Fatty acids, C18, unsatd., dimers, reaction products with N,N-dimethyl-1,3-propanediamine and 1,3-propanediamine

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented and reported study fully adequate for assessment. The study was conducted according to internationally accepted technical guidelines and in compliance with GLP in a recognized contract research organization.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

of 2002

Deviations:

yes

Remarks:

Contrary to OECD 429 of 2002, but in accordance with OECD 429 of 2010, scintillation vials were filled with 10 mL of scintillation fluid for 3H-counting. This did not compromise the validity of the study.

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)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS (including 3 animals for preliminary investigation + 16 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): Eight to eleven weeks.
- Weight at treatment start (1st induction): Minimum 17.4 g, maximum 22.4 g
- Housing: Individual housing in polycarbonate cages inside a barriered rodent facility.
- Bedding material: 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.

Vehicle:

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

Concentration:

Preliminary Range-finding Test:
Induction on Days 1 and 2 or 1, 2 and 3 at the following concentrations of WS400130 in vehicle (w/v):
10% (1 female), 25% (1 female), 50% (1 female).

Main Study:
Induction on Days 1, 2 and 3 at the following concentrations of WS400130 in vehicle (w/v):
0% (vehicle control, 4 females), 2.5% (4 females), 5% (4 females), 10% (4 females).

No. of animals per dose:

Preliminary Range-finding Test: 1 female animal per dose
Main Study: 4 female animals per dose

Details on study design:

TEST SUBSTANCE SOLUBILITY
A vehicle trial has demonstrated that WS400130 was unsuitable for dosing as supplied, due to its high viscosity, and that a 50 % (w/v) solution in the vehicle, acetone:olive oil (4:1 v/v), is the maximum practical concentration suitable for use in the LLNA. At this concentration a pale brown emulsion was formed.

TREATMENT PREPARATION AND ADMINISTRATION
- Preliminary Range-finding Test
Administration of test substance concentrations of 10, 25 or 50% w/v to one animal each in a preliminary range-finding test produced signs of irritation (reddening behind the ear) at the 25 and 50% dose levels. Based on this information 10% 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 following concentrations (w/v) of test substance in the vehicle:

Group 1 (Vehicle Control): 0%,
Group 2 (Low Dose): 2.5%,
Group 3 (Mid Dose): 5%,
Group 4 (High Dose): 10%

The test substance preparations were prepared on each day of administration 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.

Positive control results:

A stimulation index (SI) of 6.0 was attained in an about contemporaneous positive control assay with the same strain of mice (CBA/Ca) in response to 25% v/v hexyl cinnamic aldehyde in acetone:olive oil (4:1 v/v), thus demonstrating the reliability and sensitivity of this test system and assay to detect skin sensitization potential in this laboratory.

Key result

Parameter:

SI

Value:

4.1

Test group / Remarks:

2.5% test substance

Key result

Parameter:

SI

Value:

5.7

Test group / Remarks:

5% test substance

Key result

Parameter:

SI

Value:

8.9

Test group / Remarks:

10% test substance

There were no deaths, no signs of ill health or toxicity and no signs of local irritation over the treated area during the main study. Greasy fur was noted for all control and test animals post-dose from Day 1 and was still seen on Day 6.

Bodyweight loss, marginal in degree, was recorded for one main study animal of the vehicle control group and three of the 10% w/v dose group and stable bodyweight for one animal of the 5% w/v dose group. All other animals gained bodyweight during the main study.

Interpretation of results:

other: skin sensitisation sub-category 1A

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Stimulation indices of 4.1, 5.7 and 8.9 were attained after induction treatment with WS400130 at 2.5, 5 and 10% (w/v), respectively. From the stimulation indices attained at the two lower doses an EC3ex value* of 1.6% w/v was calculated by log-linear extrapolation.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In a local lymph node assay with mice, the stimulation index (SI) threshold of ≥ 3.0, indicating a positive sensitisation response, was attained in all treated groups. The EC3ex value was calculated to be 1.6% w/v. Therefore, according to EU classification rules the test substance was classified as “Skin sensitisation Category 1, subcategory 1A” [REGULATION (EC) 1272/2008].