Fatty acids, tall-oil, compds. with ethanolamine

Endpoint summary

Description of key information

The test item is not considered to be a contact sensitiser in guinea pigs; however, it is considered to be a dermal irritant at concentrations of ≥ 12.5 % in propylene glycol. The HCA historical control results demonstrate that the test design would detect potential contact sensitisers (OECD 406 and OPPTS 870.2600).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 September 2016 to 17 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

yes

Remarks:

non-naïve site used for rechallenge with no impact on results or integrity of the study (see below)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

yes

Remarks:

non-naïve site used for rechallenge with no impact on results or integrity of the study (see below)

GLP compliance:

yes (incl. QA statement)

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 13 September 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 332 grams and 338 grams. The female range-finding animals were approximately 6 weeks of age on the day prior to dosing with body weights of 312 grams and
357 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 374 grams to 449 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 326 grams to 412 grams.
- The male second range-finding animals were approximately 9 weeks of age on the day prior to dosing with body weights of 513 grams and 604 grams. The female second range-finding animals were approximately 10 weeks of age on the day prior to dosing with body weights of 463 grams and 468 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.
- As an alternative, guinea pigs were individually housed in solid bottom cages containing a hiding device and direct bedding material.
- 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.
- Control group animals were housed on a separate rack from test substance-treated animals.

ENVIRONMENTAL CONDITIONS
- Temperatures of 68 °F to 70 °F (20 °C to 21 °C) with a relative humidity of 49 % to 62 % 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 analysed 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.
- Water bottles and/or hydrogels were provided when required.
- The water is analysed 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.
- As an alternative, guinea pigs were individually housed in solid bottom cages containing direct bedding material.
- When individually housed, a hiding comfort device (PVC pipe) was provided.
- 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 or treatment were required.

Route:

epicutaneous, occlusive

Vehicle:

propylene glycol

Concentration / amount:

100 % test item (0.3 mL dose volume)

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:

polyethylene glycol

Concentration / amount:

25 % test item (0.3 mL dose volume)

Day(s)/duration:

Day 28 (six hours duration)

Adequacy of challenge:

other: challenge

No.:

#2

Route:

epicutaneous, occlusive

Vehicle:

propylene glycol

Concentration / amount:

12.5 % test item (0.3 mL dose volume)

Day(s)/duration:

Day 35 (six hours duration)

Adequacy of challenge:

other: rechallenge

No.:

#3

Route:

epicutaneous, occlusive

Vehicle:

propylene glycol

Concentration / amount:

12.5 % test item (0.3 mL dose volume)

Day(s)/duration:

Day 42 (six hours duration)

Adequacy of challenge:

other: second rechallenge

No.:

#4

Route:

epicutaneous, occlusive

Vehicle:

propylene glycol

Concentration / amount:

5.0 % test item (0.3 mL dose volume)

Day(s)/duration:

Day 49 (six hours duration)

Adequacy of challenge:

other: third rechallenge

No. of animals per dose:

- Induction phase: 10 males and 10 females
- Main phase: 10 males and 10 females

Details on study design:

CONTROL SUBSTANCE
- Identification: Propylene glycol
- Physical description: Clear colourless liquid
- Supplier: Sigma Aldrich
- Batch: MKBZ1609V
- Expiry date: 11 July 2017
- Storage conditions: Room temperature

POSITIVE CONTROL COMPONENT 1
- Identification: HCA (α-Hexylcinnamaldehyde)
- Physical description: Clear yellow liquid
- Supplier: TCI America
- Batch: FDZEJ
- Expiry date: 08 September 2017
- Storage conditions: Room temperature, protected from light and desiccated

POSITIVE CONTROL COMPONENT 2
- Identification: Ethanol
- Physical description: Clear colourless liquid
- Supplier: Decon Labs Inc
- Batch: 176611
- Expiry date: 30 June 2019
- Storage conditions: Room temperature in a flammable cabinet

POSITIVE CONTROL COMPONENT 3
- Identification: Acetone
- Physical description: Clear colourless liquid
- Supplier: Spectrum Chemical Mfg Corp
- Batch: 1EI0266
- Expiry date: 15 July 2020
- Storage conditions: Room temperature in a flammable cabinet

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 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 and positive control substance components.
- The reserve samples were maintained under the appropriate storage conditions by the testing facility.

PREPARATION OF TEST SUBSTANCE
- Trial solubility preparations were carried out prior to the start of the study to assess the suitability of the formulation procedure and to determine an appropriate vehicle for the study.
- The trial solubility preparations were discarded after assessments were completed.
- The test substance was administered as received and/or diluted with the control substance (propylene glycol) on the day of dosing.
- Selected doses were achieved by adjustment of the test substance concentration in the control substance.
- 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.

SAMPLE COLLECTION AND ANALYSIS
- No samples for analytical analysis were collected by the testing facility.

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 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. Hilltop Chambers (25 mm) backed by adhesive tape (occlusive patch) were applied to the clipped surface as quickly as possible. 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 propylene glycol, followed by dry gauze, and then wiped with gauze moistened in reverse osmosis deionized (RODI) 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 5 (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.
- Prior to the start of dosing, Animal No. 6073 (Group 7 male) was rejected from the study due to abrasions within the test site area and was replaced by a spare animal which became Animal No. 6166.
- 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 second range-finding phase was performed to aid in determining the appropriate dose levels for the challenge phase. 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 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 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 propylene glycol, 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.

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 the rechallenge dosing table below.

SECOND RECHALLENGE
- On the day prior to the second rechallenge dose administration, the test and second rechallenge
control animals were weighed and the hair was removed from the right side of the animals.
- On the day following clipping (Day 42), chambers were applied as indicated in the second rechallenge dosing table below.

THIRD RECHALLENGE
- On the day prior to the third rechallenge dose administration, the test and third rechallenge control animals were weighed and the hair was removed from the right side of the animals.
- On the day following clipping (Day 49), chambers were applied as indicated in the third rechallenge 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.
- Detailed 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, rechallenge, second rechallenge, and third rechallenge) using the Macroscopic Dermal Grading System in Appendix 5 (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, the day prior to the rechallenge dosing, the day prior to the second rechallenge dosing, and the day prior to the third rechallenge dosing for the appropriate test and challenge control animals.
- Scheduled euthanasia: Following the third rechallenge phase 48-hour scoring interval, 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, rechallenge, second rechallenge, and third rechallenge.
- Generally, dermal scores of ≥ 1 in the test animals with scores of 0 to ± noted in the controls were considered indicative of sensitisation.
- 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 sensitiser in this study design.

Challenge controls:

5 males and 5 females

Positive control substance(s):

yes

Remarks:

HCA in ethanol (5.0 % w/v); HCA in acetone (2.5 % w/v); HCA in acetone (1.0 % w/v)

Positive control results:

POSITIVE CONTROL RESULTS
- Following challenge with 2.5% w/v HCA in acetone, a dermal score of 1 was noted in 1/10 HCA test animals at the 24-hour scoring interval and in 0/10 at the 48-hour scoring interval. Dermal reactions in the HCA control animals were limited to a ± score for 1/10 animals at the 48-hour scoring interval. Group mean dermal scores were higher in the HCA test animals (0.3 and 0.1) compared to the HCA control animals (0.0).
- Following challenge with 1.0% w/v HCA in acetone, dermal scores were limited to a ± score for 1/10 animals at the 24-hour scoring interval and 3/10 animals at the 48-hour scoring interval. Dermal reactions in the HCA control animals consisted of scores of ± for 2/10 and a score of 1 for 1/10 animals at the 24-hour scoring interval with scores of 0 for all 10 animals at the 48-hour scoring interval. Group mean dermal scores were higher in the HCA control vs. test animals (0.2 vs. 0.0, respectively) at the 24-hour interval and were higher in the HCA test vs.
control animals at the 48-hour scoring interval (0.2 vs. 0.0, respectively).
- The exact details are unknown, but it was considered that the lack of response in the nonadjuvant test was attributed to the HCA material itself; therefore, following consultation with the Sponsor, the Testing Facility’s HCA historical control data from the last 6 months was included and served as the positive control reference for this study.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25 % test item

No. with + reactions:

7

Total no. in group:

20

Clinical observations:

Group mean dermal score 0.5 at 24 hours

Remarks on result:

other: dermal scores of 1 or 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25 % test item

No. with + reactions:

3

Total no. in group:

20

Clinical observations:

Group mean dermal score 0.3 at 48 hours

Remarks on result:

other: dermal scores of 1

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

12.5 % test item

No. with + reactions:

7

Total no. in group:

20

Clinical observations:

Group mean dermal score 0.8 at 24 hours

Remarks on result:

other: dermal scores of 1 or 2 (site was not naïve)

Reading:

rechallenge

Hours after challenge:

48

Group:

test chemical

Dose level:

12.5 % test item

No. with + reactions:

9

Total no. in group:

20

Clinical observations:

Group mean dermal score 1.0 at 48 hours

Remarks on result:

other: dermal scores of 1, 2, 3 or maximised erythema (site was not naïve)

Reading:

other: second rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

12.5 % test item

No. with + reactions:

2

Total no. in group:

20

Clinical observations:

Group mean dermal score 0.5 at 24 hours

Remarks on result:

other: dermal scores of 1

Reading:

other: second rechallenge

Hours after challenge:

48

Group:

test chemical

Dose level:

12.5 % test item

No. with + reactions:

1

Total no. in group:

20

Clinical observations:

Group mean score 0.4 at 48 hours

Remarks on result:

other: dermal score of 1

Reading:

other: third rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

5.0 % test item

No. with + reactions:

1

Total no. in group:

20

Clinical observations:

Group mean score 0.3 at 24 hours

Remarks on result:

other: dermal score of 1

Reading:

other: third rechallenge

Hours after challenge:

48

Group:

test chemical

Dose level:

5.0 % test item

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

Group mean score 0.1 at 48 hours

Remarks on result:

other: dermal irritation limited to ±

MORTALITY

-No mortality occurred during the study.

CLINICAL OBSERVATIONS

- Clinical observations during the study were limited to yellow fur staining of the abdominal or urogenital areas on Day 0 (prior to dose administration) and red pinna skin on Day 0 or Day 49.

- These findings are not considered test article related because they occurred prior to dose administration, occurred in multiple groups (including controls), and/or are common findings for guinea pigs.

 

BODY WEIGHTS

- No test item-related effects on body weight were observed in the test animals during the study.

- Body weight gain occurred for all animals at the time of study completion.

 

RANGE-FINDING PHASE

- The Dermal Grading System is presented in Appendix 5 (attached).

- Based on the initial range-finding phase, the 100% as received concentration was considered appropriate for conducting the induction phase based on ± (slight patchy erythema) and 1 (slight, but confluent or moderate patchy erythema) scores at 24 hours and 0 to ± scores at the 48-hour scoring interval. During induction, irritation was observed at the 100% concentration; therefore, prior to challenge, a second range-finding phase was conducted.

- Based on similar dermal irritation at the 25% concentration compared to the 100% as received concentration during the first range-finding phase, the 25% formulation was selected as the high dose for the second range-finding phase. Additional concentrations of 12.5%, 6.5%, and 1% test item in propylene glycol were selected for assessment.

- For the second range-finding phase, the 25% and 12.5% concentrations each resulted in a ± erythema score for 1 animal (Animal No. 6086) at the 24-hour interval. Irritation resolved by the 48-hour scoring interval. All 4 concentrations assessed were considered appropriate for future testing.

 

INDUCTION

- The Dermal Grading System is presented in Appendix 5 (attached).

- For the Induction Phase, test item was administered at 100% as received once weekly for 3 weeks. Following the first application, dermal scores of ± or 1 were noted for all test animals.

- By the 48-hour scoring interval for Induction 2, dermal irritation had increased to an erythema score of 2 (moderate, confluent erythema) present for 11/20 animals, a score of 3 (severe erythema with or without edema) for 3/20 animals, and maximized erythema (notable dermal lesions) for 2/20 animals. Additional dermal irritation was observed consisting of blanching, eschar, skin flaking, and/or edema. Based on the increase in dermal irritation, the test site was adjusted for Induction 3, but still remained at Site 1.

- For Induction 3, erythema scores ranged from ± to 2 by the 48-hour interval, with 1 animal (Animal No. 6097) showing no signs of dermal irritation. Very slight edema and focal and/or pinpoint blanching also occurred.

 

CHALLENGE

- Following challenge with 25% test item in propylene glycol, dermal scores of 1 or 2 were noted in 7/20 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.

- One male in the challenge control group had an erythema score of 1 at the 24-hour scoring interval.

- Group mean dermal scores were higher in the test animals (0.5 and 0.3) as compared to challenge control animals (0.3 and 0.1).

- Based on the positive result in the challenge control group, a rechallenge was conducted.

 

RECHALLENGE

- For the rechallenge phase at 12.5% test item in propylene glycol, dermal scores of 1 or 2 were noted for 7/20 test animals at the 24-hour scoring interval. At the 48-hour scoring interval, dermal scores of 1, 2, 3, or maximized erythema were noted for 9/20 animals. The challenge control animals had erythema scores of ± for 4/10 animals at the 24-hour interval with no irritation by the 48-hour interval. Group mean dermal scores were higher in the test animals (0.8 to 1.0) as compared to challenge control animals (0.2 and 0.0).

- The rechallenge phase was conducted at 12.5% test item in propylene glycol; however, the test substance was applied to the same site as the challenge phase for the test animals instead of at a naïve site. This resulted in increased dermal irritation which made it difficult to determine the effects of the 12.5% concentration; therefore, a second rechallenge was conducted at the same concentration but at a naïve test site.

 

SECOND RECHALLENGE

- Following the second rechallenge with 12.5% test item in propylene glycol, dermal scores of 1 were noted in 2/20 test animals at the 24-hour scoring interval. At the 48-hour scoring interval, a dermal score of 1 was noted in 1/20 test animals. The challenge control animals had erythema scores of ± for 5/10 animals at the 24-hour and 48-hour intervals. Group mean dermal scores were higher in the test animals (0.5 and 0.4) as compared to challenge control animals (0.3).

- Based on the results of the second rechallenge, the test substance was a borderline sensitiser; therefore, a third rechallenge was added to the study using a lower concentration of test substance to assess the sensitization potential.

 

THIRD RECHALLENGE

- Following the third rechallenge with 5.0% test item in propylene glycol, a dermal score of 1 was noted in 1/20 test animals at the 24-hour scoring interval. At the 48-hour scoring interval, dermal irritation was limited to ± scores. The challenge control animals had erythema scores of ± for 3/10 animals at the 24-hour and 48-hour intervals. Group mean dermal scores were higher in the test animals (0.3) at the 24-hour interval as compared to challenge control animals (0.2), but were lower at the 48-hour interval (0.1 vs 0.2, respectively).

- Based on these results, the 5% concentration was not considered to be a sensitiser.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of this study, the test item is not considered to be a contact sensitiser in guinea pigs; however, it is considered to be a dermal irritant at concentrations of≥12.5 % in propylene glycol. The HCA historical control results demonstrate 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 25 % test item in propylene glycol. A rechallenge was performed one week later in which 20 test and 10 untreated rechallenge control guinea pigs were topically treated with 12.5 % test item in propylene glycol. A second rechallenge was performed one week later in which 20 test and 10 untreated second rechallenge control guinea pigs were topically treated with 12.5 % test item in propylene glycol to confirm previous rechallenge results. A third rechallenge was performed one week later in which 20 test and 10 untreated third rechallenge control guinea pigs were topically treated with 5.0 % test item in propylene glycol. 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 25 % test item in propylene glycol, dermal scores of 1 or 2 were noted in 7/20 test animals at the 24-hour scoring interval. At the 48-hour scoring interval, dermal scores of 1 or 2 were noted in 4/20 test animals. One male in the challenge control group had an erythema score of 1 at the 24-hour scoring interval. Group mean dermal scores were higher in the test animals (0.5 and 0.4) as compared to challenge control animals (0.3 and 0.1). Based on positive results in the challenge control group, a rechallenge was conducted.

 

The rechallenge phase was conducted at 12.5 % test item in propylene glycol; however, the test substance was applied to the same site as the challenge phase for the test animals instead of at a naïve site. This resulted in increased dermal irritation which made it difficult to determine the effects of the 12.5 % concentration; therefore, a second rechallenge was conducted at the same concentration but at a naïve test site.

 

Following the second rechallenge with 12.5 % in propylene glycol, dermal scores of 1 were noted in 2/20 test animals at the 24-hour scoring interval. At the 48-hour scoring interval, a dermal score of 1 was noted in 1/20 test animals. The challenge control animals had erythema scores of ± for 5/10 animals at the 24-hour and 48-hour intervals. Group mean dermal scores were higher in the test animals (0.5 and 0.4) as compared to challenge control animals (0.3). Based on the results of the second rechallenge, the test substance was a borderline sensitiser; therefore, a third rechallenge was added to the study using a lower concentration of test substance to assess the sensitisation potential.

 

Following the third rechallenge with 5.0 % test item in propylene glycol, a dermal score of 1 was noted in 1/20 test animals at the 24-hour scoring interval. At the 48-hour scoring interval, dermal irritation was limited to ± scores. The challenge control animals had erythema scores of ± for 3/10 animals at the 24-hour and 48-hour intervals. Group mean dermal scores were higher in the test animals (0.3) at the 24-hour interval as compared to challenge control animals (0.2), but were lower at the 48-hour interval (0.1 vs 0.2, respectively).

 

The exact details are unknown, but it was considered that the lack of response in the nonadjuvant test was attributed to the HCA material itself; therefore, following consultation with the sponsor, the testing facility’s HCA historical control data from the last 6 months was included and served as the positive control reference for this study.

 

CONCLUSION

Based on the results of this study, the test item is not considered to be a contact sensitiser in guinea pigs; however, it is considered to be a dermal irritant at concentrations of ≥ 12.5 % in propylene glycol. The HCA historical control results demonstrate that the test design would detect potential contact sensitisers.

Reference 2

Endpoint:

skin sensitisation: in vitro

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In vitro

An in vitro or in chemico skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available.

In vivo

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.

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 wasperformed whereby the 20 test and 10 previously untreated (naïve) challenge control guinea pigs were topically treated with 25 % test item in propylene glycol. A rechallenge was performed one week later in which 20 test and 10 untreated rechallenge control guinea pigs were topically treated with 12.5 % test item in propylene glycol. A second rechallenge was performed one week later in which 20 test and 10 untreated second rechallenge control guinea pigs were topically treated with 12.5 % test item in propylene glycol to confirm previous rechallenge results. A third rechallenge was performed one week later in which 20 test and 10 untreated third rechallenge control guinea pigs were topically treated with 5.0 % test item in propylene glycol. 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.

Following challenge with 25 % test item in propylene glycol, dermal scores of 1 or 2 were noted in 7/20 test animals at the 24-hour scoring interval. At the 48-hour scoring interval, dermal scores of 1 or 2 were noted in 4/20 test animals. One male in the challenge control group had an erythema score of 1 at the 24-hour scoring interval. Group mean dermal scores were higher in the test animals (0.5 and 0.4) as compared to challenge control animals (0.3 and 0.1). Based on positive results in the challenge control group, a rechallenge was conducted.

The rechallenge phase was conducted at 12.5 % test item in propylene glycol; however, the test substance was applied to the same site as the challenge phase for the test animals instead of at a naïve site. This resulted in increased dermal irritation which made it difficult to determine the effects of the 12.5 % concentration; therefore, a second rechallenge was conducted at the same concentration but at a naïve test site.

Following the second rechallenge with 12.5 % in propylene glycol, dermal scores of 1 were noted in 2/20 test animals at the 24-hour scoring interval. At the 48-hour scoring interval, a dermal score of 1 was noted in 1/20 test animals. The challenge control animals had erythema scores of ± for 5/10 animals at the 24-hour and 48-hour intervals. Group mean dermal scores were higher in the test animals (0.5 and 0.4) as compared to challenge control animals (0.3). Based on the results of the second rechallenge, the test substance was a borderline sensitiser; therefore, a third rechallenge was added to the study using a lower concentration of test substance to assess the sensitisation potential.

Following the third rechallenge with 5.0 % test item in propylene glycol, a dermal score of 1 was noted in 1/20 test animals at the 24-hour scoring interval. At the 48-hour scoring interval, dermal irritation was limited to ± scores. The challenge control animals had erythema scores of ± for 3/10 animals at the 24-hour and 48-hour intervals. Group mean dermal scores were higher in the test animals (0.3) at the 24-hour interval as compared to challenge control animals (0.2), but were lower at the 48-hour interval (0.1 vs 0.2, respectively).

The exact details are unknown, but it was considered that the lack of response in the nonadjuvant test was attributed to the HCA material itself; therefore, following consultation with the sponsor, the testing facility’s HCA historical control data from the last 6 months was included and served as the positive control reference for this study.

Based on the results of this study, the test item is not considered to be a contact sensitiser in guinea pigs; however, it is considered to be a dermal irritant at concentrations of ≥ 12.5 % in propylene glycol. The HCA historical control results demonstrate 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 skin 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.