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Diss Factsheets

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:
Between 11 August 2004 and 16 August 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with OECD Guidelines and in compliance GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
Pamplefleur
IUPAC Name:
Pamplefleur
Test material form:
other: liquid
Details on test material:
Identification: 04-223-03
Physical Description: Clear colorless liquid
Storage Conditions: Room temperature and protected from light

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
Test System (Animals and Animal Care)
Species: Mouse
Strain/Substrain: CBA/J
Total Number: 25
Gender: Female
Age Range: 9 weeks at start of dosing; records of dates of birth for animals used in this study are retained in the Calvert archives.
Body Weight Range: 19-23 grams at the outset (Day 1) of the study.
Animal Source: Jackson Laboratories, Bar Harbor, ME 04609
Experimental History: Purpose-bred and experimentally naive at the outset of the study.
Identification: Tail marked with an indelible marker and cage card

Husbandry
Housing: Animals were group housed (5 per cage) upon receipt in compliance with National Research Council "Guide for the Care and Use of
Laboratory Animals". The room in which the animals were kept was documented in the study records. No other species were kept in the same room.
Lighting: 12 hours light/12 hours dark
Room Temperature: 21.1-25.0°C
Relative Humidity: 38-54%
Food: Animals had access to Certified Rodent Chow 7012C ad libitum. The lot number(s) and specifications of each lot used are archived at Calvert. No contaminants were known to be present in the certified diet at levels that would be expected to interfere with the results of this study. Analysis of the diet was limited to that performed by the manufacturer, records of which are maintained in the Calvert archives.
Water: Tap water was available ad libitum, via water bottles. The water is routinely analyzed for contaminants as per Calvert SOP's. No contaminants were known to be present in the water at levels that would be expected to interfere with the results of this study. Results of the water analysis are maintained in the Calvert archives.
Acclimation: Study animals were acclimated to their housing for seven days prior to their first day of dosing.

Prestudy Health Screen and Selection Criteria
All animals used in this study were assessed as to their general health by a member of the veterinary staff or other authorized personnel. During
the acclimation period, each animal was observed at least once daily for any abnormalities or for the development of infectious disease. Only animals that were determined by the veterinary staff and/or Study Director to be suitable for use were assigned to this study.

Humane Care of Animals
Treatment of animals was in accordance with the study protocol and also in accordance with Calvert SOP's which adhere to the regulations
outlined in the USDA Animal Welfare Act (9 CFR Parts 1, 2 and 3) and the conditions specified in the Guide for the Care and Use of Laboratory
Animals (ILAR publication, 1996, National Academy Press). The Calvert IACUC approved the study protocol prior to dose administration.

Study design: in vivo (LLNA)

Vehicle:
other: Diethyl phthalate/ethanol in a 3:1 ratio
Concentration:
The test article was prepared at 7.5, 15 and 30% (w/v)
No. of animals per dose:
5
Details on study design:
Dosing
Route: Topically on the dorsal surface of both ears
Frequency: Once daily for 3 consecutive days (Days 1-3).
The timing of dose administration remained consistent (± 2 hours) during the dosing phase.
Procedure: A volume of 25 ml/ear was applied using a micro pipette.

Justification for Route and Dose Levels
The dermal route was selected as this is the route required for this model of hypersensitivity.
The doses were selected by the Sponsor. The highest dose that will be used in the intended application will not exceed 20%. However, as a conservative measure, and for a higher margin of safety, 30% was chosen as the high dose.
The frequency of dosing is the convention for this type of study.

In-Life Observations and Measurements
1. Mortality/Morbidity
Frequency: Daily on Days 1 to 6.
2. Clinical Observations
Frequency: Prior to dose administration and once post-dose on Days 1 to 3. Clinical observations were performed once daily on Days 4-6. Particular
attention was given to the application sites. Any significant alterations (e.g. , erythema and edema) to the application sites, and the general appearance of the pinnae, including build up of test article, was recorded.
3. Body Weight
Frequency: Animals were weighed at the time of randomization/selection, and on Days 1 and 6.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Statistical Analysis
The mean DPM for each group was evaluated using SYSTAT version 9.01, developed by SPSS, Inc. Increases in 3H-thymidine incorporation relative to
the vehicle-treated control were derived for each group and recorded as stimulation indices (SI). The criterion for a positive response is that one or
more concentrations of a test article elicits a 3-fold or greater increase in isotope incorporation relative to the vehicle control.
Body weight data and ear thickness measurements were also evaluated.
Individual DPM values were analyzed by log transformation (base 10) of the data. The evaluation of the equality of means for the DPM, body weight and
ear thickness data was made by a one-way analysis of variance using the F distribution to assess statistical significance. If statistically significant
differences between the means are found, a Dunnett's test was used to determine the degree of significance from the control means.
If the data indicated that the test article was positive, the EC3 was calculated by 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:
Positive Control Article
Identification: Hexylcinnamaldehyde (HCA)
LoVBatch No.: 13102MO
Manufacturer: Sigma
Physical Form: Clear yellow liquid
Storage Conditions: Room temperature

The positive control, 35% (v/v) HCA, resulted in a stimulation index (SI) of 6.52. A 3-fold or greater increase in proliferative activity relative to the concurrent vehicle treated control is considered a positive response. In addition, the response with the positive control in this study was also statistically significant (p<0.001) when compared to the vehicle control group.

In vivo (LLNA)

Results
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Exposure to Pamplefleur at 7.5, 15 and 30% (w/v) resulted in stimulation indices of 1.28, 1.20 and 1.61, respectively. The positive control, 35% (v/v) HCA, resulted in a stimulation index (SI) of 6.52. A 3-fold or greater increase in proliferative activity relative to the concurrent vehicle treated control is considered a positive response. In addition, the response with the positive control in this study was also statistically significant (p<0.001) when compared to the vehicle control group.

Any other information on results incl. tables

There was no mortality and all animals appeared normal throughout the study. The application sites on the mice from the groups treated with the positive control appeared wet on Days 2-5. There were no other findings. At termination, the lymph nodes from the mice treated with the positive control were enlarged but appeared normal. The lymph nodes from the mice in the vehicle and all test article treated animals were normal in size and appearance.

Mean body weights at Day 1 and Day 6 and mean changes in body weights were evaluated

There were no statistically significant differences observed between any of the treatment groups. Therefore, the test article did not appear to cause any overt toxicity. The positive control, 35% (v/v) HCA, resulted in a stimulation index (SI) of 6.52. A 3-fold or greater increase in proliferative activity relative to the concurrent vehicle treated control is considered a positive response. In addition, the response with the positive control in this study was also statistically significant (p<0.001) when compared to the vehicle control group. Exposure to Pamplefleur at 7.5, 15 and 30% (w/v) resulted in stimulation indices of 1.28, 1.20 and 1.61, respectively. When the DPM data was log transformed to control for the variance, a statistically significant difference (p<0.05) was found between the mean DPM for the group treated with the test article at a concentration of 30% and the vehicle control. However, since the SI for this group was less than 3, it is still considered to be negative, There were no statistically significant differences found between the mean DPM of the other test article treatment groups and the control group. At termination both ears of each mouse were also measured. Statistically significant increases (p<0.001) in the ear measurements were observed in the groups treated with the positive control when compared to the vehicle group, This may be an indication that the positive control caused some irritation, A statistically significant decrease (p<0.05) in the ear measurements was noted in the group treated with 15% Pamplefleur. This is not believed to be biologically significant.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
A test material is considered to have skin sensitizing activity if, at one or more concentrations, it induces a 3-fold or greater increase in proliferative
activity relative to the concurrent vehicle treated control. Thus, a stimulation index >=3.0 is regarded as a positive response, Therefore, based on the
criteria of this study, treatment with Pamplefleur at 7.5, 15 and 30% (w/v) did not result in a stimulation index of 3 or greater and hence is not considered to have skin sensitizing activity.
Executive summary:

In an LLNA five groups of female mice were treated on the dorsal surface of both ears once per day for 3 days with 7.5%, 15% or 30% (w/v) of Pamplefleur, with the vehicle (Diethyl phthalate/ethanol in a ratio of 3:1) or with the positive control (35% Hexylcinnamaldehyde [HCAl). There was no mortality and all animals appeared normal throughout the study. At termination, the lymph nodes from the mice treated with the positive control were enlarged but appeared normal. The lymph nodes from the mice in the vehicle and all test article treated animals were normal in size and appearance. Mean body weights at Day 1 and Day 6 and mean changes in body weights were evaluated. There were no statistically significant differences observed between any of the treatment groups. Therefore, the test article did not appear to cause any overt toxicity.

Exposure to Pamplefleur at 7.5, 15 and 30% (w/v) resulted in stimulation indices of 1.28, 1.20 and 1.61, respectively. Therefore, based on the criteria of this study, treatment with Pamplefleur at 7.5, 15 and 30% (w/v) did not result in a stimulation index of 3 or greater and hence is not considered to have skin sensitizing activity.