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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:
26 July 2011 - 05 March 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
22 July 2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Commission Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).
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:
Tetraesters of pentaerythritol with 2-methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid
EC Number:
813-120-0
Cas Number:
1262967-45-2
Molecular formula:
C21H36O8 C26H46O8 C31H56O8 C36H66O8 C41H76O8
IUPAC Name:
Tetraesters of pentaerythritol with 2-methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid
Test material form:
liquid
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark and under nitrogen.
- Stability under test conditions: Assumed stable.
- Solubility and stability of the test substance in the solvent/vehicle: Diluted in acetone:olive oil (4:1 v/v)
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material): Diluted in acetone:olive oil (4:1 v/v).

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable): N/A

OTHER SPECIFICS: No

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Ltd
- Females (if applicable) nulliparous and non-pregnant: Not reported
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15.8-19.2 g
- Housing: Housed two animals per cage, in solid bottomed polycarbonate cages with a stainless steel mesh lid. Each cage contained a quantity of autoclaved wood flake bedding, additionally Nestlets and a plastic shelter were included for environmental enrichment.
- Diet (e.g. ad libitum):
- Water (e.g. ad libitum):
- Acclimation period:
- Indication of any skin lesions:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23 ºC
- Humidity (%): 40-70 %
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12:12 (light:dark)
- IN-LIFE DATES: From: 08 December 2011 To: 14 December 2011

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25, 50 and 100 % v/v
No. of animals per dose:
4
Details on study design:
PRE-SCREEN TESTS: No
- Compound solubility: A vehicle trial performed withthe test item showed that it formed a clear pale yellow liquid at 25 and 50% v/v and the test material ‘as supplied’ was a clear colourless liquid which were all satisfactory for dose administration.
- Irritation: The maximum practical concentration for dermal administration (using a micropipette) of the test substance was ‘as supplied’. Available toxicity and irritancy information indicated this would be a suitable high concentration for the study therefore no preliminary investigations were performed.
- Systemic toxicity: All animals were observed daily for signs of ill health or toxicity.
- Ear thickness measurements: The ears were examined for signs of irritation but not measured.
- Erythema scores: Not evaluated

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: Results for each treatment group were expressed as the Stimulation Index (SI). This was derived by dividing the mean dpm/mouse for each formulated treated group and the positive control group by the mean dpm/mouse in the vehicle control group and the test group dosed with the test material as supplied was derived by dividing the mean dpm/mouse with the mean value of the sham dosed control group. If the SI is 3 or more, the test substance is regarded as a skin sensitizer.

TREATMENT PREPARATION AND ADMINISTRATION:

The maximum practical concentration for dermal administration (using a micropipette) of the test substance was ‘as supplied’. Available toxicity and irritancy information indicated this would be a suitable high concentration for the study therefore no preliminary investigations were performed. The low and intermediate concentrations were selected from the concentration series given in regulatory guidelines and the concentrations administered were 25 and 50 % v/v in acetone:olive oil 4:1 and ‘as supplied’.

Groups of four mice were treated at one of three concentrations of the test substance. The mice were treated by daily application of 25 µL of the appropriate concentration of the test substance to the dorsal surface of each ear for three consecutive days (Days 1-3). The test substance was applied to the dorsal surface of each ear using an automatic micropipette and was spread over the entire dorsal surface of the ear using the tip of the pipette. Further groups of four mice received a sham dose, the vehicle alone or the positive control substance in the same manner. The sham dosed animals were used to calculate the result of animals dosed with the test material as supplied (Group 5).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The SI for the positive control substance hexyl cinnamic aldehyde (HCA) was 6.9 which demonstrates the validity of this study.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.5
Test group / Remarks:
25 % v/v test group. Mean of 4 individuals.
Key result
Parameter:
SI
Value:
1.8
Test group / Remarks:
50 % w/w test group. Mean of 4 individuals.
Key result
Parameter:
SI
Value:
1.6
Test group / Remarks:
100 % v/v test group i.e. as supplied. Mean of 4 individuals.

Any other information on results incl. tables

Table1       Group dpm/node and Stimulation Index

 

Concentration

dpm

Number of lymph nodes per group

dpm/node

Stimulation index*

Result

+ = positive

- = negative

Control

5169.85

8.0

646.23

n/a

n/a

Solvent controla

6184.15

8.0

733.02

n/a

n/a

25 % v/v

9222.45

8.0

1152.81

1.5

-

50 % v/v

10826.05

8.0

1353.26

1.8

-

Undiluted

8239.25

8.0

1029.91

1.6

-

HCAb25 % v/v

42625.75

8.0

5328.22

6.9

+

aacetone:olive oil (4:1 v/v)

bhexyl cinnamic aldehyde (positive control)

* Stimulation Index ≥3 indicates a positive result

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
As a SI of 3 or more was not recorded for any of the concentrations tested, Tetraesters of pentaerythritol with 2-methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid is not considered to have the potential to cause skin sensitization.
Executive summary:

OECD 429 (2012) - In a dermal sensitization study with undiluted Tetraesters of pentaerythritol with 2-methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid and Tetraesters of pentaerythritol with 2- methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid diluted in acetone: olive oil (4:1 v/v) in young female adult mice (CBA/CaCrl) were tested using the Local Lymph Node Assay (LLNA)

 

Following review of available toxicity and irritancy data a concentration of 100 % was selected as the highest dose investigated in the main test. Three groups, each of five animals, were treated with 25 μL of the test item (undiluted and formulated in the vehicle) at concentrations of 100, 50 and 25 % v/v. A control group (no solution applied) and a solvent control group (acetone:olive oil, 4:1 v/v only), each containing 5 individuals, were also treated. A concurrent positive control test, using a group of five animals, was also performed with the known sensitiser, α-Hexylcinnamaldehyde at a concentration of 25 % v/v in the vehicle.

 

There were no signs of systemic toxicity or dermal reactions through the test period. Bodyweight increases were within normal range. The Stimulation Index, expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group, was determined for each treatment group.

 

The test item did not elicit a Stimulation Index ≥ 3 in any of the test item treated groups. SI values of 1.6, 1.8 and 1.5 were calculated for the 100, 50 and 25 % v/v test groups, respectively. The test item was therefore considered not to be a sensitiser under the conditions of the test.

 

The positive control group elicited an SI value of 6.9, indicating a positive skin sensitisation response and demonstrating the validity of the test.

 

In this study, Tetraesters of pentaerythritol with 2-methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid was not a dermal sensitiser.

 

According to the criteria of European Commission Regulation 1272/2008, Reaction product of 3,5,5-trimethyl-hexanoic acid and 2-metheylpropanoic acid and pentaerythritol does not require classification or labelling, with regards to skin sensitisation.