2-methyl-4-phenylpentanol

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

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.

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)

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.

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.

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.

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.

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Two animal studies (LLNA and a Buehler study) and two human patch test studies are available.

In the key LLNA performed according to OECD 429 with test concentrations of 7.5, 15 and 30% SI values of 1.28, 1.20 and 1.61 were seen. As no SI values of 3 or greater were seen at these concentrations the substance is considered non sensitisating.

A Buehler study is available. A 3% concentration in ethanol was used at each stage of induction and challenge. During the induction phase slight to confluent or moderate patchy erythema to severe erythema with/without edema were observed in three males and seven females after the third induction period at 48 hours. After first challenge three positive responses were observed at 24 and 48 hours after treatment in the test group receiving the test substance at a 3% concentration. After rechallenge three positive responses were observed in the test group at 24 hours after rechallenge. No positive responses were observed at 48 hours after rechallenge. A conclusion on the skin sensitising properties is not possible because no good distinction between skin irritation and skin sensitisation can be done because also in the induction skin irritation was seen at test substance application of 3%.

Furthermore two HRIPT tests were performed. In the first study 54 volunteers completed the full term study. Patches containing approx. 0.2 ml of a 3% solution of the test substance were prepared and used throughout the study. The test substance did produce a tendency for causing localized skin damage. In the second HRIPT study 55 volunteers completed the full term study. Scattered, transient, barely perceptible non-specific patch test responses were observed on 7/55 test volunteers during the induction phase of the study. None of these non-specific responses were considered to be irritant or allergic in nature.

Information on structural activity relationships (SAR) shows that the substance does not contain any skin sensitisation alerts. The substance does not contain electrophilic groups which may lead to sensitisation. The benzyl group and the alcohol group as such are not sufficient electrophilic for sensitisation. This is confirmed when running the OECD Toolbox protein profilers for Pamplefleur (details not shown).

Migrated from Short description of key information:
Two animal studies (LLNA and a Buehler study) and two human patch test studies are available. The LLNA was identified as the key study to cover the endpoint skin sensitisation.

Justification for selection of skin sensitisation endpoint:
For the endpoint skin sensitisation, two different studies have been conducted. In 2004 a LLNA study was conducted according to OECD guideline 429 at Calvert Laboratories. This was conducted according to the appropriate guidelines and can be considered a Klimisch reliability 1 study. The second study is an OECD 406, Buehler skin sensitisation study using 3% at the inducation and challenge. The slight redness seen in 3/19 animals are questionable because irritant effects were seen in the inducation at this concentration and in 1/4 naive animals used as controls during rechallenge. Therefore the LLNA study was identified as the key study being more sensitive compared to the Buehler study and no clear results.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Pamplefleur is listed on Annex I of the DSD 67/548/EC (Index no. 603 -092 -00 -6) as a skin sensitiser (R43) based on the information available at that timepoint (i.e. without the LLNA). Using the translation table Pamplefleur will be classified H317 (skin sensitiser 1) under the CLP Regulation EC 1272/2008.

However when taking into account the LLNA which became available in 2004 it can be concluded that based on a LLNA tested at concentrations of 7.5, 15 and 30% no skin sensitization potential was found. This result overrules the weak positive result in the Buehler assay, where 3 out of 20 animals had a positive response at a dose of 3%, which are considered irritant effects instead of sensitisation. Therefore, classification with H317 (skin sensitizer 1) is not warranted.

In addition, 2 HRIPT tests did not show sensitisation at 3%, which was the highest concentration feasible because of slight irritation seen and the substance does not contain structural features indicative for sensitisation.