Trimagnesium bis(orthophosphate)

Endpoint summary

Description of key information

There is no available data for trimagnesium bis(orthophosphate). Reliable data are available for the read across substance sodium dihydrogenorthophosphate (CAS 7558 -80 -7).

Skin sensitisation (RA-A 7558 -80 -7, OECD 429): not sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 19 October 2009 and 03 November 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:

The Department of Health of the Government of the United Kingdom

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: Harlan UK Limited, Bicester, Oxon, UK.
- Females nulliparous and non-pregnant: yes
- 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: ad libitum (2014 Teklad Global Rodent diet supplied by Harlan Teklad, Blackthorn, Bicester, Oxon, UK)
- Water: ad libitum tap water
- Acclimation period: at least five days
- Indication of any skin lesions:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

propylene glycol

Concentration:

Each group was exposed to concentrations of 10%, 5% or 2.5% 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 undiluted test material 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 give in the results section (table 1).

No signs of systemic toxicity were noted.

Based on this information the dose levels selected for the main test were 2.5% , 5% and 10% w/w in propylene glycol.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
-animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card

- 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 undiluted and also freshly prepared in propylene glycol. This vehicle was chosen as it produced the most suitable formulation at the required concentration. The concentrations used are given above.

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.

Test Material Administration
Groups of four mice were treated with the test material at concentrations of 10%,5% or 2.5% w/win 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, 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%)

Positive control results:

One 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 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 are as follows:

Concentration % v/v in acetone/olive oil 4:1 Stimulation Index (SI) Result
15 10.91 Positive

Alpha-Hexylcinnamaldehyde, Tech 85% was considered to be a sensitiser under the conditions of the test.
EXAMPLE

Parameter:

SI

Value:

1.31

Test group / Remarks:

2.5% test substance

Parameter:

SI

Value:

1.01

Test group / Remarks:

5% test substance

Parameter:

SI

Value:

1.05

Test group / Remarks:

10% test substance

Parameter:

SI

Value:

8

Test group / Remarks:

positive control - 90% Phenylacetaldehyde

Cellular proliferation data / Observations:

CLINICAL OBSERVATIONS: No signs of systemic toxicity

BODY WEIGHTS: normal

Preliminary Screening Test

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 10%, 5% and 2.5% w/w in propylene glycol.

Main Test

Estimation of the Proliferative Response of Lymph Node Cells

The radioactive disintegrations per minute per lymph node and the stimulation index are given in Table 2.

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
2.5	1.31	Negative
5	1.01	Negative
10	1.05	Negative

Clinical Observations and Mortality Data

Individual clinical observations and mortality data for test and control animals are given in Table 3.

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

Bodyweight

Individual bodyweights and bodyweight changes for test and control animals are given in Table 4.

Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Table1              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
10	S-1	19	19	0	0	0	0	0	0	0	0	0

0=      No signs of systemic toxicity

Table2              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	5155.29	644.41	na	na
2.5	6770.88	846.36	1.31	Negative
5	5203.17	650.40	1.01	Negative
10	5413.31	676.66	1.05	Negative

 

Table3              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
2.5	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
5	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
10	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

Table4          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	19	21	2
	1-2	18	18	0
	1-3	21	20	-1
	1-4	19	19	0
2.5	2-1	21	21	0
	2-2	17	18	1
	2-3	19	20	1
	2-4	18	18	0
5	3-1	18	20	2
	3-2	17	19	2
	3-3	18	18	0
	3-4	20	20	0
10	4-1	18	18	0
	4-2	19	20	1
	4-3	19	19	0
	4-4	21	21	0

Interpretation of results:

GHS criteria not met

Conclusions:

The test material was considered to be a non-sensitiser under the conditions of the test. The study is considered to be reliable and acceptable for use as a key study.

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

refer to analogue justification provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Positive control results:

One 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 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 are as follows:

Concentration % v/v in acetone/olive oil 4:1 Stimulation Index (SI) Result
15 10.91 Positive

Alpha-Hexylcinnamaldehyde, Tech 85% was considered to be a sensitiser under the conditions of the test.
EXAMPLE

Key result

Parameter:

SI

Value:

1.31

Test group / Remarks:

2.5% test substance

Key result

Parameter:

SI

Value:

1.01

Test group / Remarks:

5% test substance

Key result

Parameter:

SI

Value:

1.05

Test group / Remarks:

10% test substance

Key result

Parameter:

SI

Value:

8

Test group / Remarks:

positive control - 90% Phenylacetaldehyde

Cellular proliferation data / Observations:

CLINICAL OBSERVATIONS: No signs of systemic toxicity

BODY WEIGHTS: normal

Preliminary Screening Test

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 10%, 5% and 2.5% w/w in propylene glycol.

Main Test

Estimation of the Proliferative Response of Lymph Node Cells

The radioactive disintegrations per minute per lymph node and the stimulation index are given in Table 2.

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
2.5	1.31	Negative
5	1.01	Negative
10	1.05	Negative

Clinical Observations and Mortality Data

Individual clinical observations and mortality data for test and control animals are given in Table 3.

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

Bodyweight

Individual bodyweights and bodyweight changes for test and control animals are given in Table 4.

Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Table1              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
10	S-1	19	19	0	0	0	0	0	0	0	0	0

0=      No signs of systemic toxicity

Table2              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	5155.29	644.41	na	na
2.5	6770.88	846.36	1.31	Negative
5	5203.17	650.40	1.01	Negative
10	5413.31	676.66	1.05	Negative

 

Table3              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
2.5	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
5	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
10	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

Table4          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	19	21	2
	1-2	18	18	0
	1-3	21	20	-1
	1-4	19	19	0
2.5	2-1	21	21	0
	2-2	17	18	1
	2-3	19	20	1
	2-4	18	18	0
5	3-1	18	20	2
	3-2	17	19	2
	3-3	18	18	0
	3-4	20	20	0
10	4-1	18	18	0
	4-2	19	20	1
	4-3	19	19	0
	4-4	21	21	0

Interpretation of results:

GHS criteria not met

Conclusions:

Trimagensium bis(orthophosphate) was considered to be a non-sensitiser under the conditions of the test. The study is considered to be reliable and acceptable for use as a key study.

Executive summary:

No skin senistisation properties are considered for trimagnesium bis(orthophosphate) as found in the source study performed with sodium dihydrogenorthophophsate. As explained in the justification for type of information, the differences in molecular structure between trimagnesium bis(orthophosphate) and sodium dihydrogenorthophosphate are unlikely to lead to differences in the partition coefficient that are higher than the typical experimental error of the test method.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

No studies are available with trimagnesium bis(orthophosphate) (CAS 7757 - 87 -1). Reliable data is available for sodium dihydrogenorthophosphate (CAS 7558-80-7).

Sodium dihydrogenorthophosphate and trimagnesium bis(orthophosphate) are structurally similar ionic compounds with the only differences being that sodium is replaced with magnesium, the removal of the hydrogen atoms and increasing the number of phosphate groups to account for the additional charge on the magnesium. Removing the hydrogen atoms from the anion will not enhance any sensitisation potential and the stimulation index results from the LLNA test with sodium dihydrogenorthophosphate do not indicate that increasing the exposure level has an impact on the results so increasing the levels of the phosphate are unlikely to have an impact on the results in terms of read-across.
Sodium and magnesium are both alkali metals from groups 1 and 2 and period 3, respectively. Although magnesium has a smaller ionic radius than sodium, the increased charge is likely to reduce its ability to cross the skin barrier. Dermal absorption of magnesium is anticipated to be less than sodium.
The difference between the two compounds will not have an impact on any sensitisation potential and therefore the negative LLNA results with sodium dihydrogenorthophosphate can reliably be read across to trimagnesium bis(orthophosphate). An additional consideration is that both magnesium and sodium are essential biological compounds and are unlikely to have any sensitisation potential.

 

Sodium dihydrogenorthophosphate (CAS 7558-80-7)

A LLNA study according to OECD TG 429 was performed to assess the skin sensitisation potential of sodium dihydrogenorthophosphate in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 10% 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 a suspension in propylene glycol at concentrations of 10%, 5% or 2.5% w/w. A further group of four animals was treated with propylene glycol alone. 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: SI = 1.31 with 2.5% test substance, SI = 1.01 with 5% test substance and SI = 1.05 with 10% test substance. Thus, the test material was considered to be a non-sensitiser under the conditions of the test.

 

In conclusion, since sodium hydrogenorthophosphate is a reliable read across substance and it does not show skin sensitising potential, trimagnesium bis(orthophosphate) is also considered to be not sensitising.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification is not proposed on the basis of read-across from an analogue substance (sodium dihydrogenorthophosphate). Further in vivo testing is deemed to be scientifically unjustified.