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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-06-26 to 2014-08-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
22 July 2010
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
March 2003
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2004/73/EC L152
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Appearance: clear liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: PFW120356
- Expiration date of the lot/batch: Not declared
- Purity: 98.70%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (20-23°C)




In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, Frederick, MD
- Age at study initiation:
Preliminary Irritation Screen - 7-8 weeks
Main Study - 9 weeks
- Weight at study initiation:
Preliminary Irritation Screen - 18-25 grams
Main Study - 20 to 24 grams
- Housing: Animals were group-housed 5 per cage upon receipt and during study in compliance with National Research Council "Guide for the Care and Use of Laboratory Animals".
- Diet (e.g. ad libitum): free access to Harlan Teklad Rodent Diet
- Water (e.g. ad libitum): ad libitum, via water bottles.
- Acclimation period: at least 8 days prior to their first day of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
AeroNeg 1 (9- 22 Jul 2014): 24-27°C
AeroNeg 2 (22 Jul - 11 Aug 2014): 19-27°C
- Humidity (%):
AeroNeg 1 (9-22 Jul 2014): 24-98%
AeroNeg 2 (22 Jul - 11 Aug 2014): 24-97%
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Preliminary study (irritation screen): 25%, 50% and 100% (v/v)
Second irritation screen: 2.5, 5 and 10% (v/v)
Main test: 1, 2.5 and 5% v/v
No. of animals per dose:
5 females
Details on study design:
PRE-SCREEN TESTS:
Prior to the main study, an irritation screen was performed using the substance at 25, 50 and 100% (v/v). Each concentration was applied to the dorsal surface of both ears of 2 mice once per day for 3 consecutive days. Mice were observed daily for any clinical signs of systemic toxicity. Body weights were recorded pre-test and on Day 6. Ears of each mouse were observed for erythema and scored daily using the Draize scoring system. Ear thickness measurements were taken on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose), and Day 6. Because of observed toxicity and excessive local irritation, a second irritation screen was run using the substance at 2.5, 5 and 10% (v/v).
- Irritation: excessive local irritation in the first irritation screen, therefore a second irritation screen was run using the substance at lower concentrations
- Systemic toxicity: observed in first irritation screen, therefore a second irritation screen was run using the substance at lower concentrations; in the second irritation screen, all animals were clinically normal throughout the study.
- Ear thickness measurements: The appearance of the ears, which were the application sites, appeared hard and reduced in size on Day 6 in the group treated with 25%. The ears in the group treated with 25% could not be measured on Day 6. Therefore, a second irritation screen was run with the substance at concentrations of 2.5, 5 and 10% (v/v). In the second irritation screen, the ears in the group treated with the test article at 10% had a hard feel to them on Day 6. The ear measurements indicated little or no edema in the ears of the mice treated with the test article at 2.5 and 5%, but treatment with 10% caused a 22.7% increase in ear thickness on day 3 and a 67.5% increase on Day 6, indicating the presence of edema.
- Erythema scores: first irritation screen: ; second irritation screen: There was very slight erythema on the ears of the mice treated with the test article at 2.5 and 5%. Very slight to well-defined erythema was seen on the ears of the mice treated with 10%.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: The criterion for a positive response was that one or more concentrations of a test article elicit a 3-fold or greater increase in isotope incorporation relative to the vehicle control.

TREATMENT PREPARATION AND ADMINISTRATION: The test article formulations will be prepared on each day of dosing by dissolving the appropriate amount of test article in the vehicle using a volumetric flask for each concentration. The positive control will be prepared as a 35% solution in the vehicle.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean DPM and standard error (sem) for each group was determined.
Increases in ³H-thymidine incorporation relative to the vehicle-treated control was derived for each group and recorded as stimulation indeces (SI).
Body weight, body weight changes and ear measurements were also evaluated using SYSTAT version 9.01, developed by SPSS, Inc. The evaluation of equality of means for body weight data and ear measurements was made by a one-way analysis of variance using the F distribution to assess statistical significance. If statistically significant differences between the means were found, a Dunnett's test was used to determine the degree of significance from the control means.
Individual DPM values were analyzed by log transformation (base 10) of the data. The evaluation of the equality of means for the log DPM was made by a one-way analysis of variance using the F distribution to assess statistical significance. If statistically differences between the means were found, a Dunnett's test was used to determine the degree of significance from the control means.
The percent change in ear thickness from Day 1 was determined for both ears of each mouse on Days 3 and 6. The mean percent change for each group was calculated.
The EC3 was calculated using the formula:
EC3 = c+[(3-d)/(b-d)](a-c)
where the data points lying immediately above and below the SI value of 3 have the co-ordinates (a,b) and (c,d) respectively.

Results and discussion

Positive control results:
The sensitivity and reliability of the experimental technique employed was assessed by use of a substance which is known to have skin sensitisation properties in CBA/J mice. The validation- /positive control study was performed with alpha-Hexylcinnamaldehyde in acetone:olive oil, 4:1 (v/v).
The positive control, 35% (v/v) HCA, resulted in a stimulation index (SI) of 10.5. A 3-fold or greater increase in proliferative activity relative to the concurrent vehicle control is considered a positive response. In addition, the response with HCA was also statistically significant (p<0.001) when the log DPM for this group was compared to the vehicle group. Thus, the sensitivity of the test system was demonstrated.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
2.2
Test group / Remarks:
1%
Parameter:
SI
Value:
6.9
Test group / Remarks:
2.5%
Parameter:
SI
Value:
5.1
Test group / Remarks:
5%
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
DPM (mean ± sem):
Vehicle: 1207.51 ± 55.0
1% v/v: 2704.66 ± 364.2
2.5% v/v: 8328.50 ± 55.6
5% v/v: 6204.23 ± 549.3

DETAILS ON STIMULATION INDEX CALCULATION
Concentrations of 1.0, 2.5, and 5% resulted in SI of 2.2, 6.9, and 5.1, respectively, when compared to the vehicle. There was a statistically significant difference between the groups treated with the test article at 1 % (p<0.05) and 2.5% and 5% (p < 0.001) when compared to the vehicle control group. These differences were biologically relevant since the SIs were> 3.0

EC3 CALCULATION
EC3 = 1.26%

CLINICAL OBSERVATIONS:
In the main study, there was no mortality and all animals appeared clinically normal. The ears of the mice treated with HCA appeared wet on Days 2-6. The ears of the mice treated with JEFFCAT LE-30 at 5% appeared wet on Days 2-5 while the ears of the mice treated with JEFF CAT LE-30 at 1 % or 2.5% appeared wet on days 3-5. There were no other findings.

DERMAL IRRITATION
There was no visible erythema at the application site of any of the animals in the substance 1% or 2.5% group or the vehicle treated groups. All animals treated with the substance at 5% or HCA had very slight erythema on Days 3-6.
There was also no edema at the application site of any of the mice treated with the test article at 1% or vehicle. Mean increases in ear thickness for groups treated with the sustance at 1% or vehicle were < 25% on Days 3 and 6. Edema was observed in the ears of the mice treated with the substance at 2.5 and 5% and HCA. Mean increases in ear thicness for the substance at 2.5% treated group were 12.4% on Day 3 and 26.9% on Day 6. These increases in ear thickness for the substance at 5% treated group were 31.5% on Day 3 and 38.0% on Day 6 which were statistically significant (p<0.01). Mean increases in ear thickness for the HCA treated group were 41.4% on Day 3 and 58.6% on Day 6 which were statistically significant (p<0.001).

BODY WEIGHTS
In the initial irritation screen there were no significant changes in body weights in any of the animals.
In the second irritation screen there were no significant changes in body weights in any of the animals
In the main study there were no statistically significant differences. Therefore, the test article did not appear to cause any overt toxicity at 1, 2.5 or 5%.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
Based on the criteria of the study, treatment with the substance at 2.5, and 5% did result in a stimulation index greater than 3.0 and hence the substance was considered to be a potential sensitiser and classified as skin sensitizer category 1.