Registration Dossier

Diss Factsheets

Administrative data

Description of key information

The weight of evidence on the test material demonstrates that this substance is not a skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April, 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in compliance with recognised guidelines.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
On one occasion, the animal room temperature range [67-75°F (19-24°C)] exceeded the preferred range [63-73°F (17-23°C)] during this study. This occurrence was considered to have had no adverse effect on the outcome of this study.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
yes
Remarks:
On one occasion, the animal room temperature range [67-75°F (19-24°C)] exceeded the preferred range [63-73°F (17-23°C)] during this study. This occurrence was considered to have had no adverse effect on the outcome of this study.
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of study:
Buehler test
Justification for non-LLNA method:
See discussion of supporting LLNA results (Sanders, 2012)
Species:
guinea pig
Strain:
other: Hartley-derived albino guinea pigs
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hartley-derived albino guinea pigs were received from Hilltop Lab Animals, Inc., Scottdale, Pennsylvania.
- Age at study initiation: male: ca 7 weeks; female: ca 8 weeks
- Weight at study initiation: male: 332-445 g; female: 310-385 g
- Housing: The animals were housed individually in suspended stainless steel cages
- Diet: PMI Certified Guinea Pig Chow #5026 (PMI Nutrition International) was provided ad libitum
- Water: Municipal tap water treated by reverse osmosis was available ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-24 °C
- Humidity: 35-64%
- Air changes: 10-15 air changes/hour
- Photoperiod: 12 h light / 12 h dark

IN-LIFE DATES: From: January 28, 2004; to: February 27, 2004
Route:
other: topical, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
Range-finding test: 100 %

Main test:
Induction phase: 100 %
Challenge phase: 100 %
Route:
other: topical, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
Range-finding test: 100 %

Main test:
Induction phase: 100 %
Challenge phase: 100 %
No. of animals per dose:
- Range-finding test: 2 males and 2 females
- Main test: 10 males and 10 females for test group; 5 males and 5 females for challenge control
Details on study design:
RANGE FINDING TESTS:
- On Day 0, up to 4 closed chambers at 4 different concentrations of the test substance were prepared and a 0.3 mL dose of each concentration was applied to the clipped area of each topical range-finding animal. The chambers were applied to the clipped surface as quickly as possible. The trunk of the animal was wrapped with elastic wrap which was secured with adhesive tape 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 deionized water, followed by dry gauze to remove test substance residue, and the animals were returned to their cages.


MAIN STUDY
A. INDUCTION EXPOSURE:
- No. of exposures: Three (Day 0, 7 and 14)
- Exposure period: 6 hours
- Test groups: 0.3 ml of the test substance was placed on a 25 mm Hilltop chamber backed by adhesive tape (occlusive patch).
- Control group: None
- Site: Left side of test animals
- Frequency of applications: Once
- Duration: on Day 0, 7 and 14
- Concentrations: 100 %
- Evaluation (hr after induction): 24 and 48 hours

B. CHALLENGE EXPOSURE:
- No. of exposures: One
- Day(s) of challenge: Day 28
- Exposure period: 6 hours
- Test groups: 0.3 mL dose of the appropriate test substance was placed on a 25 mm Hilltop chamber backed by adhesive tape (occlusive patch).
- Control group: 0.3 mL dose of the appropriate test substance was placed on a 25 mm Hilltop chamber backed by adhesive tape (occlusive patch).
- Site: Right side of test animals
- Concentrations: 100 %
- Evaluation (hr after challenge): 24 and 48 hours


OTHER:
- Mortality/moribundity checks: Any unusual observations were recorded. The animals were observed for general health/mortality twice daily, once in the morning and once in the afternoon
- Dermal Observations: The test sites were graded for irritation at approximately 24 and 48 hours following chamber application (induction) or chamber removal (challenge) using the Dermal Grading System
- Body Weights: Individual body weights were obtained for all sensitization study animals on the day prior to the first induction (day -1) and for the appropriate test and challenge control animals on the day prior to challenge dosing
- Scheduled Euthanasia: Each sensitization study animal was euthanized by carbon dioxide inhalation following its final scoring interval. Gross necropsy examinations were not required for these animals
Challenge controls:
10 previously untreated (naive) challenge control guinea pigs were topically treated with 100 % test material
Positive control substance(s):
yes
Remarks:
HCA (Study no. 999.201)
Positive control results:
Study No. 999.201, an HCA positive control study, demonstrated that a valid test was performed and indicated that the test design would detect potential contact sensitizers.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
100 % induction and 100 % challenge application
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 100 % induction and 100 % challenge application . No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
100 % induction and 100 % challenge application
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100 % induction and 100 % challenge application . No with. + reactions: 0.0. Total no. in groups: 20.0.

Table 7.4.1/1: Individual challenge data

 

    Dermal Score  
  ANIMAL NO./ SEX 24h 48h
Test Group G7378/M 0 0
   G7379/M 0 0
  G7380/M 0 0
  G7381 /M ± 0
  G7382/M ± 0
  G7383/M ± ±
  G7384/M 0 0
  G7385/M 0 0
  G7386/M ± 0
  G7387/M ± 0
       
  G7505/F 0 0
  G7506/F 0 0
  G7507/F 0 0
  G7508/F ± 0
  G7509/F 0 0
  G7510/F ± 0
  G7511 IF 0 0
  G751 2/F ± 0
  G7513/F ± 0
  G7514/F ± 0
       
       
Challenge Control G7388/M  0 0
  G7389/M 0 0
  G7390/M  0 0
  G7391 /M  0 0
  G7392/M  0 0
       
  G7515/F  ± 0
  G7516/F  0 0
  G7517/F  0 0
  G7518/F  0 0
  G7519/F 0 0

For purposes of calculation, ± = 0.5;

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test substance is not sensitizing under these test conditions.
Executive summary:

Test Guidance

In a Guinea Pigs-Modified Buehler test performed according to OECD Guideline 406 and in compliance with GLP.

The dermal sensitization potential of the test material was evaluated in Hartley-derived albino guinea pigs. Ten male and ten female guinea pigs were topically treated with 100% test material, once per week, for three consecutive weeks. Following a two-week rest period, a challenge was performed whereby the 20 test and 10 previously untreated (naive) challenge control guinea pigs were topically treated with 100% test material. Challenge responses in the test animals were compared with those of the challenge control animals.

Test Group

Following challenge with 100% test material, dermal reactions in the test and challenge control animals were limited to scores of 0 to ±. Group mean dermal scores were noted to be similar in the test animals as compared to the challenge control animals.

HCA

Using a-Hexylcinnamaldehyde (HCA) as a positive control, Charles River Laboratories, Inc., Spencerville, Ohio, has completed a study during the past six months which provided historical control data for contact sensitization to this agent utilizing the test system described herein (Modified Buehler Design). Following induction at 5% w/v HCA in ethanol, challenge at levels of 2.5% and 1% w/v HCA in acetone and rechallenge at a level of 2.5% w/v HCA in acetone, a contact sensitization response was observed, thereby demonstrating the susceptibility of the test system to this sensitizing agent.

Conclusion

Number of Test Animals with Score > 1/Number Dosed

 

24h

48h

Test Group

0/20

0/20

 

Note: All challenge control animals had scores of ± and 0.

The test substance is not sensitizing under these test conditions.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September, 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in compliance with recognised guidelines.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
On one occasion, the animal room relative humidity range (41-72%) exceeded the preferred range (30-70%) during this study. This occurrence was considered to have had no adverse effect on the outcome of this study
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
yes
Remarks:
On one occasion, the animal room relative humidity range (41-72%) exceeded the preferred range (30-70%) during this study. This occurrence was considered to have had no adverse effect on the outcome of this study
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of study:
Buehler test
Justification for non-LLNA method:
See discussion of supporting LLNA results (Sanders, 2012)
Species:
guinea pig
Strain:
other: Hartley-derived albino guinea pigs.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hilltop Lab Animals, Inc., Scottdale, Pennsylvania
- Age at study initiation: Male: ca 6 weeks; female: ca 9 weeks); Main test (male: ca 6 weeks; female: ca 7 weeks)
- Weight at study initiation: Male: 310 to 353 g; female: 301 to 365 g
- Housing: The animals were housed individually in suspended stainless steel cages.
- Diet: PMI Certified Guinea Pig Chow #5026 (PMI Nutrition International), ad libitum
- Water: Municipal tap water treated by reverse osmosis was available ad libitum throughout the study
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-22 °C
- Humidity: 41-72 %
- Air changes: 10 -15 air changes per hour
- Photoperiod: 12 h light / 12 h dark

IN-LIFE DATES: From June 22, 2004 to July 22, 2004
Route:
other: topical, occlusive
Vehicle:
other: mineral oil, USP
Concentration / amount:
Induction phase: 100 % (w/v)
Challenge phase: 75 % (w/v)
Route:
other: topical, occlusive
Vehicle:
other: mineral oil, USP
Concentration / amount:
Induction phase: 100 % (w/v)
Challenge phase: 75 % (w/v)
No. of animals per dose:
Group No. of Animals
Male Female
Test 10 10
Challenge Control 5 5
HCA Test 5 5
HCA Challenge Control 5 5
Details on study design:
MAIN STUDY
A. INDUCTION EXPOSURE:
- No. of exposures: Three
- Exposure period: 6 hours (± 20 minutes)
- Test groups: 0.3 ml (100 % concentration only) of the test substance was placed on a 25 mm Hilltop chamber backed by adhesive tape (occlusive patch).
- Control group: None
- Site: Left side of test animals
- Frequency of applications: Once per week
- Duration: on Day 0, 7 and 14
- Concentrations: 100 %
- Evaluation (hr after induction): 24 and 48 hours

B. CHALLENGE EXPOSURE:
- No. of exposures: One
- Day(s) of challenge: Day 28
- Exposure period: 6 hours
- Test groups: 0.3 mL dose of the appropriate test substance was placed on a 25 mm Hilltop chamber backed by adhesive tape (occlusive patch).
- Control group: 0.3 mL dose of the appropriate test substance was placed on a 25 mm Hilltop chamber backed by adhesive tape (occlusive patch).
- Site: Right side of test animals
- Concentrations: 75 %
- Evaluation (hr after challenge): 24 and 48 hours


OTHER:
- Mortality/moribundity checks: Animals were observed for general health/mortality and moribundity twice daily, once in the morning and afternoon, throughout the study.
- Dermal Observations: Test sites were graded for irritation at approximately 24 and 48 hours after chamber application (induction) or chamber removal (challenge) using the Macroscopic Dermal Grading System.
- Body Weights: Individual body weights were obtained for all sensitization study animals on the day prior to the first induction (day -1) and for the appropriate test and challenge control animals on the day prior to challenge dosing.
- Scheduled Euthanasia: Each sensitization study animal was euthanized by carbon dioxide inhalation following each animal's final scoring interval. Gross necropsy examinations were not required for these animals.
Challenge controls:
Previously untreated (naive) challenge control (guinea pigs 5 male and 5 female) were topically treated with 75 % test material (w/w) in mineral oil.
Positive control substance(s):
yes
Remarks:
HCA (Study no. LPW00005)
Positive control results:
HCA positive control group demonstrated that a valid test was performed and indicated that the test design would detect potential contact sensitizers.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
100 % w/w induction and 75 % w/w challenge application
No. with + reactions:
2
Total no. in group:
20
Clinical observations:
dermal scores of 1 were noted
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 100 % w/w induction and 75 % w/w challenge application . No with. + reactions: 2.0. Total no. in groups: 20.0. Clinical observations: dermal scores of 1 were noted .
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
100 % w/w induction and 75 % w/w challenge application
No. with + reactions:
2
Total no. in group:
20
Clinical observations:
dermal scores of 1 were noted
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100 % w/w induction and 75 % w/w challenge application . No with. + reactions: 2.0. Total no. in groups: 20.0. Clinical observations: dermal scores of 1 were noted .

Table: Individual challenge data

Dermal Score
24h 48h
Test Male G9369 0 0
Male G9370 0 0
Male G9371 0 0
Male G9372 ± ±
Male G9373 0 0
Male G9374 0 0
Male G9375 0IT 0
Male G9376 0IT 0IT
Male G9377 0 0
Male G9378 1 1
Female G9467 0 0
Female G9468 0 0
Female G9470 0 0
Female G9472 0 0
Female G9473 ±IT 0
Female G9474 1IT 1
Female G9475 0 0
Female G9476 0 0
Female G9478 0 0
Female G9479 0 0
Challenge Control Male G9380 0IT 0
Male G9381 0IT 0
Male G9382 ±IT 0IT
Male G9383 0IT 0
Male G9384 0IT 0IT
Female G9480 ±IT ±
Female G9481 0IT 0
Female G9482 0 0
Female G9483 0 0
Female G9484 0IT 0IT

±: Slightly patchy erythema

IT: Dermal irritation outside of the test site

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test material is not sensitizing under these test conditions.
Executive summary:

Test Guidance

In a Guinea Pigs-Modified Buehler test performed according to OECD Guideline 406 and in compliance with GLP.

The dermal sensitization potential of the test material was evaluated in Hartley-derived albino guinea pigs. Ten male and ten female guinea pigs were topically treated with 100% test material, once per week, for three consecutive weeks. Following a two-week rest period, a challenge was performed whereby the 20 test and 10 previously untreated (naive) challenge control guinea pigs were topically treated with 75% w/v test material in mineral oil, USP. Challenge responses in the test animals were compared with those of the challenge control animals.

An a-Hexylcinnamaldehyde (HCA) positive control group consisting of ten HCA test and ten HCA control guinea pigs was included in this study. The HCA test animals received 5.0% w/v HCA in ethanol for induction and 2.5% and 1.0% w/v HCA in acetone for challenge.

Test Group

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

a-Hexylcinnamaldehyde

Following challenge with 2.5% w/v HCA in acetone, dermal scores of 1 were noted in 5/1 0 HCA test animals at the 24-hour scoring interval. Dermal scores of 1 were noted in 3/10 HCA test animals at the 48-hour scoring interval. Dermal reactions were limited to scores of 0 to ± in the remaining HCA test and HCA challenge control animals. Group mean dermal scores were noted to be higher in the HCA test animals as compared to the HCA challenge control animals.

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

Conclusion

Number of Test Animals with Score > 1/Number Dosed

StudyPhase

TestArticleIdentification

24Hour

48Hour

Challenge

75%w/v Test material

2/20

2/20

HCA

2.5%w/vHCA

5/10

3/10

HCA

l.0%w/vHCA

1/10

1/10

 

Notes:All challenge control animals had scores of ± and 0.

The test material is not sensitizing under these test conditions.

Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
The study was performed between 18 October 2011 and 15 November 2011.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
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:
other: CBA/Ca (CBA/CaOlaHsd)
Sex:
female
Details on test animals and environmental conditions:
Female CBA/Ca (CBA/CaOlaHsd) strain mice were used. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non pregnant. After an acclimatisation period of at least five 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 eight to twelve weeks old. The animals were individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes. Free access to mains tap water and food was allowed throughout the study. The temperature and relative humidity were controlled to remain within target ranges of 19 to 25°C and 30 to 70%, respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve 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.
Vehicle:
acetone/olive oil (4:1 v/v)
Remarks:
Please see below for Vehicle Determination Record
Concentration:
Each group was exposed to concentrations of 100% (undiluted), 50% or 25% v/v (in acetone/olive oil 4:1)
No. of animals per dose:
Groups of five mice were treated
Details on study design:
Preliminary Screening Test
Using available information regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µl of the undiluted test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local irritation was scored daily according to the scale included as Appendix 3. Any clinical signs of toxicity, if present, were also recorded. The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and on Day 6. The thickness of each ear was measured using an Oditest micrometer (Dyer, PA), pre dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 to 3 and Days 1 to 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 sensitisation.

Main Test
Test Item Administration
Groups of five mice were treated with the undiluted test item or the test item at concentrations of 50% or 25% v/v in acetone/olive oil 4:1. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. The mice were treated by daily application of 25 µl of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface 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, α Hexylcinnamaldehyde, tech., 85%, at a concentration of 25% v/v in acetone/olive oil 4:1 was applied to the dorsal surface of each ear. The thickness of each ear of each animal from the test groups and the vehicle and positive control groups was measured using an Oditest micrometer (Dyer, PA), on Days 1, 3 and 6. Any changes in the ear thickness were noted. Group mean ear thickness changes were calculated between time periods Days 1 to 3 and Days 1 to 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 sensitisation.

3H-Methyl Thymidine Administration
Five days following the first topical application of the test item, vehicle control or positive control item (Day 6) all mice were injected via the tail vein with 250 µl of phosphate buffered saline (PBS) containing 3H methyl thymidine (3HTdR:80µCi/ml, specific activity 2.0 Ci/mmol, ARC UK Ltd) giving a total of 20 µCi to each mouse.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
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 by analysis of homogeneity of variance followed by one way analysis of variance (ANOVA). In the event of a significant result from the ANOVA, pairwise comparisons were performed between control and treated groups. For homogenous datasets Dunnett’s Multiple Comparison test was used and for non homogenous datasets Dunnett’s T3 Multiple Comparison Method was 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:
A concurrent positive control test, using a group of five animals, was also performed with the known sensitiser, α Hexylcinnamaldehyde tech., 85%, at a concentration of 25% v/v in acetone/olive oil 4:1.

Identification : α-Hexylcinnamaldehyde, tech., 85%
Description : yellow liquid
Batch number: MKAA2596
Purity : 85%
Date received: 09 June 2011
Expiry date : 08 June 2012
Storage conditions: room temperature

Preparation of Positive Control Item
The positive control item was freshly prepared as a 25% v/v dilution in acetone/olive oil 4:1.

Parameter:
SI
Remarks on result:
other: Concentration Stimulation index 25% 1.23 50% 1.54 100% 3.19
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The radioactive disintegrations per minute (dpm) per lymph node and the stimulation index (SI) are given in Table 2.

Interpretation of Results

The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute from each individual animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index). The test item will be regarded as a sensitiser if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitiser".

The EC3 value was also calculated. The EC3 value is the concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation. The equation used for the calculation of EC3 is:

aEC3= c + [[(3-d)/(b-d)] x (a-c)]


a=     lowest concentration giving stimulation index >3

b =     actual stimulation index caused by ‘a’

c =     highest concentration failing to produce a stimulation index of 3

d =     actual stimulation index caused by ‘c’

Preliminary Screening Test

Clinical observations, bodyweight and mortality data are given in Table 1 and local skin irritation is given in Table 2. The ear thickness measurements and mean ear thickness changes are given in Table 3. No signs of systemic toxicity or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted. Very slight erythema was noted on both ears on Day 3. Based on this information the undiluted test item and the test item at concentrations of 50% and25% v/v in acetone/olive oil 4:1were selected for the main test.

Main Test

Estimation of the Proliferative Response of Lymph Node Cells

The radioactive disintegrations per minute per animal and the stimulation index are given in Table 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

Stimulation Index

Result

Test Item

25%v/v in
acetone/olive oil4:1

1.23

Negative

50%v/v in
acetone/olive oil4:1

1.54

Negative

100%

3.19

Positive

Positive Control Item

25%v/v in
acetone/olive oil 4:1

5.04

Positive

Additional statistical analysis was conducted by the Sponsor, following published guidance, to better understand the confidence intervals around the SI values, and the results show that the 95% confidence interval for the SI at 100% are 2.3 for the lower bound and 4.45 for the upper bound.  This additional statistical evaluation included Dunnett’s type comparisons and others following guidance from Hothorn and Vohr 2010, “Statistical Evaluation of the Local Lymph Node Assay”,Regulatory Toxicology and Pharmacology, 56:352-356.

Clinical Observations and Mortality Data

Individual clinical observations and mortality data for test and control animals are given in Table 5 and local skin irritation is given in Table 6 (attachment 1). The ear thickness measurements and mean ear thickness changes are given in Table 7 (attachment 2).

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

Very slight erythema was noted on Days 3 and 4 on the ears of all test animals treated with the undiluted test item.

Bodyweight

Individual bodyweights and bodyweight changes for test and control animals are given in Table 8 (attachment 3).

A greater than expected bodyweight loss (4g) was noted in one animal treated with the positive control item. Bodyweight changes of the remaining test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

CALCULATION OF EC3VALUE

EC3= c + [[(3-d)/(b-d)] x (a-c)]a

a

=

100

b

=

3.19

c

=

50

d

=

1.54

EC3= 50 + [[(3-1.54)/(3.19-1.54)] x (100-50)] =94

The concentration of test item expected to cause a 3 fold increase in3HTdR incorporation (EC3value) was calculated to be 94%.


a=     lowest concentration giving stimulation index >3

b =     actual stimulation index caused by ‘a’

c =     highest concentration failing to produce a stimulation index of 3

d =     actual stimulation index caused by ‘c’

Table 1: Clinical Observations, Bodyweight and Mortality Data – Preliminary Screening Test

Concentration

Animal Number

Bodyweight (g)

Day

1

2

3

4

5

6

Day 1

Day 6

Pre-Dose

Post Dose

Pre-Dose

Post Dose

Pre-Dose

Post Dose

100%

S-1

17

19

0

0

0

0

0

0

0

0

0


0 =     No signs of systemic toxicity

Table 2: Local Skin Irritation – Preliminary Screening Test

Concentration

Animal Number

Local Skin Irritation

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

pre dose

post dose

post dose

left

right

left

right

left

right

left

right

left

right

left

right

100%

S-1

0

0

0

0

1

1

0

0

0

0

0

0

Table 3: Measurement of Ear Thickness and Mean Ear Thickness Changes – Preliminary Screening Test

Concentration

Animal Number

Ear Thickness Measurement (mm)

Day 1

Day 3

Day 6

pre‑dose

post dose

left

right

left

right

left

right

100%

S-1

0.235

0.230

0.240

0.230

0.235

0.240

overall mean (mm)

0.233

0.235

0.238

overall mean
ear thickness change (%)

na

1.075

2.151


na =     Not applicable

Table 4: Individual Disintegrations per Minute and Stimulation Indices

Treatment

Animal Number

dpm/
Animal
a

Mean dpm/Animal
(Standard Deviation)

Stimulation Indexb

Result

Vehicle
acetone/olive oil 4:1

1-1

5204.44

4244.05
(±523.51)

na

na

1-2

3743.52

1-3

5238.84

1-4

3207.67

1-5

3825.80

Test Item
25% v/v in
acetone/olive oil 4:1

2-1

5339.96

5207.16
(±1484.97)

1.23

Negative

2-2

6211.19

2-3

4282.38

2-4

6960.85

2-5

3241.42

Test Item
50% v/v in
acetone/olive oil 4:1

3-1

8879.38

6526.93
(±1425.00)

1.54

Negative

3-2

5319.29

3-3

6783.64

3-4

5623.58

3-5

6028.76

Test Item
100%

4-1

18551.28

13542.12**
(±2931.06)

3.19

Positive

4-2

12705.60

4-3

10809.88

4-4

12634.20

4-5

13009.62

Positive Control Item

25% v/v in
acetone/olive oil 4:1

5-1

14837.41

21393.96*
(±6193.58)

5.04

Positive

5-2

15252.69

5-3

22598.40

5-4

28759.09

5-5

25522.23


dpm = Disintegrations per minute

a =     Total number of lymph nodes per animal is 2

b =     Stimulation Index of 3.0 or greater indicates a positive result

na =     Not applicable

** =     Significantly different from control group p<0.01

* =      Significantly different from control group p<0.05

Table 5: Individual Clinical Observations and Mortality Data

Treatment

Animal Number

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Pre-Dose

Post Dose

Pre-Dose

Post Dose

Pre-Dose

Post Dose

Vehicle
acetone/olive oil 4:1

1-1

0

0

0

0

0

0

0

0

0

1-2

0

0

0

0

0

0

0

0

0

1-3

0

0

0

0

0

0

0

0

0

1-4

0

0

0

0

0

0

0

0

0

1-5

0

0

0

0

0

0

0

0

0

Test Item
25% v/v in
acetone/olive oil4:1

2-1

0

0

0

0

0

0

0

0

0

2-2

0

0

0

0

0

0

0

0

0

2-3

0

0

0

0

0

0

0

0

0

2-4

0

0

0

0

0

0

0

0

0

2-5

0

0

0

0

0

0

0

0

0

Test Item
50% v/v in
acetone/olive oil4:1

3-1

0

0

0

0

0

0

0

0

0

3-2

0

0

0

0

0

0

0

0

0

3-3

0

0

0

0

0

0

0

0

0

3-4

0

0

0

0

0

0

0

0

0

3-5

0

0

0

0

0

0

0

0

0

Test Item
100%

4-1

0

0

0

0

0

0

0

0

0

4-2

0

0

0

0

0

0

0

0

0

4-3

0

0

0

0

0

0

0

0

0

4-4

0

0

0

0

0

0

0

0

0

4-5

0

0

0

0

0

0

0

0

0

Positive Control Item

25% v/v in
acetone/olive oil4:1

5-1

0

0

0

0

0

0

0

0

0

5-2

0

0

0

0

0

0

0

0

0

5-3

0

0

0

0

0

0

0

0

0

5-4

0

0

0

0

0

0

0

0

0

5-5

0

0

0

0

0

0

0

0

0

0= No signs of systemic toxicity

Appendix 3: Scale for Erythema

Observation

Score

No erythema

0

Very slight erythema (barely perceptible)

1

Well-defined erythema

2

Moderate to severe erythema

3

Severe erythema (beef redness) to eschar formation preventing grading of erythema

4

 


Interpretation of results:
other: The concentration of test item expected to cause a 3 fold increase in3HTdR incorporation (EC3value) was calculated to be 94%.
Conclusions:
The concentration of test item expected to cause a 3 fold increase in3HTdR incorporation (EC3value) was calculated to be 94%. Additional statistical analysis was conducted by the Sponsor, following published guidance, to better understand the confidence intervals around the SI values, and the results show that the 95% confidence interval for the SI at 100% are 2.3 for the lower bound and 4.45 for the upper bound.  This additional statistical evaluation included Dunnett’s type comparisons and others following guidance from Hothorn and Vohr 2010, “Statistical Evaluation of the Local Lymph Node Assay”,Regulatory Toxicology and Pharmacology, 56:352-356.
Executive summary:

Introduction

A study was performed to assess the skin sensitisation potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed to be compatible with the following:

OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted22 July 2010) and

Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Regulation (EC) No. 440/2008

Methods

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 100%, 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 undiluted test item or the test item as a solution in acetone/olive oil 4:1 at concentrations of 50% or 25% 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 sensitiser,α-Hexylcinnamaldehyde tech., 85%, 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

25%v/v in
acetone/olive oil4:1

1.23

Negative

50%v/v in
acetone/olive oil4:1

1.54

Negative

100%

3.19

Positive

Positive Control Item

25%v/v in
acetone/olive oil4:1

5.04

Positive

The concentration of test item expected to cause a 3 fold increase in3HTdR incorporation (EC3value) was calculated to be 94%.

Additional statistical analysis was conducted by the Sponsor, following published guidance, to better understand the confidence intervals around the SI values, and the results show that the 95% confidence interval for the SI at 100% are 2.3 for the lower bound and 4.45 for the upper bound. This additional statistical evaluation included Dunnett’s type comparisons and others following guidance from Hothorn and Vohr 2010, “Statistical Evaluation of the Local Lymph Node Assay”,Regulatory Toxicology and Pharmacology, 56:352-356.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:
  • The first key Guinea Pigs-Modified Buehler test was performed according to OECD406 (Charles River, 2004 a). 

Range-Finding Study: Exposure to the test material at concentrations of 25, 50, 75 and 100 % resulted in dermal scores of 0, ± or 1. Therefore, induction was determined to be acceptable at 100 % (as received). A second range-finding study conducted at concentrations of 10, 20, 30 and 50 % to determine an appropriate challenge level resulted in dermal scores of 0. The results of the range-finding study indicated that a test article concentration of 100% was considered appropriate for induction and challenge since 100% was the highest nonirritating concentration.

Main Study:10 male and 10 female Hartley derived albino guinea pigs were topically treated with 100% (as received) test material once per week, for 3 consecutive weeks. Following a 2-week rest period, a challenge was performed whereby the 20 test and 10 previously untreated (naive) challenge control guinea pigs were topically treated with 100% test material. Challenge responses in the test animals were compared to those of the challenge control animals. No mortality was observed during the study. Following challenge with 100 % test material dermal scores of 0 or ± were noted in test animals at the 24 -hour and 48 –hour scoring interval. Historical data on positive control (HCA) demonstrated a validity of the test.

Under these test conditions, test item is not classified as sensitizing to the skin according to the CLP Regulation (EC) No.1272/2008.

 

  • The second key Guinea Pigs-Modified Buehler test was performed according to OECD 406 (Charles River, 2004 b).

10 male and 10 female Hartley derived albino guinea pigs were topically treated with 100% (as received) test material once per week, for 3 consecutive weeks. Following a 2-week rest period, a challenge was performed whereby the 20 test and 10 previously untreated (naive) challenge control guinea pigs were topically treated with 75 % (w/w) in mineral oil, USP. Challenge responses in the test animals were compared to those of the challenge control animals. No mortality was observed during the study. Following challenge with 75 % test material in mineral oil USP, dermal scores of 1 were noted in 2/20 test animals at the 24 -hour and 48 -hour scoring intervals. Historical data on positive control (HCA) demonstrated a validity of the test.

Under these test conditions, test item is classified as sensitizing to the skin according to the CLP Regulation (EC) No.1272/2008.

 

  • The supporting study was performed according to OECD 429 and Method B42 (LLNA study, individual animal node approach) (Harlan 2012b).

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 100%, three groups each of 5 animals were treated with the undiluted test item or the test item as a solution in acetone/olive oil. The EC3 value was calculated to be 94%. Additional statistical analysis was conducted in house, following published guidance, to better understand the confidence intervals around the SI values. The results show that the 95% confidence interval for the SI at 100% are 2.3 for the lower bound and 4.4 for the upper bound (Rose A, , 2012). This additional statistical evaluation included Dunnett’s type comparisons and other robust statistical methods following guidance from Hothorn and Vohr 2010. The LLNA is reported to be overly sensitive and subject to false positive interpretations (Hans-Werner and Jurgen 2005; Basketter and Kimber 2010). In addition, at 100% concentration, some local skin irritation was observed with some animals getting post dose scores of 1. It is possible skin irritation and non-specific inflammation confounded the 100% result increasing the potential for a false positive. In addition this substance’s poor lipophilicity and very low water solubility limit dermal absorption. Low absorption potential is also supported by the dermal LD50 between 4 and 8 g/kg and the oral LD50 >10 g/kg. Furthermore, the OECD Toolbox predicts no protein binding and the CAESAR QSAR model predicts that this substance is non-sensitizing.

Discussion

The weight of evidence on the test material demonstrates that this substance is not a skin sensitizer.


Migrated from Short description of key information:
Not sensitising; study performed in line with OECD Guideline 406 and 429

Justification for selection of skin sensitisation endpoint:
The available studies are reliable and can support the development of a chemical safety assessment.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The substance does not require classification in accordance with the criteria specified in either Regulation 1272/2008 or Directive 67/546/EEC.