Phosphorodithioic acid, O,O-bis(2-methylpropyl) ester, compd. with N,N-dimethylmethanamine (1:1)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 August - 17 September, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Also according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

O,O-bis(2-methylpropyl) sulfanidylphosphonothioate; trimethylazanium

EC Number:

700-910-4

Cas Number:

1354201-99-2

Molecular formula:

C11H28NO2PS2

IUPAC Name:

O,O-bis(2-methylpropyl) sulfanidylphosphonothioate; trimethylazanium

Test material form:

solid

Remarks:

pale yellow glassy solid

Specific details on test material used for the study:

- Name of test material (as cited in study report): S-10713
- Substance type: Straw color, lump solid
- Physical state: solid
- Lot/batch No.: AQ11216TZ
- Expiration date of the lot/batch: July 2013
- Stability: Stable under normal conditions
- Storage condition of test material: Room temperature; in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

- Source: Janvier, Le Genest-Saint-Isle, France
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 9-10 weeks old
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean (range 21-24g)
- Housing: Animals were group housed in labeled makrolon cages
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 – 24
- Humidity (%): 40 - 70
- Air changes (per hr): approx 15
- Photoperiod (hrs dark / hrs light): 12/12

Temporary deviations from the minimum and maximum daily mean relative humidity occurred. The study integrity was not adversely affected by the deviations.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

0, 10, 40, 80% w/w

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
Two test substance concentrations were tested: 50% and 80% concentration. The highest concentration was the maximum concentration that could technically be applied and that could be prepared homogeneously.

List of protocol deviations:
1. The clinical signs were observed slightly too early on Day 3: at 2 hours and 21 minutes after dosing instead of 3-4 hours after dosing.
Evaluation: Sufficient information was available; the slight deviation in observation time on Day 3 was considered not to have adversely affected the dose level selection.

The study integrity was not adversely affected by the deviations.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group. If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer (according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011) and the Regulation (EC) No 1272/2008 of the European Parliament
- The EC3 was determined according to Basketter et al: "A comparison of statistical approaches to the derivation of EC3 values from local lymph node assay dose responses", J Appl Toxicol 1999;19:261-266

ANIMAL ASSIGNMENT
Three groups of five animals were treated with one test substance concentration per group. One group of five animals was treated with vehicle.

TREATMENT PREPARATION AND ADMINISTRATION:
Test substance preparation: The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. Homogeneity was obtained to visually acceptable levels.
Rationale for vehicle: The vehicle was selected based on trial formulations performed at WIL Research and on test substance data supplied by the sponsor.

Induction - Days 1, 2 and 3; Excision of nodes - Day 6; Tissue processing for radioacitivity - Day 6; Radioactivity measurements - Day 7; Performed according to test guidelines.

Observations:
Mortality/Viability: Twice daily.
Body weights: On Day 1 (pre-dose) and Day 6 (prior to necropsy).
Clinical signs: Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
Irritation: Once daily on Days 1-6 (on Days 1 - 3 immediately after dosing) according to the numerical scoring system as descibed in the guideline. Furthermore, a description of all other (local) effects was recorded according to guidelines.

Necropsy: No necropsy was performed according to protocol.

Positive control substance(s):

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

Statistics:

Not performed.

Results and discussion

Positive control results:

The results of a reliability test with three concentrations of Hexylcinnamaldehyde in Acetone/Olive oil (4:1 v/v), performed not more than 6 months previously and using the same materials, animal supplier, animal strain and essential procedures was available. For both scientific and animal welfare reasons, no concurrent positive control group was added to the study. An extensive database is available with reliability checks performed each half year during at least the recent 9 years showing reproducible and consistent positive results.

In vivo (LLNA)

Results

Results 1

Parameter:

EC3

Value:

> 0 - < 10

Results 2

Parameter:

SI

Value:

3.3

Test group / Remarks:

10%

Results 3

Parameter:

SI

Value:

3.6

Test group / Remarks:

40%

Results 4

Parameter:

SI

Test group / Remarks:

80%

Remarks on result:

not determinable

Remarks:

Based on the clinical signs, this concentration was not used for interpretation of the sensitization potential

Results 5

Parameter:

other: disintegrations per minute (DPM)

Value:

2 032

Test group / Remarks:

20%

Results 6

Parameter:

other: disintegrations per minute (DPM)

Value:

2 229

Test group / Remarks:

40%

Results 7

Parameter:

other: disintegrations per minute (DPM)

Value:

6 727

Test group / Remarks:

80%

Any other information on results incl. tables

Results Pre-screen test:

Very sight erythema was noted in both animals at 50% and 80% on Days 4 and 5. No signs of systemic toxicity were noted in the animals examined. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values.

Based on these results, the highest test substance concentration selected for the main study was an 80% concentration.

 

Other results - main study:

Slight irritation of the ears was shown in all animals treated at 40% and 80% between Days 3 and/or 5. One animal at 80% showed slight irritation on Day 2, 4 and 5 and well defined erythema on Day 3. One animal at 10% showed scabs on one ear on Day 4 only. The irritation in these animals was considered not to have a toxicologically significant effect on the activity of the nodes.

 

The auricular lymph nodes of the animals treated at 10%, 40% or 80% appeared larger in size as compared to the other treated groups. The largest auricular lymph nodes were found in the highest dose group. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

     

No mortality occurred. No clinical signs of systemic toxicity were observed at 10 and 40% concentration. On Day 3 lethargy, ptosis and abnormal gait were noted in one animal at the 80% concentration. Based on the clinical signs the highest concentration of 80% cannot be used for interpretation of the sensitization potential.

Body weights and body weight gain of experimental animals at 10 and 40% remained in the same range as controls over the study period. One animal at 80% showed very slight body weight loss.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Based on the results of a LLNA skin sensitisation study, the substance is considered to be a skin sensitiser, as the SI appeared to be ≥ 3 when tested at 10% with and EC3 value < 10% .

Executive summary:

An LLNA skin sensitisation study was performed according to OECD TG 429 and following GLP prinicples, with three concentrations: 10, 40 and 80%. Slight irritation of the ears was shown in all animals treated at 40% and 80% between Days 3 and/or 5. One animal at 10% showed scabs on one ear on Day 4 only.

The auricular lymph nodes of animals in all dose groups appeared larger in size as compared to the other treated groups. No macroscopic abnormalities of the surrounding area were noted in any of the animals. No mortality or unexpected weight loss occurred at 10 and 40%. Due to observation of clinical signs in the highest dose group (80%), data from these animals were not used for interpretation of the sensitization potential.

The SI values calculated for the substance concentrations 10 and 40% were 3.3 and 3.6 respectively, the EC3 value (the estimated test substance concentration that will give an SI =3) was established to be between 0 and 10%. Based on these results, the test material has to be regarded as a skin sensitizer.