HCA control animals were limited to scores of 0 or ±. Group mean dermal scores were higher in the HCA test animals compared to the HCA control animals.

 

Conclusion

Based on the results of this study, the test material is not considered to be a contact sensitizer in guinea pigs, as the criterion for sensitization (dermal scores ≥ 1 in at least 15% of the test animals) was not met. The results of the HCA positive control study demonstrated that a valid test was performed and indicated that the test design would detect potential contact sensitizers.

 

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In accordance with the CLP Regulation, No 1272/2008, a substance should be classified as a skin sensitiser if there are positive results from an appropriate animal test. Based on the results of in vivo studies, the test material was not considered to be a sensitiser and therefore does not meet the criteria for classification..