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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:
08 January to 14 January 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
read-across: supporting information
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
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
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under 'Attached justification' and 'Cross-reference'.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
4-methyl-4-phenylpentan-2-ol (source chemical) is the hydrolysis product of 1,3-dimethyl-3-phenylbutyl acetate (target chemical). Hydrolysis of 3-dimethyl-3-phenylbutyl acetate is expected to occur in the human body, with enzyme activity playing an important role. As the hydrolysis is expected to occur, read across from the hydrolysis product product 4-methyl-4-phenylpentan-2-ol is considered to be appropriate.

2. SOURCE AND TARGET CHEMICAL(S)
4-methyl-4-phenylpentan-2-ol is expected to be one of the hydrolysis products of 1,3-dimethyl-3-phenylbutyl acetate, along with acetic acid (acetate ion). The target substance is the acetate of the test substance 4-methyl-4-phenylpentan-2-ol. In 1,3-dimethyl-3-phenylbutyl acetate, the hydroxyl group in the 2 position of the pentane backbone is replaced by an acetate group (O-C(CH3)=O).
These two substances have similar melting point, boiling point, density, surface tension and vapour pressure properties. Although there is a difference in water solubility, both substances are soluble to some extent. Partition coefficient values are similar, at 2.82 and 3.55 for the source and target substance respectively. Neither of the substances would be considered to be bioaccumulative. As both substances have flash points >60 ºC they are not considered to be flammable.
3. ANALOGUE APPROACH JUSTIFICATION
The read across justification is based on the fact that 4-methyl-4-phenylpentan-2-ol is expected to be one of the hydrolysis products of 1,3-dimethyl-3-phenylbutyl acetate, along with acetic acid (acetate ion). Hydrolysis is only expected to occur at high and low pH values. Initial studies have shown low hydrolysis at pH 4 (< 3 % after 120 hours) and moderate hydrolysis at pH 9 (ca 27 % after 120 hours). The test item showed a moderate hydrolysis rate (t1/2 ≤ 30 d) at pH 9 and 50 °C. At pH 9 at 20 and 30 °C only a slow hydrolysis (t1/2 > 30 d) was observed and at 20 °C the half live was > 1 year indicating no significant hydrolysis of the test item in a study according to OECD Guideline 111 and EC Method C.7 (Lange 2015).
While no data is available for the hydrolysis of 1,3-dimethyl-3-phenylbutyl acetate at gastric pH values (pH 1.2), hydrolysis data is available for two related esters, namely butyl acetate (CAS No. 123-86-4, EC no. 204-658-1) and phenylethyl acetate (CAS no. 103-45-7, EC 203-113-5) in artificial gastric fluid at 37 ºC (Longland et al, 1977). The acid hydrolysis half-life for these two esters was 318 and 300 minutes for butyl acetate and phenylethyl acetate, respectively. In the same study, hydrolysis in artificial pancreatic juice adjusted to pH 7.5 was measured to be 66 and 29.7 minutes respectively, for butyl acetate and phenylethyl acetate. In rat liver and small intestinal mucosa preparations the hydrolysis half-life for butyl acetate was 8.13 minutes and 1.8 minutes respectively. The study showed that enzyme activity was a major contributing factor in the hydrolysis of the two esters and that studies employing liver and small intestine preparations reflect more accurately the hydrolytic fate of esters in in vitro toxicological evaluations.
1,3-dimethyl-3-phenylbutyl acetate would react similarly to butyl acetate and phenylethyl acetate and therefore would be expected to hydrolyse rapidly to 4-methyl-4-phenylpentan-2-ol and acetate ion. A review of the human health data from the sodium acetate registration dossier indicates that there are no reported hazards associated with exposure to a variety of acetate ions for any of the toxicological endpoints. Consequently, it can be concluded that the acetate ion would not be expected to contribute to any of the potential toxicological endpoints required under REACH.

4. DATA MATRIX
See 'Attached justification'.
Reason / purpose for cross-reference:
read-across source
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 sensitization, as the stimulation index was below 3.0 for each concentration tested.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2003

Materials and methods

Test guidelineopen allclose all
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)

Test material

Constituent 1
Chemical structure
Reference substance name:
4-methyl-4-phenylpentan-2-ol
EC Number:
218-002-7
EC Name:
4-methyl-4-phenylpentan-2-ol
Cas Number:
2035-93-0
Molecular formula:
C12H18O
IUPAC Name:
4-methyl-4-phenylpentan-2-ol

In vivo test system

Test animals

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

Study design: in vivo (LLNA)

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

Results and discussion

In vivo (LLNA)

Results
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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.