Registration Dossier

Administrative data

Description of key information

A regulatory (OECD 429) Local Lymph Node Assay (LLNA) was conducted in mice with the read-across substance at three dose levels of 100%, 50% and 10%. This study was supported by a standard sensitsation test in guinea-pigs with the test substance at a concentration of 5%. No sensitizing properties were found in any of the studies.

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:
08 January to 14 January 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelman GmBH, D-33178, Borchen
- Age at study initiation: 7-12 weeks
- Weight at study initiation:
- Housing: barrier maintained in an air conditioned room.
- Diet (e.g. ad libitum):ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: described as "adequate"

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22+/-3
- Humidity (%): 55+/-10%
- Air changes (per hr): at least 10/hr
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:
other: Acetone/Olive oil (3+1 v/v)
Concentration:
10, 50, and 100%
No. of animals per dose:
5 female mice per dose group
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: Not determined in this study
- Irritation: Not determined in this study
- Systemic toxicity: Not determined in this study
- Ear thickness measurements: Not determined in this study
- Erythema scores: Not determined in this study

MAIN STUDY
- Topical Application
Each mouse was treated by topical application of 25ul of the selected solution to the entire dorsal surface of each ear.
Topical applications were performed once daily over three consecutive days.
- Administration of 3H-methyl thymidine
Five days after the first topical application treatment all mice were dosed with 20tCi 3H-methyl thymidine by intravenous injection (tail vein) of 250ul of 3H-methyl thymidine, diluted to a working concentration of 80 uCi/ml
- Preparation of cell Suspension
Approximately 5 hours after 3H-methyl thymidine-injection all mice were sacrificed. The draining „auricular lymph nodes" were excised, weighed individually, pooled for each animal (2 lymph nodes per animal) and collected in PBS. A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamid gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated twice. After the final wash each pellet was resuspended in approx. 3 ml 5% TCA at approx. 4°C overnight for precipitation of macromolecules. Each precipitate was recovered by centrifugation, resuspended in 1 ml 5% TCA and transfered into scintillation vials.
- Determination of incorporated 3H-methyl thymidine
The 3H-methyl thymidine incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually for each animal.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) and as the ratio of 3H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for control group animals (STIMULATION INDEX). Before DPM/NODE values were determined, background values were substracted.
EC3 values, calculated concentrations which induce stimulation indices of three, are determined by linear interpolation {EC3 = c + [(3-d) / (b-d)] x (ac)}, between two points of the stimulation indices axis, one above (a,b) and one below (c,d) the stimulation index of three. If all measured points are above or below the stimulation index of three, no EC3 value can be stated. A substance is regarded as a 'sensitizer' in the LLNA if at least one concentration of the test item results in a 3 fold or greater increase in 3Hmethyl thymidine - incorporation into lymph node cells of the lymph nodes of the test group animals, relative to that recorded for the lymph nodes of control group animals (Stimulation Index equal to or greater than 3.0.).

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was assessed in three concentrations of 100 %, 50 % and 10 % (w/w) respectively.
The concentrations were prepared immediately prior to each dosing and dosing was via application of 25 ul volumes to the dorsal surface of each ear.
Positive control substance(s):
not specified
Statistics:
Not specified
Key result
Parameter:
SI
Value:
>= 0.8 - <= 1.6
Test group / Remarks:
Low dose level group (10%)to high dose level group (100%)
Remarks on result:
other: EC3 value could not be stated
Remarks:
All values were below the stimulation index of 3.0
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION:
SI 1.6 at 100% concentration
SI 1.4 at 50 % concentration
SI 0.8 at 10% concentration

EC3 CALCULATION:
Could not be determined by linear extrapolation since all measured points were below the Stimulation Index of 3.0

CLINICAL OBSERVATIONS:
Not specified

BODY WEIGHTS:
No treatment related changes

Mean Stimulation index (SI)

Control = 1.0 (baseline)

100% = 1.6 (SD 0.6)

50% = 1.4 (SD 0.6)

10% = 0.8 (SD 0.3)

Mean lymph node weights of test groups

Control : 2.5mg

High dose : 3.7mg

Mid dose : 3.4mg

Low dose : 3.0 mg

Interpretation of results:
GHS criteria not met
Conclusions:
The stimulation index was below 3.0 for all concentrations tested, and so the read-across substance, 4-methyl-4-phenylpentan-2-ol, is not identified as a skin sensitiser.

The proliferative response of lymph node cells was calculated as the ratio of 3H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for control group animals. A stimulation index, ratio of test item / negative control, was calculated for each concentration.

The stimulation index at a concentration of 100 % was 1.6
The stimulation index at a concentration of 50 % was 1.4
The stimulation index at a concentration of 10 % was 0.8

The EC3 value (derived by linear interpolation) could not be determined as all measured points were below the stimulation index of three.
Executive summary:

The read-across substance, 4-methyl-4-phenylpentan-2-ol, was assayed at three concentrations of 100 %, 50 % and 10 % (w/w) respectively. The vehicle was A00 (3+1 (v/v) Acetone/Olive Oil). Each mouse was treated by topical application with the prepared test item to the entire dorsal surface of each ear once daily over three consecutive days. Five days after the first topical application all mice were injected intravenously with 3H-methyl thymidine. Approximately 5 hours after 3H-methyl thymidine-injection all mice were sacrificed and the draining "auricular lymph nodes" were excised and weighed individually, in order to prepare a single cell suspension of the lymph node cells for each animal. The 3H-methyl thymidine-incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Determination of radioactivity was performed individually for each animal. The proliferative response of lymph node cells was calculated as the ratio of 3H-methyl thymidine-incorporation into lymph node cells of test group animals relative to that recorded for control group animals. A stimulation index, ratio of test item / negative control, was calculated for each concentration.

The stimulation index at a concentration of 100 % was 1.6

The stimulation index at a concentration of 50 % was 1.4

The stimulation index at a concentration of 10 % was 0.8

The EC3 value (derived by linear interpolation) could not be determined as all measured points were below the stimulation index of three.

Conclusions

Considering the reported data of this sensitization test it can be stated that the test item causes no reactions identified as sensitization, as the stimulation index was below 3.0 for each concentration tested.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The local lymph node assay conducted with the structurally related alcohol, 4-methyl-4-phenylpentan-2-ol, produced no evidence of a sensitising response in mice.

The test item was assayed at three concentrations of 100 %, 50 % and 10 % (w/w) respectively. The vehicle was A00 (3+1 (v/v) Acetone/Olive Oil). Each mouse was treated by topical application with the prepared test item to the entire dorsal surface of each ear once daily over three consecutive days. Five days after the first topical application all mice were injected intravenously with 3H-methyl thymidine. Approximately 5 hours after 3H-methyl thymidine-injection all mice were sacrificed and the draining "auricular lymph nodes" were excised and weighed individually, in order to prepare a single cell suspension of the lymph node cells for each animal. The 3H-methyl thymidine-incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Determination of radioactivity was performed individually for each animal. The proliferative response of lymph node cells was calculated as the ratio of 3H-methyl thymidine-incorporation into lymph node cells of test group animals relative to that recorded for control group animals. A stimulation index, ratio of test item / negative control, was calculated for each concentration.

The stimulation index at a concentration of 100 % was 1.6

The stimulation index at a concentration of 50 % was 1.4

The stimulation index at a concentration of 10 % was 0.8

The EC3 value (derived by linear interpolation) could not be determined as all measured points were below the stimulation index of three. Considering the reported data of this sensitization test it can be stated that the test item causes no reactions identified as sensitization, as the stimulation index was below 3.0 for each concentration tested.

Patch testing with human volunteers with the same substance also elicited no evidence of sensitisation in 56 volunteers at 10% in DEP:EtOH(3:1).

In the Buehler assay, test material was applied under a semi occlusive patch three times per week for 3 weeks. Challenge dose applied 14 days later and evaluated for skin reactions after a further 24 and 48 hours. A 5% formulation of 1,3-dimethyl-3 phenylbutyl acetate in vaseline resulted in no skin reactions in 20 guinea pigs, 24 and 48 hours after a challenge dose, supporting the negative results obtained for the read-across substance.


Short description of key information:
When tested in accordance with OECD test guideline 429 the following stimulation indices (SI) were recorded at the 3 concentrations tested in mice:
At 100 % the Stimulation Index (SI) was 1.6 ; at 50 % the SI was 1.4 ; at 10 % the SI was 0.8. The EC3 value (derived by linear interpolation) could not be determined as all measured points were below the stimulation index of three. It is concluded that the read-across substance, 4-methyl-4-phenylpentan-2-ol, causes no reactions identified as sensitization, as the stimulation index was below 3.0 for each concentration tested.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The local lymph node assay conducted with the hydrolysis product, 4-methyl-4-phenylpentan-2-ol, produced no evidence of a sensitising response in mice, even at 100% concentration. The read-across substance is not classified as a skin sensitiser because the stimulation index at all concentrations tested, including 100% concentration, was below 3.

Patch testing with human volunteers with the same substance also elicited no evidence of sensitisation in 56 volunteers.

These data were supported by a Buehler assay, a 5% formulation of 1,3-dimethyl-3 phenylbutyl acetate in vaseline, which resulted in no skin reactions in 20 guinea pigs.

These data with the read-across substance, together with a supporting negative result in a guinea-pig Buehler assay with 1,3-dimethyl-3-phenylbutyl acetate, indicates that the latter substance should not be classified as a skin sensitiser.