Registration Dossier
Registration Dossier
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EC number: 946-398-8 | CAS number: -
Endpoint summary
Administrative data
Description of key information
The skin sensitisation potential of the test item was assessed in accordance with OECD Guideline 406. Based on the results of this study, the test substance is considered to be a contact sensitizer in guinea pigs. The criterion for sensitization (dermal scores ≥ 1 in at least 15% of the test animals) was met at challenge as 80% of the test animals responded with a dermal score of 2. There was a clear demonstration of sensitization potential in the guinea pig based on increasing irritation during induction with a similar dermal response at challenge, as would be expected in the guinea pig when sensitized to a test substance. 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.
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, in accordance with OECD Guideline 429. Following a preliminary screening test in which no clinical signs of toxicity or skin irritation were noted at a concentration of 10% v/v, 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 10%, 5% or 2.5% v/v. 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, a-Hexylcinnamaldehyde, 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: 0.7, 1.18, 1.39 and for the positive control, 5.81.
The test item was considered to be a non-sensitizer under the conditions of the test. The positive control a-Hexylcinnamaldehyde 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
- Endpoint:
- skin sensitisation: in vivo (non-LLNA)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- The experimental start dates were 16 Feb 2016 (OECD) and 23 Feb 2016 (EPA), the experimental completion date was 04 Apr 2016 (OECD) and 01 Apr 2016 (EPA).
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 406 (Skin Sensitisation)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.2600 (Skin Sensitisation)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- guinea pig maximisation test
- Justification for non-LLNA method:
- The surface tension of test item prepared at 90 % saturation in water was determined to be 32.0 mN/m at 22.0 ± 0.5°C; literature data suggested LLNA study may give false positive result, thus a confirmatory Buehler test was conducted.
- Species:
- guinea pig
- Strain:
- Hartley
- Remarks:
- Hartley-derived albino guinea pigs
- Sex:
- male/female
- Details on test animals and environmental conditions:
- Receipt
On 16 Feb 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 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.
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 372 grams and 380 grams. The female range-finding animals were approximately 6 weeks of age on the day prior to dosing with body weights of 325 grams and 348 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 343 grams to 481 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 337 grams to 424 grams.
The male second range-finding animals were approximately 9 weeks of age on the day prior to dosing with body weights of 510 grams and 561 grams. The female second range-finding animals were approximately 10 weeks of age on the day prior to dosing with body weights of 447 grams and 473 grams.
The male third range-finding animals were approximately 10 weeks of age on the day prior to dosing with body weights of 609 grams and 650 grams. The female third range-finding animals were approximately 11 weeks of age on the day prior to dosing with body weights of 493 grams and 515 grams.
The disposition of all animals was documented in the Study Records.
Husbandry
Housing
The animals were pair housed (2 animals of the same sex and same dosing group together) throughout the study in polycarbonate cages containing direct bedding material. 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 70°F to 73°F (21°C to 23°C) with a relative humidity of 40% to 52% 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 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 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. 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 were required or medicinal treatments were administered during the study. - Route:
- epicutaneous, occlusive
- Vehicle:
- unchanged (no vehicle)
- Concentration / amount:
- 100%
- Day(s)/duration:
- once per week, for 3 consecutive weeks
- Adequacy of induction:
- other: There was a clear demonstration of sensitization potential in the guinea pig based on increasing irritation during induction
- No.:
- #1
- Route:
- epicutaneous, semiocclusive
- Vehicle:
- propylene glycol
- Remarks:
- 75% test substance in propylene glycol
- Concentration / amount:
- Following a 2-week rest period after the end of the Induction, a challenge was performed whereby the 20 test and 10 previously untreated (naïve) challenge control guinea pigs were topically treated with 75% test substance in propylene glycol
- Day(s)/duration:
- 48
- Adequacy of challenge:
- other: At 48 h dermal scores of 1 or 2 were noted in 20/20 animals. Challenge control animals showed no signs of irritation with scores of 0. Group mean dermal scores were higher in the test animals (1.5 to 1.8) as compared to challenge control animals (0.0).
- No. of animals per dose:
- In the induction phase, 10 male and 10 female guinea pigs were topically treated
The challenge was performed whereby the 20 test and 10 previously untreated (naïve) challenge control guinea pigs were topically treated - Details on study design:
- This study was performed in accordance with the United States Code of Federal Regulations, Title 40, Parts 792: Good Laboratory Practice (GLP) Standards and as accepted by Regulatory Authorities throughout the European Union (OECD Principles of Good Laboratory Practice), and Japan (MAFF and METI), and other countries that are signatories to the OECD Mutual Acceptance of Data Agreement.
Exceptions from the above regulations are listed below.
• Characterization of the test substance was performed by the Sponsor or Sponsor subcontractor according to established SOPs, controls, and approved test methodologies to ensure integrity and validity of the results generated; these analyses were not conducted in compliance with the GLP or Good Manufacturing Practice (GMP) regulations.
• Concentration, stability, and homogeneity of the test substance formulations were not determined in this study.
• Concentration, stability, and homogeneity of the α-Hexylcinnamaldehyde (HCA) formulations were not determined in this study.
This study was conducted in accordance with the procedures described herein. All deviations authorized/acknowledged by the Study Director are documented in the Study Records. The report represents an accurate and complete record of the results obtained.
There were no deviations from the above regulations that affected the overall integrity of the study or the interpretation of the study results and conclusions. - Challenge controls:
- Following challenge with 75% test substance in propylene glycol, dermal scores of 1 or 2 were noted in 18/20 test animals at the 24-hour scoring interval. At the 48-hour scoring interval, dermal scores of 1 or 2 were noted in 20/20 test animals. The challenge control animals showed no signs of irritation with scores of 0. Group mean dermal scores were higher in the test animals (1.5 to 1.8) as compared to challenge control animals (0.0).
- Positive control substance(s):
- yes
- Remarks:
- α-Hexylcinnamaldehyde (HCA)
- Positive control results:
- 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.
- Key result
- Reading:
- 1st reading
- Hours after challenge:
- 24
- Group:
- test chemical
- Dose level:
- 75%
- No. with + reactions:
- 18
- Total no. in group:
- 20
- Clinical observations:
- Following challenge with 75% test substance in propylene glycol, dermal scores of 1 or 2 were noted in 18/20 test animals at the 24-hour scoring interval.
- Remarks on result:
- positive indication of skin sensitisation
- Remarks:
- Based on the results of this study, the test substance is considered to be a contact sensitizer in guinea pigs. The criterion for sensitization (dermal scores ≥ 1 in at least 15% of the test animals) was met at challenge as 80% of the test animals responded with a dermal score of 2.
- Key result
- Reading:
- 2nd reading
- Hours after challenge:
- 48
- Group:
- test chemical
- Dose level:
- 75%
- No. with + reactions:
- 20
- Total no. in group:
- 20
- Clinical observations:
- Following challenge with 75% test substance in propylene glycol, at the 48-hour scoring interval, dermal scores of 1 or 2 were noted in 20/20 test animals.
- Remarks on result:
- positive indication of skin sensitisation
- Remarks:
- Based on the results of this study, the test substance is considered to be a contact sensitizer in guinea pigs. The criterion for sensitization (dermal scores ≥ 1 in at least 15% of the test animals) was met at challenge as 80% of the test animals responded with a dermal score of 2.
- Interpretation of results:
- Category 1 (skin sensitising) based on GHS criteria
- Conclusions:
- The skin sensitisation potential of the test item was assessed in accordance with OECD Guideline 406. Based on the results of this study, the test substance is considered to be a contact sensitizer in guinea pigs. The criterion for sensitization (dermal scores ≥ 1 in at least 15% of the test animals) was met at challenge as 80% of the test animals responded with a dermal score of 2. There was a clear demonstration of sensitization potential in the guinea pig based on increasing irritation during induction with a similar dermal response at challenge, as would be expected in the guinea pig when sensitized to a test substance. 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:
The dermal sensitization potential of the test substance was evaluated in Hartley-derived albino guinea pigs. In the induction phase, 10 male and 10 female guinea pigs were topically treated with the test substance, 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% 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.
Test item
Following challenge with 75%the test item in propylene glycol, dermal scores of 1 or 2 were noted in 18/20 test animals at the 24-hour scoring interval. At the 48-hour scoring interval, dermal scores of 1 or 2 were noted in 20/20 test animals. The challenge control animals showed no signs of irritation with scores of 0. Group mean dermal scores were higher in the test animals (1.5 to 1.8) as compared to challenge control animals (0.0).
α-Hexylcinnamaldehyde (HCA)
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. Dermal reactions in the HCA control animals were limited to scores of ± in 3/10 at the 24-hour interval and none had irritation (erythema 0) by the 48-hour interval. Group mean dermal scores were higher in the HCA test animals (0.4 to 1.6) compared to the HCA control animals (0.2 and 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. Dermal reactions in the HCA control animals were limited to scores of ± in 4/10 and 1 in 1/10 by the 48-hour interval. Group mean dermal scores were higher in the HCA test animals (0.4 to 1.5) compared to the HCA control animals (0.2 to 0.3).
Conclusion
Based on the results of this study,the test item is considered to be a contact sensitizer in guinea pigs. The criterion for sensitization (dermal scores ≥ 1 in at least 15% of the test animals) was met at challenge as 80% of the test animals responded with a dermal score of 2. There was a clear demonstration of sensitization potential in the guinea pig based on increasing irritation during induction with a similar dermal response at challenge, as would be expected in the guinea pig when sensitized to a test substance. 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:
- skin sensitisation: in vivo (LLNA)
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- Experimental start date 08 August 2016 Experimental completion date 22 November 2016
- 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/Ca
- Sex:
- female
- Details on test animals and environmental conditions:
- Animal Information
Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Envigo RMS B.V., Inc., Horst, The Netherlands. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non-pregnant. After an acclimatization period of at least 5 days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals were in the weight range of 15 to 23 g, and were 8 to 12 weeks old.
Animal Care and Husbandry
The animals were housed in suspended solid floor polypropylene cages furnished with softwood woodflakes. Free access to mains tap water and food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study.
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 at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study. - Positive control substance(s):
- yes
- Remarks:
- a-Hexylcinnamaldehyde
- Vehicle:
- acetone/olive oil (4:1 v/v)
- Remarks:
- For the purpose of the study, the test item was used undiluted and freshly prepared as a solution in acetone/olive oil 4:1. This vehicle was chosen as it producedthe most suitable formulation at the required concentration.
- Concentration:
- Test item concentrations 10%, 5% or 2.5% v/v in acetone/olive oil 4:1.
- No. of animals per dose:
- Groups of five mice
- Details on study design:
- For the purpose of the study, the test item was used undiluted and freshly prepared as a solution in acetone/olive oil 4:1. This vehicle was chosen as it produced the most suitable formulation at the required concentration. The concentrations used are given in the procedure section. The vehicle determination record is attached as Annex 3.
The test item was formulated within 2 hours of being applied to the test system. It is assumed that the formulation was stable for this duration.
No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.
Positive Control Item
Information as provided by the supplier.
Identification: a-Hexylcinnamaldehyde
Batch: MKBT2800V
Purity: 97.3%
Physical state / Appearance: colorless to dark yellow liquid
Supplier: Sigma-Aldrich
Expiry Date: 30 August 2017
Storage Conditions: room temperature
Reference Item Preparation
The positive control item was freshly prepared as a 25% v/v dilution in acetone/olive oil 4:1.
Preliminary Screening Test
As no toxicological information was available regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using four mice, one mouse per test item concentration. The mice were treated by daily application of 25 µL of the undiluted test item or the test item at concentrations of 50%, 25% or 10% v/v in acetone/olive oil 4:1, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mice were observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and
6. Local skin irritation was scored daily according to the scale attached as Annex 4. Any clinical signs of toxicity, if present, were also recorded. The body weight of each mouse was recorded on Day 1 (prior to dosing) and on Day 6.
The thickness of each ear was measured using a Mitutoyo 547-300S gauge (Mitutoyo Corporation), pre-dose and post-dose on Day 1, post-dose on Days 2 and 3 and on Days 4, 5 and 6, except for the animal treated with the undiluted test item where, due to technician error, measurements were not performed on Days 2 to 4 and the animal treated with the test item at a concentration of 25% where similarly the measurements were not performed on Day
4. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization.
The highest suitable concentration that did not produce systemic toxicity or local skin irritation was selected as the maximum concentration in the main test.
Main Test
Test Item Administration
Groups of five mice were treated with the test item at concentrations of 10%, 5% or 2.5% v/v in acetone/olive oil 4:1. The preliminary screening test suggested that the test item would not produce systemic toxicity or local skin 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 of the 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, a-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.
Local skin irritation was scored daily according to the scale attached as Annex 4. The thickness of each ear was measured and recorded pre and post-dose on Day 1, post-dose on Days 2 and 3 and on Days 4, 5 and 6.
3 H-Methyl Thymidine Administration
Five days following the first topical application of the test item, vehicle control item or positive control item (Day 6) all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 3 H-methyl thymidine (3HTdR: 80 µCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 µCito each mouse.
Observations
Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
Body Weights: The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).
Terminal Procedures
Termination: Five hours following the administration of3 HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. For each individual animal of each group the draining auricular lymph nodes were excised and processed. For each individual animal 1 mL of PBS was added to the lymph nodes.
Preparation of Single Cell Suspension: A single cell suspension of the lymph node cells for each individual animal was prepared by gentle mechanical disaggregation through a
200-mesh stainless steel gauze. 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 centrifuge tube. The lymph node cells were pelleted at 1400 rpm (approximately 190 g) for 10 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).
Determination of3 HTdR Incorporation: After approximately 18 hours incubation at approximately 4 °C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, re-suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3 HTdR incorporation was measured by -scintillation counting. The "Poly Q™" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left to stand in darkness for approximately 20 minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately 20 minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA). - Positive control substance(s):
- other: a-Hexylcinnamaldehyde
- Statistics:
- Statistical Analysis
Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships. Data was first assessed for suitability by analysis of normality and homogeneity of variance. If the assumptions that the data are both normally distributed and has homogeneity of variances, then parametric one way analysis of variance (ANOVA) and Dunnett's multiple comparison procedure were used to determine statistical significance. If the assumptions were not met, non-parametric Kruskal-Wallis Rank Sum and Mann-Whitney U test procedures were used.
Probability values (p) are presented as follows:
P<0.001 ***
P<0.01 **
P<0.05 *
P::::0.05 (not significant) - Positive control results:
- Stimulation Index = 5.81 = positive result
- Key result
- Parameter:
- SI
- Value:
- 0.7
- Variability:
- 2.5% vlv in acetone/olive oil 4:1
- Test group / Remarks:
- Treatment Group: Test Item
- Remarks on result:
- other: Negative
- Key result
- Parameter:
- SI
- Value:
- 1.18
- Variability:
- 5% vlv in acetone/olive oil 4:1
- Test group / Remarks:
- Treatment Group: Test Item
- Remarks on result:
- other: Negative
- Key result
- Parameter:
- SI
- Value:
- 1.39
- Variability:
- 10% v/v in acetone/olive oil 4:1
- Test group / Remarks:
- Treatment Group: Test Item
- Remarks on result:
- other: Negative
- Key result
- Parameter:
- SI
- Value:
- 5.81
- Variability:
- 25% v/v in acetone/olive oil 4:1
- Test group / Remarks:
- Treatment Group: Positive Control Item
- Remarks on result:
- other: Positive
- 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-sensitizer under the conditions of the test.
- 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 or skin irritation were noted at a concentration of 10% v/v, 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 10%, 5% or 2.5% v/v. 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,a-Hexylcinnamaldehyde, at a concentration of 25% v/v in acetone/olive oil 4:1.
Results
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:
Treatment Group
Concentration
Stimulation Index
Result
Test Item
2.5% v/v in
acetone/olive oil 4:1
0.70
Negative
5% v/v in
acetone/olive oil 4:1
1.18
Negative
10% v/v in
acetone/olive oil 4:1
1.39
Negative
Positive Control Item
25% v/v in
acetone/olive oil 4:1
5.81
Positive
Conclusion
The test item was considered to be a non-sensitizer under the conditions of the test.
The positive control a-Hexylcinnamaldehyde 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.
Referenceopen allclose all
RESULTS
Mortality
No mortality occurred during the study.
Range-Finding Phases
(Table 1, Table 2, and Table 3)
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) scores through 48 hours. During induction, irritation was observed at the 100% concentration; therefore, prior to challenge, a second range-finding phase was conducted by repeating the 75%, 50%, and 25% concentrations with the addition of a 12.5% concentration. The second range‑finding phase produced increased dermal irritation (±, 1, and 2 erythema scores) at the lower concentrations with only 1 animal showing an erythema score of 1 at the 75% concentration. Based on these results, a third range-finding phase was added at the same concentrations as the second range-finding phase to assess the variability between the first and second range-finding phase results. For the third range-finding phase, the test substance was warmed in an attempt to produce a more homogeneous bulk test substance prior to removal for dilution. The third range-finding phase produced erythema scores of ± at all 3 concentrations and it was determined that the 75% concentration was appropriate as the highest non-irritating concentration for the challenge phase.
Table 1
Topical range finding data
GROUP |
ANIMAL NO./SEX BODY WEIGHT (G) |
RANGE-FINDING DERMAL SCORES |
|||||||
100%a |
75%b |
50%b |
25%b |
||||||
24 HOURS |
48 HOURS |
24 HOURS |
48 HOURS |
24 HOURS |
48 HOURS |
24 HOURS |
48 HOURS |
||
RANGE-FINDING |
6064/M |
± |
± |
0 |
0 |
0 |
0 |
0 |
0 |
(380) |
|
|
|
|
|
|
|
|
|
6065/M |
± |
± |
0 |
0 |
0 |
0 |
0 |
0 |
|
(372) |
|
|
|
|
|
|
|
|
|
6066/F |
0 |
± |
0 |
0 |
0 |
0 |
0 |
0 |
|
(348) |
|
|
|
|
|
|
|
|
|
6067/F |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
(325) |
|
|
|
|
|
|
|
|
|
aAS RECEIVED. bTHE VEHICLE USED WAS PROPYLENE GLYCOL. |
|||||||||
Table 2
Second
Topical Range-Finding Data
GROUP |
ANIMAL NO./SEX BODY WEIGHT (G) |
SECOND RANGE-FINDING DERMAL SCORES |
|||||||
75%a |
50%a |
25a |
12.5a |
||||||
24 HOURS |
48 HOURS |
24 HOURS |
48 HOURS |
24 HOURS |
48 HOURS |
24 HOURS |
48 HOURS |
||
SECOND RANGE-FINDING |
6022/M |
1 |
1 |
2 |
0 |
2 |
2 |
2 |
2 |
(510) |
|
|
|
|
|
|
|
|
|
6023/M |
0 |
0 |
1 |
1 |
1 |
1 |
1 |
1 |
|
(561) |
|
|
|
|
|
|
|
|
|
6108/F |
0 |
0 |
1 |
1 |
0 |
0 |
1 |
1 |
|
(447) |
|
|
|
|
|
|
|
|
|
6109/F |
0 |
0 |
± |
± |
± |
± |
0 |
1 |
|
(473) |
|
|
|
|
|
|
|
|
|
aTHE VEHICLE USED WAS PROPYLENE GLYCOL. |
|||||||||
Table 3
Third
Topical Range-Finding Data
GROUP |
ANIMAL NO./SEX BODY WEIGHT (G) |
THIRD RANGE-FINDING DERMAL SCORES |
|||||||
75%a |
50%a |
25a |
12.5a |
||||||
24 HOURS |
48 HOURS |
24 HOURS |
48 HOURS |
24 HOURS |
48 HOURS |
24 HOURS |
48 HOURS |
||
THIRD RANGE-FINDING |
6062/M |
0 |
0 |
0 |
0 |
± |
0 |
± |
0 |
(609) |
|
|
|
|
|
|
|
|
|
6063/M |
± |
0 |
0 |
0 |
± |
0 |
± |
± |
|
(650) |
|
|
|
|
|
|
|
|
|
6106/F |
0 |
0 |
± |
0 |
± |
0 |
0 |
0 |
|
(515) |
|
|
|
|
|
|
|
|
|
6107/F |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
|
(493) |
|
|
|
|
|
|
|
|
|
aTHE VEHICLE USED WAS PROPYLENE GLYCOL. |
|||||||||
Induction Phase
(Table 4)
Dermal scores of ± (slight patchy erythema) or 1 (slight, but confluent or moderate patchy erythema) were present at Induction 1 for all test animals. One animal (male Animal No. 6025) had an erythema score of 0 during Induction 2, while all others had erythema scores of 1 or 2 with 3 animals also showing signs of very slight edema. By Induction 3, male Animal No. 6025 had an erythema score of 1 with all others showing signs of erythema 2 or maximized erythema grade 3 (notable dermal lesions). Additional dermal irritation was observed consisting of blanching, eschar, and/or edema for the test animals.
Table 4
Individual
Induction Data
GROUP |
ANIMAL NO./SEX |
DERMAL SCORES |
|||||
INDUCTION 1 |
INDUCTION 2 |
INDUCTION 3 |
|||||
100%a |
100%a |
100%a |
|||||
24 HOURS |
48 HOURS |
24 HOURS |
48 HOURS |
24 HOURS |
48 HOURS |
||
TEST |
6024/M |
1 |
1 |
1 |
1 |
M-3BLA-2, ED-2 |
2BLA-1, ED-1 |
6025/M |
1 |
1 |
0 |
0 |
1 |
1 |
|
6026/M |
1 |
1 |
2ED-1 |
2ED-1 |
M-3BLA-3, ED-2 |
M-3BLA-1, ED-2, ES-2 |
|
6027/M |
1 |
1 |
2ED-1 |
2ED-1 |
M-3BLA-3, ED-2 |
M-3BLA-3, ED-2 |
|
6028/M |
1 |
1 |
2 |
2 |
2ES-1, BLA-1, ED-2 |
M-3ED-2, ES-3 |
|
6029/M |
1 |
1 |
2 |
2 |
2 |
2ED-1 |
|
6030/M |
1 |
1 |
2 |
2 |
M-3BLA-3, ED-2 |
M-3ED-2, ES-4 |
|
6031/M |
1 |
± |
1 |
2 |
2ED-1 |
2ED-1 |
|
6032/M |
± |
± |
2 |
2 |
M-3BLA-2, ED-1 |
M-3ED-2, ES-3 |
|
6033/M |
1 |
1 |
± |
± |
2ED-1 |
M-3BLA-4, ED-2 |
|
6068/F |
1 |
1 |
2ED-1 |
2ED-1 |
2ED-1, ES-1 |
M-3BLA-3, ED-2, ES-1 |
|
6069/F |
1 |
1 |
2 |
2 |
2 |
2ED-1 |
|
6070/F |
± |
± |
2 |
2 |
M-3ED-2, ES-3 |
M-3ED-3, ES-4 |
|
6071/F |
1 |
1 |
2 |
2 |
M-3ED-2, ES-3 |
M-3ED-2, ES-3 |
|
6072/F |
1 |
1 |
1 |
1 |
2BLA-1, ED-1 |
M-3BLA-2, ED-2 |
|
6073/F |
± |
± |
2 |
2 |
M-3ES-2, BLA-1, ED-2 |
M-3ES-3, BLA-1, ED-2 |
|
6074/F |
1 |
1 |
2 |
2 |
M-3ES-4, ED-2 |
M-3ES-4, ED-2 |
|
6075/F |
1 |
1 |
2 |
2 |
M-3ES-4, ED-2 |
M-3ES-4, ED-2 |
|
6076/F |
± |
± |
2 |
2 |
M-3ES-4, ED-3 |
M-3ES-4, ED-3 |
|
6077/F |
1 |
1 |
2 |
2 |
2ES-1, BLA-1, ED-2 |
M-3ES-4, ED-3 |
|
aAS RECEIVED. |
|||||||
GROUP |
ANIMAL NO./SEX |
DERMAL SCORES |
|||||
INDUCTION 1 |
INDUCTION 2 |
INDUCTION 3 |
|||||
5.0%a |
5.0%a |
5.0%a |
|||||
24 HOURS |
48 HOURS |
24 HOURS |
48 HOURS |
24 HOURS |
48 HOURS |
||
HCA TEST |
6034/M |
±BLA-1 |
1BLA-1 |
M-3ES-3 |
M-3ES-3 |
2BLA-1 |
2BLA-1 |
6035/M |
± |
1 |
M-3ES-3 |
M-3ES-3 |
2 |
2BLA-1 |
|
6036/M |
2 |
2 |
M-3ES-2, BLA-1 |
M-3ES-2, BLA-1 |
2BLA-1, ED-1 |
2BLA-1, ED-1 |
|
6037/M |
2 |
2 |
M-3ES-3 |
M-3ES-3 |
2BLA-1, ED-1 |
2BLA-1, ED-1 |
|
6038/M |
2BLA-1 |
2BLA-1 |
M-3ES-3 |
M-3ES-3 |
2 |
2BLA-1 |
|
6078/F |
2 |
2ED-1 |
M-3ES-3 |
M-3ES-3 |
2BLA-1 |
2BLA-1 |
|
6079/F |
2BLA-1 |
2BLA-1 |
M-3ES-3 |
M-3ES-3 |
2 |
2 |
|
6080/F |
2 |
2 |
M-3ES-2 |
M-3ES-2 |
2BLA-1, ED-1 |
2BLA-1, ED-1 |
|
6081/F |
1BLA-1 |
1BLA-1 |
M-3ES-3 |
M-3ES-3 |
2BLA-1 |
2BLA-1 |
|
6082/F |
1 |
1 |
M-3ES-3 |
M-3ES-3 |
2BLA-1 |
2BLA-1 |
|
aTHE VEHICLE USED WAS ETHANOL. |
|||||||
Challenge Phase
(Table 5)
Following challenge with 75% test substance in propylene glycol, dermal scores of 1 or 2 were noted in 18/20 test animals at the 24-hour scoring interval. At the 48-hour scoring interval, dermal scores of 1 or 2 were noted in 20/20 test animals. The challenge control animals showed no signs of irritation with scores of 0. Group mean dermal scores were higher in the test animals (1.5 to 1.8) as compared to challenge control animals (0.0).
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. Dermal reactions in the HCA control animals were limited to scores of ± in 3/10 at the 24-hour interval and none had irritation (erythema 0) by the 48-hour interval. Group mean dermal scores were higher in the HCA test animals (0.4 to 1.6) compared to the HCA control animals (0.2 and 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. Dermal reactions in the HCA control animals were limited to scores of ± in 4/10 and 1 in 1/10 by the 48-hour interval. Group mean dermal scores were higher in the HCA test animals (0.4 to 1.5) compared to the HCA control animals (0.2 to 0.3).
Table 5
Individual
Challenge Data
GROUP |
ANIMAL NO./SEX |
DERMAL SCORES |
|
|
|||
75%a |
|
|
|||||
24 HOURS |
48 HOURS |
|
|
|
|
||
TEST |
6024/M |
0 |
1 |
|
|
|
|
6025/M |
1 |
1 |
|
|
|
|
|
6026/M |
2ED-1 |
2ED-1 |
|
|
|
|
|
6027/M |
1 |
2 |
|
|
|
|
|
6028/M |
2ED-1 |
2ED-1 |
|
|
|
|
|
6029/M |
2ED-1 |
2 |
|
|
|
|
|
6030/M |
0 |
1 |
|
|
|
|
|
6031/M |
1 |
1 |
|
|
|
|
|
6032/M |
1 |
2 |
|
|
|
|
|
6033/M |
1 |
2 |
|
|
|
|
|
6068/F |
2ED-1 |
2ED-1 |
|
|
|
|
|
6069/F |
2ED-1 |
2 |
|
|
|
|
|
6070/F |
2ED-1 |
2ED-1 |
|
|
|
|
|
6071/F |
2ED-1 |
2ED-1 |
|
|
|
|
|
6072/F |
2 |
2 |
|
|
|
|
|
6073/F |
2 |
2 |
|
|
|
|
|
6074/F |
1 |
2 |
|
|
|
|
|
6075/F |
2 |
2 |
|
|
|
|
|
6076/F |
2 |
2 |
|
|
|
|
|
6077/F |
2 |
2 |
|
|
|
|
|
|
|
|
|
|
|
|
|
MEAN |
1.5 |
1.8 |
|
|
|
|
|
aTHE VEHICLE USED WAS PROPYLENE GLYCOL. |
|||||||
GROUP |
ANIMAL NO./SEX |
DERMAL SCORES |
|
|
|||
75%a |
|
|
|||||
24 HOURS |
48 HOURS |
|
|
|
|
||
CHALLENGE CONTROL |
6039/M |
0 |
0 |
|
|
|
|
6040/M |
0 |
0 |
|
|
|
|
|
6041/M |
0 |
0 |
|
|
|
|
|
6042/M |
0 |
0 |
|
|
|
|
|
6043/M |
0 |
0 |
|
|
|
|
|
6083/F |
0 |
0 |
|
|
|
|
|
6084/F |
0 |
0 |
|
|
|
|
|
6085/F |
0 |
0 |
|
|
|
|
|
6086/F |
0 |
0 |
|
|
|
|
|
6087/F |
0 |
0 |
|
|
|
|
|
|
|
|
|
|
|
|
|
MEAN |
0 |
0 |
|
|
|
|
|
aVEHICLE USED WAS PROPYLENE GLYCOL |
|||||||
GROUP |
ANIMAL NO./SEX |
DERMAL SCORES |
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
2.5%a |
1.0a |
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
24 HOURS |
48 HOURS |
24 HOURS |
48 HOURS |
|
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
HCA TEST |
6034/M |
± |
1 |
± |
1 |
|
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
6035/M |
0 |
2 |
0 |
1 |
|
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
6036/M |
0 |
2 |
0 |
2 |
|
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
6037/M |
2 |
2 |
2 |
2 |
|
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
6038/M |
± |
2 |
± |
2 |
|
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
6078/F |
0 |
1 |
0 |
1 |
|
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
6079/F |
0 |
1 |
0 |
1 |
|
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
6080/F |
± |
2 |
± |
2 |
|
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
6081/F |
0 |
1 |
0 |
1 |
|
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
6082/F |
± |
2 |
± |
2 |
|
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
|
|
|
|
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
MEAN |
0.4 |
1.6 |
0.4 |
1.5 |
|
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
FOR PURPOSES OF CALCULATION, ± = 0.5. aTHE VEHICLE USED WAS ACETONE.
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Body Weights
(Appendix 4)
No test item - 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.
One test animal (male Animal No. 6032) showed a slight body weight loss (2 grams) between Day 28 and Day 34. The Day 34 body weight was collected in preparation for a rechallenge phase that was not needed based on the study requirements were met.
Appendix 4
Individual
Body Weight Data
GROUP |
ANIMAL NO./SEX |
BODY WEIGHT (G) |
|
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
DAY -1 |
DAY 28 |
34a |
|
|
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TEST |
6024/M |
450 |
570 |
603 |
|
|
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
6025/M |
454 |
615 |
641 |
|
|
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
6026/M |
402 |
552 |
582 |
|
|
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
6027/M |
431 |
638 |
663 |
|
|
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
6028/M |
449 |
702 |
740 |
|
|
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
6029/M |
444 |
691 |
730 |
|
|
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
6030/M |
443 |
672 |
710 |
|
|
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
6031/M |
419 |
618 |
652 |
|
|
|
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6032/M |
416 |
588 |
586 |
|
|
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
6033/M |
409 |
636 |
654 |
|
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|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
6068/F |
383 |
536 |
566 |
|
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|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
6069/F |
388 |
583 |
607 |
|
|
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
6070/F |
384 |
533 |
538 |
|
|
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
6071/F |
399 |
592 |
616 |
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6072/F |
366 |
534 |
555 |
|
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6073/F |
385 |
567 |
604 |
|
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6074/F |
386 |
550 |
559 |
|
|
|
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6075/F |
386 |
577 |
618 |
|
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6076/F |
374 |
486 |
498 |
|
|
|
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6077/F |
421 |
557 |
591 |
|
|
|
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HCA TEST |
6034/M |
419 |
622 |
- |
|
|
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6035/M |
444 |
647 |
- |
|
|
|
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6036/M |
459 |
707 |
- |
|
|
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
6037/M |
430 |
642 |
- |
|
|
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
6038/M |
442 |
513 |
- |
|
|
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
6078/F |
424 |
593 |
- |
|
|
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
6079/F |
381 |
520 |
- |
|
|
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
6080/F |
398 |
550 |
- |
|
|
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
6081/F |
354 |
460 |
- |
|
|
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
6082/F |
380 |
580 |
- |
|
|
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
NOTE: - = NOT APPLICABLE. aANIMALS WERE WEIGHED FOR POSSIBLE RECHALLENGE PHASE DOSING.
|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Preliminary Screening Test
Clinical observations, body weight and mortality data are given in the attached Appendix 1 and local skin irritation is given in the attached Appendix 2. The ear thickness measurements and mean ear thickness changes are given in the attached Appendix 3.
No signs of systemic toxicity were noted.
Very slight erythema was noted on both ears of animals treated with the undiluted test item or the test item at concentrations of 50% or 25% v/v in acetone/olive oil 4:1. No visual local skin irritation was noted in the animal treated with the test item at a concentration of 10% v/v in acetone/olive oil 4:1.
A greater than 25% increase in mean ear thickness was noted in the animal treated with the undiluted test item.
No irritation indicated by an equal to or greater than 25% increase in mean ear thickness was noted in animals treated with the test item at concentrations of 50%, 25% or 10 v/v in acetone/olive oil 4:1.
Based on the 10% concentration being non-irritant, the dose levels selected for the main test were 10%, 5% and 2.5% v/v in acetone/olive oil 4:1.
Main Test
Estimation of the Proliferative Response of Lymph Node Cells
The radioactive disintegrations per minute per lymph node and the stimulation index are given in the attached Appendix 4.
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:
Treatment Group |
Concentration |
5.81 |
Result |
Test Item |
2.5%vlv in acetone/olive oil 4:1 |
0.70 |
Negative |
5%vlv in acetone/olive oil 4:1 |
1.18 |
Negative |
|
10%v/v in acetone/olive oil 4:1 |
1.39 |
Negative |
|
Positive Control Item |
25%v/v in acetone/olive oil 4:1 |
5.81 |
Positive |
Clinical Observations and Mortality Data
Individual clinical observations and mortality data for test and control animals are given in the attached Appendix 5 and local skin irritation is given in Appendix 6. The ear thickness measurements and mean ear thickness changes are given in the attached Appendix 7.
There were no deaths. No signs of systemic toxicity, local skin irritation or marked increase in ear thickness were noted in the test or control animals during the test.
Body Weight
Individual body weights and body weight change for test and control animals are given in the attached Appendix 8.
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.
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (sensitising)
- Additional information:
The results of the two studies are contradictory (the OECD 406 giving a positive result and the OECD 429 a negative result). This is considered to be the result of the different vehicles and dose levels used in the two studies and overall the substance is considered to be a sensitizer. The OECD 429 study data do indicate a dose-related increase in the stimulation index even though the threshold for a positive response was not exceeded. In this case the EC3 may be calculated by linear extrapolation to be 17.5% and this value is considered to be suitable for classification and labelling and for the setting of an SCL.
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 an in vivo study in the guinea pig, the test material was considered to be a sensitiser and therefore meets the criteria for classification. The data of the LLNA assay indicate that the potency is low and that the EC3 was calculated to be 17.5% and therefore the substance may be classified as skin sensitizer Sub-category1B.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
