Triethyl phosphonoacetate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

between 2012-01-18 to 2012-01-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in compliance with international standard guidelines under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

2011-07-19

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Triethyl phosphonoacetate

EC Number:

212-757-6

EC Name:

Triethyl phosphonoacetate

Cas Number:

867-13-0

Molecular formula:

C8H17O5P

IUPAC Name:

ethyl 2-(diethoxyphosphoryl)acetate

Test material form:

liquid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: 8 weeks
- Weight at study initiation: 19.2 to 21.3 g.
- Fasting period before study: not applicable
- Housing: 5/cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5-6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C,
- Humidity: 50 ± 20%,
- Light/dark cycle: 12 h/12 h (7:00 - 19:00),
- Ventilation: approximately 12 cycles/hour of filtered, non-recycled air.

IN-LIFE DATES: from 11 January 2012 to 27 January 2012

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

Preliminary test: 10; 25; 50 ; 100%
Main study: 0; 25; 50; 100%

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: A heterogeneous suspension was obtained at the concentration of 50% in AOO. However, a homogeneous solution was obtained at the concentration of 50% in dimethylformamide (DMF). Therefore DMF was chosen as a vehicle.
- Irritation: No local reactions were noted in any animals and no notable increase in ear thickness was observed at any tested concentrations. The highest concentration retained for the main test was therefore 100%.
- Lymph node proliferation response: not examined

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: upon arrival at CIT, the animals were allocated to the groups using a manual randomization procedure. A larger number of animals than necessary were acclimated to permit the selection and/or replacement of individuals.
- Criteria used to consider a positive response: The test item is considered as a skin sensitizer when the SI for a dose group is ≥ 3 together with consideration of a statistical significance when compared to the vehicle control group and a statistically significant dose-response relationship. Other relevant criteria such as ear thickness will be also taken into account to evaluate the data.

TREATMENT PREPARATION AND ADMINISTRATION: On days 1, 2 and 3, a dose-volume of 25 μL of the control or dosage form preparations was applied to the dorsal surface of both ears, using an adjustable pipette fitted with a plastic tip. In order to avoid licking and to ensure an optimized application of the test materials, the animals were placed under light isoflurane anesthesia during the administration. No massage was performed but the tip was used to spread the preparation over the application site. No rinsing was performed.

Positive control substance(s):

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

Statistics:

Statistical analysis was performed using the software SAS software version 9.2 (SAS Institute Inc).

Assessment of a dose-response relationship was performed using a Kendall test.

Pairwise comparisons
Two separate statistical comparisons were performed on dpm values:
- negative control group versus positive control group (first comparison),
- test item-treated groups versus negative control group (second comparison).

Results and discussion

Positive control results:

Positive control HCA
SI: 5.61
DPM: 2093.6
As the SI of the positive control was higher than 3, the test was considered as valid.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

0.29

Test group / Remarks:

Test item 25%

Results 2

Parameter:

SI

Value:

0.21

Test group / Remarks:

Test item 50%

Results 3

Parameter:

SI

Value:

0.35

Test group / Remarks:

Test item 100%

Results 4

Parameter:

other: disintegrations per minute (DPM)

Value:

106.4

Test group / Remarks:

Test item 25%

Results 5

Parameter:

other: disintegrations per minute (DPM)

Value:

78.9

Test group / Remarks:

Test item 50%

Results 6

Parameter:

other: disintegrations per minute (DPM)

Value:

131

Test group / Remarks:

Test item 100%

Any other information on results incl. tables

There were no unscheduled deaths and no clinical signs were observed during the observation period. No cutaneous reactions and no notable increase in ear thickness were observed at any tested concentrations.

The results are presented in the following table:

Treatment	Concentration (%)	Irritation level	Stimulation Index (SI)
Test item	25	I	0.29
Test item	50	I	0.21
Test item	100	I	0.35
HCA	25	-	5.61

 

No notable lymphoproliferation was noted with the test item at any tested concentrations.

 

No statistically significant dose-relationship was demonstrated between concentrations of test item and the number of disintegration per minute data.

 

No statistically significant differences were observed in the number of disintegration per minute between test item-treated groups and negative control group.

 

No notable proliferation of lymphocytes was noted with the test item at any test item-tested concentrations.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Triethyl phosphono acetate was not a skin sensitiser under the conditions of this study.

Executive summary:

In a local lymph node assay performed according to the OECD guideline 429 and in compliance with GLP, 8-weeks old female CBA/J mice, were exposed to triethyl phosphono acetate (purity of 99%) undiluted or diluted in Dimethylformamide (DMF).

A preliminary test was first performed in order to define the test item concentrations to be used in the main test. Two groups of two mice received the test item by topical route to the dorsal surface of both ears (one concentration per ear) on days 1, 2 and 3 at concentrations of 10, 25, 50 or 100% under a dose-volume of 25 µL. From day 1 to day 3 then on day 6, the thickness of both ears of each animal was measured and the local reactions were recorded. Each animal was observed at least once a day for mortality and clinical signs. Body weight was recorded once during the acclimation period, and then on days 1 and 6. No local reactions were noted in any animals and no notable increase in ear thickness was observed at any tested concentrations. The highest concentration retained for the main test was therefore 100%.

 

In the main study, 4 mice/concentration were topically treated with the test item at concentrations in DMF of 0; 25; 50 and 100 %. The topical application was performed onto the dorsal surface of both ears on days 1, 2 and 3 under a dose-volume of 25 µL. Additionally, one positive control group of four females received the positive control, α‑hexylcinnamaldehyde (HCA), at 25% in a mixture acetone/olive oil (4/1; v/v) under the same experimental conditions. From day 1 to day 3 then on day 6, the thickness of the left ear of each animal was measured, except in animals of the positive control group, and the local reactions were recorded. Each animal was observed at least once a day for mortality and clinical signs. Body weight was recorded once during the acclimation period, and then on days 1 and 6.

 

After 2 days of resting, on day 6, the animals received a single intravenous injection of tritiated methyl thymidine (3H-TdR). Approximately 5 hours later, the animals were sacrificed and the auricular lymph nodes were excised. The proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of 3H-TdR.

 

The results were expressed as disintegrations per minute (dpm) per group and as dpm/node. The obtained values were used to calculate Stimulation Indices (SI).

 

No unscheduled death occurred during the study and no clinical signs were observed in any animals. No cutaneous reactions and no notable increase in ear thickness were observed at any tested concentrations. The threshold positive value of 3 for the SI was reached in the positive control group (SI = 5.61). The experiment was therefore considered valid. The stimulation index of the treated animals was clearly lower than 3 at all tested concentrations.

 Therefore under the test conditions, Triethyl phosponoacetate is not classified as a skin sensitiser in a Local lymph node assay in mouse according to the criteria of the Regulation (EC) No. 1272/2008 (CLP).

 

This study is considered as acceptable and satisfies the requirement for the skin sensitisation endpoint.