Registration Dossier

Administrative data

Description of key information

Based on the results of a modified Buehler test, the test item was considered a cumulative irritant but was not considered to be a contact sensitiser in guinea pigs. 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 sensitisers (OECD 406 and EPA Health Effects Test Guideline OPPTS 870.2600).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 April 2016 to 02 June 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
with no impact on overall integrity of the study (see below)
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
yes
Remarks:
with no impact on overall integrity of the study (see below)
GLP compliance:
yes
Type of study:
Buehler test
Justification for non-LLNA method:
Expert toxicological opinion was that skin sensitisation potential of the test item would be best assessed using the modified Buehler design.
Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
RECEIPT OF ANIMALS
- On 19 April 2016, 44 male and 44 female Hartley-derived albino guinea pigs were received from Charles River Laboratories, Inc., St. Constant, QC.
- 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 characterise the effects of the test substance.
- The 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.

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 pre-test 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 367 grams and 381 grams. The female range-finding animals were approximately 6 weeks of age on the day prior to dosing with body weights of 359 grams and 368 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 307 grams to 450 grams. The female main phase animals were approximately 7 weeks of age on the day prior to Induction 1 dosing with body weights ranging from 303 grams to 440 grams.
- The disposition of all animals was documented in the Study Records.

ANIMAL HOUSING
- The animals were group housed (2 animals of the same sex and same dosing group together) 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.
- Animals were separated during designated procedures/activities.
- Each cage was clearly labelled with a colour-coded cage card indicating study, group, animal number, and sex.
- Cages were arranged on the racks in group order.

ENVIRONMENTAL CONDITIONS
- Temperatures of 71°F to 73°F (22 °C to 23 °C) with a relative humidity of 42 % to 53 % 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.

ANIMAL 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 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.
- Hydrogel was provided when required.
- Periodic analysis of the water is performed and 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 study.
- No veterinary examinations were required and no medicinal treatments were administered during the study.
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100 % test item
Day(s)/duration:
Days 0, 7 and 14 (6 hours duration)
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:
(Fischer Scientific; batch 1552215; expiry date 05 October 2016)
Concentration / amount:
75 % or 25 % test item
Day(s)/duration:
Day 28 (6 hours duration)
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
- Induction phase: 10 males and 10 females
- Main phase: 10 males and 10 females
Details on study design:
POSITIVE CONTROL COMPONENT 1
- Identification: HCA (α-Hexylcinnamaldehyde)
- Supplier: TCI America
- Batch: FDZEJ
- Expiry dates: 08 Jun 2016, 30 Nov 2016, 14 Dec 2016, and 15 Dec 2016

POSITIVE CONTROL COMPONENT 2
- Identification: Ethanol
- Supplier: Decon Labs Inc
- Batch: 208512
- Expiry date: 05 August 2016

POSITIVE CONTROL COMPONENT 3
- Identification: Acetone
- Supplier: Spectrum Chemical Mfg Corp
- Batch: 1EI0266
- Expiry date: 15 July 2020

TEST SUBSTANCE CHARACTERISATION
- 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 (1 mL), control substance (1 g), and positive control substance components (0.1 mL of HCA, 1 mL of ethanol and acetone).
- The reserve samples were maintained under the appropriate storage conditions by the testing facility.

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 phase, induction, and challenge.
- Selected doses were achieved by adjustment of test substance concentration in the controlsubstance.
- Details of the preparation and dispensing of the test substance were retained in the Study Records.

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 were retained in the Study Records.

RANGE-FINDING PHASE
- Experimental design of the range-finding phase is shown in the table below.
- The dermal route of exposure was selected because this is the intended potential route of human exposure.
- Four graded levels were utilised for the procedure.
- Optimally, the range-finding study was expected to produce no systemic toxicity and a spectrum of dermal responses that included Grades 0, ±, 1, and 2 unless the test substance was not dermally irritating at 100%.
- On the day prior to dosing, the guinea pigs selected for the topical range-finding study 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 Day 0, four concentrations of the test substance were prepared and a 0.3 mL dose of each concentration was applied to the clipped area of each topical range-finding animal. The trunk of the animal was wrapped with elastic wrap to prevent removal of the chambers 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, and then wiped with 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 were performed for all range-finding animals (see below).
- The animals were observed for general health/mortality and moribundity twice daily, once in the morning and afternoon, throughout the study.
- The animals were removed from the cage and examined in detail before dosing on Day 0.
- 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 Appendix 3 (attached) according to Buehler.
- Each topical range-finding animal was weighed on the day prior to dosing (Day -1).
- Following the 48-hour scoring interval, all range-finding animals were euthanised by carbon dioxide inhalation and discarded.

MAIN PHASE
- Experimental design of the main phase is shown in the table below.
- 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 ±.

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 applied to the appropriate animals. 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 the induction dosing table below.
- 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 the challenge dosing table below.

IN-LIFE PROCEDURES, OBSERVATIONS AND MEASUREMENTS
- Mortality/Moribundity: The animals were observed for general health/mortality and moribundity twice daily, once in the morning and afternoon, throughout the study.
- Clinical observations: The animals were removed from the cage and examined in detail before dosing on Day 0.
- 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) using the Macroscopic Dermal Grading System in Appendix 3 (attached) according to Buehler.
- 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 dosing for the appropriate test and challenge control animals.
- Scheduled euthanasia: Following the 48-hour scoring interval at challenge, all main study animals were euthanized by carbon dioxide inhalation and discarded.

COMPUTERISED SYSTEMS
- Critical computerized systems used in the study are listed in the table attached.
- All computerized systems used in the conduct of the 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.

STATISTICAL ANALYSIS
- The sensitization potential of the test substance was based on the dermal responses observed on the test and control animals at challenge.
- Generally, dermal scores of ≥ 1 in the test animals with scores of 0 to ± noted in the controls are considered indicative of sensitisation.
- Dermal scores of 1 in both the test and control animals are generally considered equivocal unless a higher dermal response (≥ grade 2) is noted in the test animals.
- Group mean dermal scores were calculated for challenge. A response of at least 15% in a nonadjuvant test was expected for a mild to moderate sensitiser in this study design.
Challenge controls:
5 males and five females
Positive control substance(s):
yes
Remarks:
5.0% (w/v) α-hexylcinnamaldehyde (HCA) in ethanol; 2.5% (w/v) α-hexylcinnamaldehyde (HCA) in acetone; 1.0% (w/v) α-hexylcinnamaldehyde (HCA) in acetone
Positive control results:
- Following challenge with 2.5% w/v HCA in acetone, dermal scores of 1 or 2 were noted in 10/10 HCA test animals at the 48-hour scoring interval with no dermal irritation in the HCA control animals. Group mean dermal scores were higher in the HCA test animals (1.8) compared to the HCA control animals (0.0).
- Following challenge with 1.0% w/v HCA in acetone, dermal scores of 1 or 2 were noted in 10/10 HCA test animals at the 48-hour scoring interval with limited dermal reactions in the HCA control animals of ± in 1/10 animals. Group mean dermal scores were higher in the HCA test animals (1.3) compared to the HCA control animals (0.1).
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
75 % test item
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
Group mean dermal scores 0.0
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
75 % test item
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
Group mean dermal scores 0.0
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
other: challenge control
Dose level:
75 % test item
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Group mean dermal scores 0.0
Remarks on result:
other: naive animals
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
other: challenge control
Dose level:
75 % test item
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Group mean dermal scores 0.0
Remarks on result:
other: naive animals
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
25 % test item
No. with + reactions:
6
Total no. in group:
20
Clinical observations:
Group mean dermal scores 0.3 at 24 hours
Remarks on result:
other: Erythema 1 reported for 2/20 test animals at 24 hours
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
25 % test item
No. with + reactions:
6
Total no. in group:
20
Clinical observations:
Group mean dermal scores 0.1 at 24 hours
Remarks on result:
other: Erythema ± scores reported in 3/20 animals at 48 hours
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
other: challenge control
Dose level:
25 % test item
No. with + reactions:
5
Total no. in group:
10
Clinical observations:
Group mean dermal scores 0.3 at 24 hours
Remarks on result:
other: naive animals
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
other: challenge control
Dose level:
25 % test item
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Group mean dermal scores 0.0 at 48 hours
Remarks on result:
other: naive animals

MORTALITY

-No mortality occurred during the study.

 

RANGE-FINDING PHASE

- The Dermal Grading System is presented in Appendix 3 (attached).

- The range-finding phase was conducted at 100% (as received) and at 75%, 50%, and 25% concentrations in mineral oil. There was no dermal irritation for any of the 4 concentrations in the 48 hours after application.

- Based on the range-finding phase, the 100% as received concentration was considered appropriate for conducting the induction phase.

 

INDUCTION

- The Dermal Grading System is presented in Appendix 3 (attached).

- For Induction 1, there was no dermal irritation in the 48 hours after chamber application.

- For Induction 2, all 20 test animals had positive dermal irritation by 24 hours after application. Dermal scores of ± (slight patchy erythema) were noted in 3/20 animals, erythema 1 (slight, but confluent or moderate patchy erythema) was present in 1/20 animals, and erythema 2 (moderate, confluent erythema) was observed in 16/20 animals. Other dermal observations at the 24-hour interval were edema 1 (very slight edema, barely perceptible) in 16/20 animals, edema 3 (moderate edema, raised approximately 1 millimeter) in 1/20 animals, and eschar 1 (focal and/or pinpoint areas up to 10% of test site) in 1/20 animals. By the 48-hour interval, the ± score was

present in 1/20 animals, erythema 1 in 10/20 animals, and erythema 2 in 9/20 animals. Other dermal findings consisted of edema 1 in 4/20 animals, edema 2 (slight edema, edges of area well defined by definite raising) in 1/20 animals, and blanching 1 (focal and/or pinpoint areas up to 10% of the test site) in 1/20 animals.

- For Induction 3, all 20 test animals had positive dermal irritation by 24 hours after application. A dermal score of ± was noted in 1/20 animals, erythema 1 was present in 1/20 animals, and erythema 2 was observed in 18/20 animals. The only other dermal observation at the 24-hour interval was edema 1 in 2/20 animals. By the 48-hour interval, erythema 1 was present in 3/20 animals and erythema 2 was present in 17/20 animals. Edema 1 was still noted in the same 2/20 animals.

- The 100% concentration was irritating and was not deemed acceptable for the challenge control phase.

 

CHALLENGE

- The challenge phase was performed as a double-patch application with 75% and 25% concentrations in mineral oil.

- There was no dermal irritation present in the 48 hours after removal of the 75% concentration in mineral oil for the 20 test animals or 10 challenge control group (naïve) animals. Group mean dermal scores were equivalent at 0.0 in both the test and challenge control groups by the end of the challenge phase.

- The 25% concentration in mineral oil resulted in ± scores in 6/20 test animals and erythema 1 in 2/20 test animals at 24 hours that decreased to only ± scores in 3/20 animals by 48 hours. The challenge control group had ± scores in 5/10 animals at 24 hours and cleared in all animals by 48 hours. Group mean dermal scores were higher in the test group (0.1) compared to the challenge control group (0.0) by the 48-hour interval at the end of the challenge phase.

 

BODY WEIGHTS

- No test item-related effects on body weight were observed in the test animals during the study (see Appendix 4, attached).

- Weight gain in the animals throughout the study 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 item was considered a cumulative irritant but was not considered to be a contact sensitiser in guinea pigs. 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 sensitisers.
Executive summary:

GUIDELINE

The dermal sensitisation potential of the test item was evaluated in Hartley-derived albino guinea pigs using a modified Buehler design in accordance with OECD 406 and EPA Health Effects Test Guideline OPPTS 870.2600.

 

METHODS

During the induction phase, 10 male and 10 female guinea pigs were topically treated with test item, as received, 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 75 % and 25 % test item in mineral oil. 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.

 

RESULTS

Following challenge with 75 % test item in mineral oil, there was no dermal irritation present in the 48 hours after removal for the 20 test animals or 10 challenge control group (naïve) animals. Group mean dermal scores were equivalent at 0.0 in both the test and challenge control groups by the end of the challenge phase. The 25 % concentration in mineral oil resulted in ± scores in 6/20 test animals and erythema 1 in 2/20 test animals at 24 hours that decreased to only ± scores in 3/20 animals by 48 hours. The challenge control group had ± scores in 5/10 animals at 24 hours and cleared in all animals by 48 hours. Group mean dermal scores were higher in the test group (0.1) compared to the challenge control group (0.0) by the 48-hour interval at the end of the challenge phase.

 

Following challenge with 2.5 % w/v HCA in acetone, dermal scores of 1 or 2 were noted in 10/10 HCA test animals at the 48-hour scoring interval with no dermal irritation in the HCA control animals. Group mean dermal scores were higher in the HCA test animals (1.8) compared to the HCA control animals (0.0). Following challenge with 1.0% w/v HCA in acetone, dermal scores of 1 or 2 were noted in 10/10 HCA test animals at the 48-hour scoring interval with limited dermal reactions in the HCA control animals of ± in 1/10 animals. Group mean dermal scores were higher in the HCA test animals (1.3) compared to the HCA control animals (0.3 and 0.0).

 

CONCLUSION

Based on the results of this study, the test item was considered a cumulative irritant but was not considered to be a contact sensitiser in guinea pigs. 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 sensitisers.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

GUIDELINE

The dermal sensitisation potential of the test item was evaluated in Hartley-derived albino guinea pigs using a modified Buehler design in accordance with OECD 406 and EPA Health Effects Test Guideline OPPTS 870.2600.

 

METHODS

During the induction phase, 10 male and 10 female guinea pigs were topically treated with test item, as received, 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 75 % and 25 % test item in mineral oil. 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.

 

RESULTS

Following challenge with 75 % test item in mineral oil, there was no dermal irritation present in the 48 hours after removal for the 20 test animals or 10 challenge control group (naïve) animals. Group mean dermal scores were equivalent at 0.0 in both the test and challenge control groups by the end of the challenge phase. The 25 % concentration in mineral oil resulted in ± scores in 6/20 test animals and erythema 1 in 2/20 test animals at 24 hours that decreased to only ± scores in 3/20 animals by 48 hours. The challenge control group had ± scores in 5/10 animals at 24 hours and cleared in all animals by 48 hours. Group mean dermal scores were higher in the test group (0.1) compared to the challenge control group (0.0) by the 48-hour interval at the end of the challenge phase.

 

Following challenge with 2.5 % w/v HCA in acetone, dermal scores of 1 or 2 were noted in 10/10 HCA test animals at the 48-hour scoring interval with no dermal irritation in the HCA control animals. Group mean dermal scores were higher in the HCA test animals (1.8) compared to the HCA control animals (0.0). Following challenge with 1.0% w/v HCA in acetone, dermal scores of 1 or 2 were noted in 10/10 HCA test animals at the 48-hour scoring interval with limited dermal reactions in the HCA control animals of ± in 1/10 animals. Group mean dermal scores were higher in the HCA test animals (1.3) compared to the HCA control animals (0.3 and 0.0).

 

CONCLUSION

Based on the results of this study, the test item was considered a cumulative irritant but was not considered to be a contact sensitiser in guinea pigs. 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 sensitisers.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on results from the modified Buehler assay, the criterion for sensitisation was not met (≤ 15% of the test animals responded when topical induction dose was > 20 %). In accordance with ECHA Guidance on the Application of the CLP Criteria (Version 4.1; June 2015), classification as a skin sensitiser is not required under the terms of Regulation (EC) No 1272/2008.