Condensation products of triethanolamine with addition products of fatty acids, C18 (unsaturated) alkyl with maleic anhydride

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

of 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

of 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS (including 2 animals for preliminary investigation + 20 main study animals).
- Mouse (healthy females only), strain: CBA/Ca with appropriate range of bodyweight at study start.
- Source: Harlan UK.
- Age at treatment start (1st induction): 8 to 12 weeks.
- Weight at treatment start (1st induction): Minimum 16.8 g, maximum 19.0 g
- Housing: 2 animals per cage in polycarbonate cages inside a barriered rodent facility.
- Bedding material: Autoclaved woodflake bedding.
- Cage enrichment: Nestlets and plastic shelter
- Diet (ad libitum): Standard rodent diet (Rat and Mouse No. 1 Maintenance Diet) containing no added antibiotic,
chemotherapeutic or prophylactic agent.
- Water (ad libitum): Tap water
- Acclimation period: At least 5 days before treatment start under laboratory conditions.

Analysis of the batch of diet used and water did not provide evidence of contamination that might have prejudiced the study.

ENVIRONMENTAL CONDITIONS

Air conditioned room kept at positve pressure without re-circulation of the filtered fresh air supplied to the room.
Controlled environment, environmental conditions were set at:
- Air changes per hour in the animal room: ca. 15
- Temperature (°C): 21 ± 2°C
- Relative Humidity (%): 40 to 70%
- Photoperiod (artificial lighting): 12 hrs day / 12 hrs night
There was no mentioning of any deviations from these ranges, which compromised the integrity or validity of the study.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

Induction administrations on Days 1, 2 and 3 at the following concentrations of WS400123 in vehicle (% w/v):
- Pre-screen Test (2 females): 100
- Main Study (4 females/dose level): 25, 50, 100

Induction administration at the following concentration of Hexyl cinnamic aldehyde (positive control) in vehicle (% v/v):
- Main Study (4 females/dose level): 25

No. of animals per dose:

Pre-screen Test: 2 female animals (1 dose group)
Main Study: 4 female animals per dose level
Positive Control: 4 female animals (1 dose group)

Details on study design:

TEST SUBSTANCE SOLUBILITY
A vehicle trial has demonstrated that WS400123 was unsuitable for dosing as supplied, due to its high viscosity, and that it was imiscible with the vehicle, acetone:olive oil (4:1 v/v). At 100% w/v in dimethylformamide (DMF), WS400123 formed a pale yellow/orange liquid suitable for dose administration.

TREATMENT PREPARATION AND ADMINISTRATION
- Pre-screen Test
Administration of WS400123 at 100% w/v did not produce death, signs of ill health, toxicity, relevant increases in ear thickness or local irritation over the treated area. Greasy fur on the head following each dosing occasion was related to unoccluded dermal administration of a liquid and not attributable to the test substance itself. A clear sticky residue on the ears on Days 2 to 5 was considered to represent residual test substance, slight hair loss noted on Day 6 probably resulted from grooming of the animals to remove the residue. Based on this information 100% w/v was selected as high dose level for the main study.

- Main Study
On three consecutive days, groups of 4 female mice were treated by topical application to the entire dorsal surface of both ears with 25 μL/ear/day at the test or positive control substance concentrations listed above in the field LLNA – Concentration. All formulations were prepared on each day of administrationusing DMF as the vehicle and dosed within 4 hours of preparation.

OBSERVATIONS, MEASUREMENTS AND ENDPOINTS (POOLED TREATMENT GROUP APPROACH) DURING THE MAIN STUDY

All animals were checked daily for signs of ill health or toxicity. The ears were also examined daily for signs of irritation. In addition, bodyweights were recorded on Days 1 (prior to treatment) and 6 (three days after the third induction administration). On Day 6, all animals were injected into the tail vein 3H-methyl thymidine diluted in phosphate buffered saline at a nominal dose of 20 µCi per mouse, in order to measure lymphocyte proliferation by radioactive labelling. Five hours afterwards the draining (auricular) lymph nodes were excised and pooled for each experimental group. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed on Day 7. Radioactivity was expressed as the number of radioactive disintegrations per minute (dpm). The ratio of the proliferation (reflected by the magnitude of measured dpm/node) in treated groups to that in the vehicle control group, termed the stimulation index (SI) or test/control ratio, was subsequently calculated for each group.

Criteria Used to Consider a Positive Response:

The test substance is regarded as a sensitizer if at least one concentration of the test substance produces a stimulation index (SI) ≥ 3.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Data were not statistically analysed.

Results and discussion

Positive control results:

A stimulation index (SI) of 15.2 was attained in a concomittant positive control assay with the same strain of mice (CBA/Ca) in response to 25% v/v hexyl cinnamic aldehyde in dimethylformamide (DMF), thus demonstrating the reliability and sensitivity of this test system and assay to detect skin sensitization potential in this laboratory.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

3.9

Test group / Remarks:

25%

Results 2

Key result

Parameter:

SI

Value:

6.4

Test group / Remarks:

50%

Results 3

Key result

Parameter:

SI

Value:

10.1

Test group / Remarks:

100%

Any other information on results incl. tables

There were no deaths, no signs of ill health or toxicity and no signs of local irritation over the treated area during the pre-screen and main tests. Ear thickness was also unaffected during the pre-screen test. Wet/greasy fur on the head following each dosing occasion and in some animals persisting until Day 5 was not attributable to the test substance itself but was considered to be related to unoccluded dermal administration of a liquid formulation/vehicle. A clear sticky residue on the ears on Days 2 to 5 in all animals dosed with 100% w/v was considered to represent residual test substance, slight hair loss noted on Day 6 in the pre-screen animals probably resulted from grooming of the animals to remove the residue.

Bodyweight gain was unaffected by treatment with the test substance.

Applicant's summary and conclusion

Interpretation of results:

other: sensitising