Disodium fluorophosphate

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

Study period:

The study was performed between 08 December 2009 and 22 December 2009

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of GLP inspection: 15 September 2006 Date of Signature on GLP certificate: 26 November 2009

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/Ca (CBA/CaOlaHsd)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source:
Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Harlan Laboratories UK Limited, Bicester, Oxon, UK.

- Age at study initiation:
At the start of the study the animals were eight to twelve weeks old.

- Weight at study initiation:
At the start of the study the animals were in the weight range of 15 to 23g.

- Housing:
The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.

- Diet (e.g. ad libitum):
ad libitum (2014 Teklad Global Rodent diet supplied by Harlan Teklad, Blackthorn, Bicester, Oxon, UK)

- Water (e.g. ad libitum):
ad libitum.

- Acclimation period:
At least five days.


ENVIRONMENTAL CONDITIONS

- Temperature (°C):
The temperature was controlled to remain within the target ranges of 19 to 25 deg C.

- Humidity (%):
The humidity was controlled to remain within the target ranges of 30 to 70%.

- Air changes (per hr):
The rate of air exchange was approximately fifteen changes per hour.

- Photoperiod (hrs dark / hrs light):
The lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.

IN-LIFE DATES:
From: Day 1 To: Day 6

Vehicle:

propylene glycol

Remarks:

Please see below for Vehicle Determination Record

Concentration:

Each group was exposed to concentrations of 25%, 10% or 5% w/w in propylene glycol.

No. of animals per dose:

Groups of four mice were treated.

Details on study design:

RANGE FINDING TESTS:
Using available information regarding the systemic toxicity/irritancy potential of the test material, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µl of the test material at a concentration of 25% w/w in propylene glycol, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Any signs of toxicity or excessive local irritation noted during this period were recorded. The bodyweight was recorded on Day 1 (prior to dosing) and on Day 6.

- Lymph node proliferation response:
Clinical observations, bodyweight and mortality data are given in Table 1.
No signs of systemic toxicity were noted.
Based on this information the dose levels selected for the main test were 25%, 10% and 5% w/w in propylene glycol.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT

- Name of test method:
Local Lymph Node Assay in the Mouse. The assay has undergone extensive inter-laboratory validation and has been shown to reliably detect test materials that are moderate to strong sensitisers.

- Criteria used to consider a positive response:
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node(dpm/node) and as the ratio of 3HTdR incorporation in lymph node cells of test nodes relative to that recorded for the control nodes (stimulation Index).

The test material will be regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitier".

TREATMENT PREPARATION AND ADMINISTRATION:
For the purpose of the study, the test material was used at concentrations of 25%, 10% or 5% w/w in propylene glycol. This vehicle was chosen as it produced the most suitable formulation at the required concentration. The concentrations used are given above. The vehicle determination record is included as Appendix 3.

Determination, by analysis, of the concentration, homogeneity and stability of the test material preparations was not appropriate because it was not specified in the Study Plan and is not a requirement of the Test Guidelines.

Groups of four mice were treated with the test material at concentrations of 25%, 10% or 5% w/w in propylene glycol. The preliminary screening test suggested that the test material would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. The mice were treated by daily application of 25 µl of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (Days 1, 2 and 3). The test material 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 four mice received the vehicle alone in the same manner.

3H-Methyl Thymidine Administration:
Five days following the first topical application of the test material (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, GE Healthcare UK Ltd) giving a total of 20 µCi to each mouse.

Positive control substance(s):

other: Phenylacetaldehyde (90%) as a solution in propylene glycol at a concentration of 2.5% v/v

Statistics:

None provided.

Positive control results:

A group of five animals was treated with 50 µl (25 µl per ear) of Phenylacetaldehyde (90%) as a solution in propylene glycol at a concentration of 2.5% v/v. A further control group of five animals was treated with propylene glycol alone.

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

Concentration (% v/v) in
propylene glycol Stimulation Index Result
2.5 4.20 Positive

Conclusion. Phenylacetaldehyde (90%) was considered to be a sensitiser under the conditions of the test.

Key result

Parameter:

SI

Value:

0.99

Test group / Remarks:

5% w/w (in propylene glycol)

Key result

Parameter:

SI

Value:

1.59

Test group / Remarks:

10% w/w (in propylene glycol)

Key result

Parameter:

SI

Value:

2.88

Test group / Remarks:

25% w/w (in propylene glycol)

Table 1 - Clinical Observations, Bodyweight and Mortality Data – Preliminary Screening Test

Concentration (%w/w) in propylene glycol	Animal Number	Bodyweight (g)		Day
				1		2		3		4	5	6
		Day 1	Day 6	Pre-Dose	Post Dose	Pre-Dose	Post Dose	Pre-Dose	Post Dose
25	S-1	19	18	0	0	0	0	0	0	0	0	0

0=      No signs of systemic toxicity

Table 2 - Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration (%w/w) in propylene glycol	dpm	dpm/Node^a	Stimulation Index^b	Result
Vehicle	6458.22	807.28	na	na
5	6388.33	798.54	0.99	Negative
10	10266.43	1283.30	1.59	Negative
25	18595.53	2324.44	2.88	Negative

dpm=  Disintegrations per minut

a=      Disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 (total number of lymph nodes)

b=      Stimulation Index of 3.0 or greater indicates a positive result

na =    Not applicable

Table 3 - Individual Clinical Observations and Mortality Data

Concentration (% w/w) in propylene glycol	Animal Number	Day 1		Day 2		Day 3		Day 4	Day 5	Day 6
		Pre-Dose	Post Dose	Pre-Dose	Post Dose	Pre-Dose	Post Dose
Vehicle	1-1	0	0	0	0	0	0	0	0	0
	1-2	0	0	0	0	0	0	0	0	0
	1-3	0	0	0	0	0	0	0	0	0
	1-4	0	0	0	0	0	0	0	0	0
5	2-1	0	0	0	0	0	0	0	0	0
	2-2	0	0	0	0	0	0	0	0	0
	2-3	0	0	0	0	0	0	0	0	0
	2-4	0	0	0	0	0	0	0	0	0
10	3-1	0	0	0	0	0	0	0	0	0
	3-2	0	0	0	0	0	0	0	0	0
	3-3	0	0	0	0	0	0	0	0	0
	3-4	0	0	0	0	0	0	0	0	0
25	4-1	0	0	0	0	0	0	0	0	0
	4-2	0	0	0	0	0	0	0	0	0
	4-3	0	0	0	0	0	0	0	0	0
	4-4	0	0	0	0	0	0	0	0	0

0=      No signs of systemic toxicity

Table 4 - Individual Bodyweights and Bodyweight Changes

Concentration (% w/w) in propylene glycol	Animal Number	Bodyweight (g)		Bodyweight Change (g)
		Day 1	Day 6
Vehicle	1-1	17	19	2
	1-2	19	20	1
	1-3	20	19	-1
	1-4	18	19	1
5	2-1	20	19	-1
	2-2	19	19	0
	2-3	20	19	-1
	2-4	20	20	0
10	3-1	20	21	1
	3-2	21	21	0
	3-3	17	18	1
	3-4	20	20	0
25	4-1	18	17	-1
	4-2	20	20	0
	4-3	19	19	0
	4-4	19	18	-1

Current Positive Control Study for the Local Lymph Node Assay

Introduction. A study was performed to assess the sensitivity of the strain of mouse used at these laboratories to a known sensitiser. The methodology for the LLNA is detailed in the OECD Guideline for the Testing of Chemicals, No. 429, and Method B.42 of CommissionRegulation (EC) No. 440/2008. The study described in this document is based on these test methods but has been refined in order to reduce the number of animals required. The reduced LLNA (rLLNA) has been endorsed by the non‑Commission members of the European Centre for the Validation of Alternative Method (ECVAM) Scientific Advisory Committee (ESAC) at its 26^thmeeting held on 26 – 27 April 2007 at ECVAM,Ispra,Italy.

Test Material: Phenylacetaldehyde (90%)

Project number: 0039/1115

Study dates:        05 November 2009 to 11 November 2009

Methods. A group of five animals was treated with 50 µl (25 µl per ear) of Phenylacetaldehyde (90%) as a solution in propylene glycol at a concentration of 2.5% v/v. A further control group of five animals was treated with propylene glycol alone.

Results. The Stimulation Index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group is as follows:

Concentration (% v/v) in propylene glycol	Stimulation Index	Result
2.5	4.20	Positive

Conclusion. Phenylacetaldehyde (90%) was considered to be a sensitiser under the conditions of the test.

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

The test material was considered to be a non-sensitiser under the conditions of the test.

This study is conducted according to an appropriate guideline and under the conditions of GLP, the study is therefore considered to be acceptable and to adequately satisfy both the guideline requirement and the regulatory requirement as a key study for this endpoint.

Executive summary:

Introduction. A study was performed to assess the skin sensitisation potential of the test material in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed to meet the requirements of the following:

OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted24 April 2002)

Method B42 Skin Sensitisation (Local Lymph Node Assay) of CommissionRegulation (EC) No. 440/2008

Methods. Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of25% w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 µl (25 µl per ear) of the test material as asuspensioninpropylene glycolat concentrations of25%,10% or5% w/w. A further group of four animals was treated withpropylene glycolalone.

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 (%w/w) in propylene glycol	Stimulation Index	Result
5	0.99	Negative
10	1.59	Negative
25	2.88	Negative

Conclusion. The test material was considered to be anon-sensitiserunder the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Migrated from Short description of key information:
One key study is available (Bradshaw J - 2009) for the skin sensitisation endpoint. This study is considered to be a reliability 1 study (according to the Klimisch scale) as it has been conducted to the appropriate guideline (OECD 429, Local Lymph Node Assay) and under the conditions of GLP. The study reports that disodium fluorophosphate is a non-sensitiser under the conditions of the study.

Justification for selection of skin sensitisation endpoint:
One study available.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

Justification for selection of respiratory sensitisation endpoint:
No further testing required for this endpoint.

Justification for classification or non-classification

Skin sensitisation: Disodium fluorophosphate is not considered to be classified for skin sensitisation in accordance with Regulation (EC) No. 1272/2008 (EU CLP). The key study is considered to be adequate and reliable for the purposes of classification and therefore further testing is not considered to be scientifically justified.