Dialkyl C18 and C18-unsaturated phosphonates

Endpoint summary

Description of key information

- Skin sensitisation: not sensitising (OECD 429, Kr.1, GLP).

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

Study period:

02 November to 24 November 2017.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Alkenyl phosphonate
- Expiration date of the lot/batch: 09 September 2017
- Purity test date: 08/06/2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25 ºC, below 70 RH%)
- Stability under test conditions: yes

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo. San Pietro al Natisone (UD). Zona Industriale Azzida, 57, 33049 Italy
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Young adults, 8-12 weeks old (age-matched, within one week)
- Weight at study initiation: The weight variation in animals involved in the study will not exceed ± 20 % of the mean weight (17 - 27 g)
- Housing :Group caging / Mice will be provided with glass tunnel-tubes
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 15-20 air exchange/hour
- IN-LIFE DATES: From: To:

Vehicle:

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

Concentration:

1, 2.5, 5 and 10% in AOO.

No. of animals per dose:

4 females.

Details on study design:

PRELIMINARY TEST:
To assess the irritant potential of the test substance (through ear thickness measurement), a preliminary test was performed on 4 animals, as follows:
- the test substance was prepared at the concentrations of (to complete).
- for three consecutive days, the animals received applications of 25 µL of the dosage form preparations to the external surface of both ears (one concentration per ear),
- measurement of the ear thickness (using a micrometer) was performed each day before treatment and 24 hours after the last application.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Kimber and Dearman
- Criteria used to consider a positive response: The test substance was considered as a skin sensitizer when the Stimulation Indices (SI) for a dose group is ≥ 3. Other relevant criteria such as cellularity, radioactivity levels and ear thickness were also taken into account for the interpretation of results.

TREATMENT PREPARATION AND ADMINISTRATION:
In the main test, twenty female CBA/Ca mice were allocated to five groups:
- three treated groups of four animals receiving the test substance Alkenyl phosphonate at the concentrations 1, 2.5, 5 and 10%
- one negative control group of four animals receiving the vehicle
- one positive control group of four animals receiving the reference item, alpha-hexylcinnamaldehyde (HCA), a moderate sensitizer, at the concentration of 25%

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 substance, the animals were placed under light isoflurane anesthezia during the administration. No massage was performed but the tip was used to spread the preparation over the application sites. No rinsing was performed between each application.
On day 6, all animals of all groups received a single intravenous injection of 250 µL of 0.9% NaCl containing 20 µCi of tritiated methyl-thymidine (3H-TdR) via the tail vein. Lymph node cell proliferative responses were measured on day 6. The lymph nodes were pooled for each experimental group.
The obtained values were used to calculate stimulation indices (SI).
The irritant potential of the test substance was assessed in parallel by measurement of ear thickness on days 1, 2, 3 and 6.

Positive control substance(s):

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

Positive control results:

In the positive control group given HCA at the concentration of 25% in AOO, a moderate increase in cellularity and a stimulation index exceeding the threshold positive value of 3 (SI = 12.8) were noted. The study was therefore considered valid.

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

Negative control

Key result

Parameter:

SI

Value:

2

Test group / Remarks:

Alkenyl phosphonate 10 (w/v)% in AOO

Key result

Parameter:

SI

Value:

1.1

Test group / Remarks:

Alkenyl phosphonate 5 (w/v)% in AOO

Key result

Parameter:

SI

Value:

1.4

Test group / Remarks:

Alkenyl phosphonate 2.5 (w/v)% in AOO

Key result

Parameter:

SI

Value:

0.8

Test group / Remarks:

Alkenyl phosphonate 1 (w/v)% in AOO

Key result

Parameter:

SI

Value:

12.8

Test group / Remarks:

Positive control (25 % HCA) in AOO

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA: The appearance of the lymph nodes was normal in the negative control group and in all test item treated dose groups. Larger than normal lymph nodes were observed in the positive control group.

EC3 CALCULATION: Not applicable

CLINICAL OBSERVATIONS: No signs of systemic toxicity were observed during the study.

BODY WEIGHTS: No treatment related marked body weight loss (≥5%) was observed on the mean body weight changes of the groups.

Other: No mortality was observed during the study. No test item precipitate was observed on the ears of the experimental animals. Slight erythema was observed (score 1) for all animals of the 10% (w/v) dose group on Days 2-3 and for all animals of the 5% (w/v) dose group on Day 3.

Table 1: DPM, DPN and Stimulation Index Values for all Groups

 

Test Group	Measured		No. of		Stimulation
Name	DPM/group	DPM	Nodes	DPN	Index Values
Background	33	-	-	-	-
(5 (w/v) % TCA )
Negative control	6478	6445.0	8	805.6	1.0
(in AOO)
Alkenyl phosphonate	13009	12976.0	8	1622.0	2.0
10 (w/v)%
in AOO
Alkenyl phosphonate	7342	7309.0	8	913.6	1.1
5 (w/v)%
in AOO
Alkenyl phosphonate	9369	9336.0	8	1167.0	1.4
2.5 (w/v)%
in AOO
Alkenyl phosphonate	5436	5403.0	8	675.4	0.8
1 (w/v)%
in AOO
Positive control	82721	82688.0	8	10336.0	12.8

Notes:

1.DPM (Disintegrations Per Minute)

2.DPN (Disintegrations Per Node) = DPM divided by the number of lymph nodes.

3.Stimulation Index = DPN of a treated group divided by DPN of the appropriate control group.

 

Interpretation of results:

GHS criteria not met

Conclusions:

under the conditions of the present assay, Alkenyl phosphonate, tested in a suitable vehicle, was shown to have no sensitisation potential (non-sensitizer) in the Local Lymph Node Assay. Therefore, no classification is required according to EU criteria.

Executive summary:

In a dermal sensitization study using the method of Local Lymph Node assay (LLNA), (CiToxLAB, 2017) with Alkenyl phosphonate in AOO, 9-week CBA/J mice (4 females per group) were tested according to the guideline OECD 429, at the concentrations of 1, 2.5, 5 and 10%. The positive control group received alpha-hexylcinnamaldehyde (25% in AOO).

No mortality and no systemic clinical signs were observed during the study. No test item precipitate was observed on the ears of the experimental animals. Slight erythema was observed (score 1) for all animals of the 10% (w/v) dose group on Days 2-3 and for all animals of the 5% (w/v) dose group on Day 3.

The appearance of the lymph nodes was normal in the negative control group and in all test item treated dose groups. Larger than normal lymph nodes were observed in the positive control group. The stimulation Index Values were 2.0, 1.1, 1.4 and 0.8 at 10%, 5%, 2.5 and 1% Alkenyl phosphonate in AOO respectively.

Under the test conditions of this study, Alkenyl phosphonate is not considered as a skin sensitizer and therefore no classification is required according to EU and UN GHS criteria.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

1- Skin sensitisation:

1 -1 In vitro tests:

Due to the insolubility of the test item, the study OECD 442D was stopped at this stage. Under the experimental conditions of this test and based on the solubility results, the test item was considered incompatible with the design of the study, indeed solubility is a limitation of this protocol. Consequently the potential to activate theNrf2 transcription factor of the test item could not be evaluated with this KeratinoSens test.

As Alkenyl phosphonate is poorly soluble in water and an UVCB, the in vitro methods are not applicable. Therefore, in vivo test is recommended (LLNA test, OECD 429) in order to conclude on the skin sensitization classification for alkenyl phosphonate (ECHA Recommendations, 2016).

1 -2 In vivo Test: LLNA test (OECD 429, Kr.1, GLP):

One study was available and considered as the key study (Kr.1, GLP). In a dermal sensitization study using the method of Local Lymph Node assay (LLNA), with Alkenyl phosphonate in AOO, 9-week CBA/J mice (4 females per group) were tested according to the guideline OECD 429, at the concentrations of 1, 2.5, 5 and 10%.

No mortality and no systemic clinical signs were observed during the study. The stimulation Index Values were 2.0, 1.1, 1.4 and 0.8 at 10%, 5%, 2.5 and 1% Alkenyl phosphonate in AOO respectively. Based on these results, Alkenyl phosphonate was not considered as a skin sensitizer.

Justification for classification or non-classification

Harmonized classification:

No harmonized classification is available according to the Regulation (EC) No 1272/2008.

Self classification:

1-Skin sensitisation: based on the results of the key study (OECD 429, Kr.1, GLP), Alkenyl phosphonate is not considered as a skin sensitizer. Therefore, no classification is required according to EU criteria.

2- Respiratory sensitisation: no classification is proposed due to lack of data.