Dibutyl [(dipropoxyphosphinothioyl)thio]succinate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 January 2018 to 03 October 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Dibutyl 2-[(dipropoxyphosphorothioyl)sulfanyl]succinate

Cas Number:

68413-47-8

Molecular formula:

C18H35O6PS2

IUPAC Name:

Dibutyl 2-[(dipropoxyphosphorothioyl)sulfanyl]succinate

Test material form:

liquid

Specific details on test material used for the study:

- Description: Light yellow, clear liquid
- Storage: Room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., Inc., Horst, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: [yes/no/not specified]: Yes
- Age at study initiation: 8 to 12 weeks old.
- Weight at study initiation: 15 to 23 g
- Housing: Suspended solid floor polypropylene cages with stainless steel mesh lids and furnished with softwood flake bedding (Datesand Ltd., Cheshire, UK).
- Diet: Free access to tap water and food (2014C Teklad Global Certified Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK)
- Water: See above

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25C
- Humidity (%): 30 to 70%
- Air changes (per hr): At least fifteen changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours continuous light and 12 hours darkness

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

10%, 25%, and 50%

No. of animals per dose:

Preliminary: 2 animals per dose
Main test: 5 animals per dose

Details on study design:

Preliminary Screening Test
A preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µL of the undiluted test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observedfor local skin irritation, ear thickness, clinical signs of toxicity, and body weight.
Main Test
Test Item Administration
Groups of five mice were treated with the test item at concentrations of 50%, 25% or 10% v/v in acetone/olive oil 4:1. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. A further group of five mice received the vehicle alone in the same manner.
3H-Methyl Thymidine Administration
Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 3H methyl thymidine (3HTdR: 80 µCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 µCi to each mouse.
Observations
Clinical Observations: All animals were observed twice daily (pre- and post-dose) on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6 for any signs of toxicity or ill health.
Body Weights: The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).
Sample Preparation: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. For each individual animal of each group the draining auricular lymph nodes were excised and processed.
Preparation of Single Cell Suspension: A single cell suspension of the lymph node cells for each individual animal was prepared by gentle mechanical disaggregation through a 200 mesh stainless steel gauze. The lymph node cells were rinsed with PBS into a petri dish labeled with the study number and dose concentration. The lymph node cell suspension was transferred to a centrifuge tube and cells were pelleted at 1400 rpm (approximately 190 g) for 10 minutes. The pellet was re suspended in PBS and re pelleted. The pellet was re suspended in 5% Trichloroacetic acid (TCA).
Determination of 3HTdR Incorporation: After approximately 18 hours incubation at approximately 4 C, the precipitates were recovered by centrifugation, re suspended in TCA and transferred to scintillation fluid. 3HTdR incorporation was measured by  scintillation counting.
Data Evaluation
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).
The test item was regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation was classified as a "non sensitizer".

Positive control substance(s):

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

Statistics:

Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships. Data was first assessed for suitability by analysis of normality and homogeneity of variance. If the assumptions that the data are both normally distributed and has homogeneity of variances, then parametric one way analysis of variance (ANOVA) and Dunnett’s multiple comparison procedure were used to determine statistical significance. If the assumptions were not met, non parametric Kruskal Wallis Rank Sum and Mann Whitney U test procedures were used.

Results and discussion

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

3.26

Test group / Remarks:

10 v/v in acetone/ olive oil 4:1

Results 2

Key result

Parameter:

SI

Value:

8.35

Test group / Remarks:

25 v/v in acetone/ olive oil 4:1

Results 3

Key result

Parameter:

SI

Value:

8.71

Test group / Remarks:

50 v/v in acetone/ olive oil 4:1

Any other information on results incl. tables

Preliminary Screening Test Results:

Following preliminary screening tests in mice treated with the undiluted test item and the test item at a concentration of 50% v/v in acetone/olive oil 4:1, in which the undiluted test item caused a greater than 25% increase in mean ear thickness but no signs of systemic toxicity. No signs of systemic toxicity, excessive irritation or greater than 25% increase in mean ear thickness were noted in the mouse treated with the test item at a concentration of 50% v/v in acetone/olive oil 4:1, therefore this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of five female animals, were treated with 50 µL (25 µL per ear) of the test item, as a solution in acetone/olive oil 4:1, at concentrations of 50%, 25% or 10% v/v. A further group of five female animals was treated with acetone/olive oil 4:1 alone in the same manner.

 

Main Test Results:

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (%v/v) in acetone/olive oil 4:1	Stimulation Index	Result
10	3.26	Positive
25	8.35	Positive
50	8.71	Positive

The concentration of the test item expected to cause a 3 fold increase in³HTdR incorporation (EC₃value) was calculated to be 9.5% (extrapolated).

Clinical Observations and Mortality Data

All animals survived to the scheduled euthanasia. No signs of systemic toxicity were noted in the test or control animals during the test.

Body Weight

Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

The test item is classified as a contact sensitizer (Category 1B) according to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures and the United Nations Globally Harmonized System of Classification and Labelling of Chemicals.

Executive summary:

A study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

 

Following preliminary screening tests in mice treated with the undiluted test item and the test item at a concentration of50% v/vinacetone/olive oil 4:1, in which the undiluted test item caused a greater than 25% increase in mean ear thickness but no signs of systemic toxicity. No signs of systemic toxicity, excessive irritation or greater than 25% increase in mean ear thickness were noted in the mouse treated with the test item at a concentration of 50%v/vinacetone/olive oil 4:1, therefore this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of five female animals, were treated with 50 µL (25 µL per ear) of the test item, as asolutioninacetone/olive oil 4:1, at concentrations of 50%, 25% or 10% v/v. A further group of five female animals was treated withacetone/olive oil 4:1alone in the same manner.

The sensitivity of the test system was confirmed periodically using the positive control substance α-Hexylcinnamaldehyde.

 

The test item, Butanedioic acid, 2-[(dipropoxyphosphinothioyl)thio]-, 1,4-dibutyl ester, was considered to be a moderate skin sensitizer with an EC3 of 9.5% under the conditions of the test.