Trilithium orthophosphate

Endpoint summary

Description of key information

A mouse local lymphnode assay (LLNA) was performed with the read-across substance sodium dihydrogenorthophosphate. Under the conditions of this test, the test item was considered to be a non-sensitiser.

A skin sensitisation test in Hartley guinea pigs was performed with lithium carbonate according to OECD 406. Under the conditions of this test, the test item was considered to be a non-sensitiser.

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:

From 2009-10-19 to 2009-11-03

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:

UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)). These Regulations are in accordance with GLP standards published as OECD Principles on Good Laboratory Practice (revised 1997, ENV/MC/CHEM

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 444/09

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark over silica gel

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 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 23 g.

- 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.

- Acclimation period:
At least five days.

ENVIRONMENTAL CONDITIONS

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

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 only.

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.

Key result

Parameter:

SI

Value:

1.31

Test group / Remarks:

2.5% test material

Key result

Parameter:

SI

Value:

1.01

Test group / Remarks:

5% test material

Key result

Parameter:

SI

Value:

1.05

Test group / Remarks:

10% test material

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.

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

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.

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

 

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.

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

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.

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

Please see attachment "Appendix1 Current Positive Control Study for the Local Lymph Node Assay”
Please see attachment "Appendix2 Summary of Positive Control Data for the Local Lymph Node Assay”

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.

Executive summary:

Introduction: 

A study according to OECD Guideline 429 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. 

Methods: 

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 only.

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
2.5	1.31	Negative
5	1.01	Negative
10	1.05	Negative

Conclusion: 

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

Reference 2

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1993-10-18 to 1993-11-11

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

1987

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 81-6 (Skin Sensitisation)

Version / remarks:

1984

Deviations:

no

Principles of method if other than guideline:

NA

GLP compliance:

yes

Type of study:

Buehler test

Justification for non-LLNA method:

A valid skin sensitisation study according to OECD guideline 406 with lithium carbonate is available and additionally an LLNA study with the read-across substance Sodium dihydrogenorthophosphate.

Species:

guinea pig

Strain:

Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: HRP, Inc., Denver, PA
- Age at study initiation: Young adult
- Weight at study initiation: 301 g - 379 g
- Housing: individually housed in suspended polycarbonate cages
- Diet (e.g. ad libitum): ad libitum, Purina Guinea Pig Chow 5025
- Water (e.g. ad libitum): ad libitum, fresh tap water


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.9°C - 23.3 °C
- Humidity (%): 37 % - 89 %
- Photoperiod (hrs dark / hrs light): 12 hours fluorescent light/ 12 hours dark

Route:

epicutaneous, occlusive

Vehicle:

other: ethanol, acetone

Concentration / amount:

0.3 g undiluted test material

Day(s)/duration:

Once weekly until a total of three applications/ 6 h

Route:

epicutaneous, occlusive

Vehicle:

other: ethanol, acetone

Concentration / amount:

0.3 g undiluted test material

No. of animals per dose:

Test group: 20 (10 male, 10 female)
Positive control group: 10 (5 male, 5 female)
Challenge group: 10 (5 male, 5 female)

Details on study design:

The test material (0.3 g) was applied undiluted to each of 20 Hill Top Chambers®. In addition, 0.3 g test material plus 0.3 mL of 0.15 % DNCB in 80 % ethanol was applied to the test sites (left shoulder), and secured with hypoallergenic tape. Each animal was then wrapped with an elastic, plastic-lined bandage.
Six hours later, the bandage and chambers were removed and the test sites were wiped with clean gauze moistened with methanol. The test sites were then rinsed with tap water. The guinea pigs were dosed in this manner once weekly until a total of three applications had been administered. Following a 14 day rest period, the guinea pigs were challenged on a virgin site on the right shoulder in the manner described above.

Challenge controls:

An additional 10 naive animals each received 0.3 g of the test material for comparison.

Positive control substance(s):

yes

Remarks:

1-chloro-2,4-dinitrobenzene (DNCB)

Positive control results:

Animals in the positive control group had slight erythema and edema following the initial induction application and all but one had well defined to moderate erythema and slight to mild edema following challenge.
No irritation was noted on any of the test or challenge control animals at any time during the study.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

0.3 g test substance

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

None

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

other: challenge group

Dose level:

0.3 g test substance

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

None

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

0.3 g test substance

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

None

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

other: challenge group

Dose level:

0.3 g test substance

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

None

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

0.3 g test substance and 0.3 mL of 0.15 % DNCB in 80 % ethanol

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

None

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

0.3 g test substance and 0.3 mL of 0.15 % DNCB in 80 % ethanol

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

None

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

other: positive control challenge

Dose level:

0.3 g test substance and 0.3 mL of 0.15 % DNCB in 80 % ethanol

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

None

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

other: positive control challenge

Dose level:

0.3 g test substance and 0.3 mL of 0.15 % DNCB in 80 % ethanol

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

None

Group:

negative control

Remarks on result:

not measured/tested

The challenge results for erythema are summarized below:

Erythema Scores^a
Group		0	1	2	3	4	Incidence^b	Severity^c
Test Material	(24hr)	0	0	0	0	0	0/20	0
	(48hr)	0	0	0	0	0	0/20	0
Challenge Control	(24hr)	0	0	0	0	0	0/10	0
	(48hr)	0	0	0	0	0	0/10	0
Positive Control	(24hr)	0	1	1	8	0	9/10	2.7
	(48hr)	0	1	5	4	0	9/10	2.3

a: Number animals exhibiting each score.

b: Number animals having scores greater than 1/ Total number animals challenged

c: Sum of (Number animals exhibiting each score x score)/ Total number animals challenged

Interpretation of results:

not sensitising

Conclusions:

Under the conditions of this study, the test material is non-sensitising when topically applied to Hartley guinea pigs. Based on this result, it can be assumed that lithium carbonate is not a sensitiser.

Executive summary:

A skin sensitisation test in Hartley guinea pigs was performed according to OECD 406 and EU method B.6. Lithium carbonate Pharmaceutical Grade (0.30 g) was applied undiluted topically to the left shoulders (previously clipped free of hair) of 10 male and 10 female Hartley guinea pigs. The test material was left in contact with the skin for approximately six hours. The animals received three induction treatments one week apart. A concurrent positive control group of 10 animals was treated in a similar manner with DNCB (0.15 % weight/volume). 14 days after the third induction treatment, the animals were challenged with the test material at a virgin skin site. An additional 5 male and 5 female naive animals received 0.30 g of the undiluted test material (challenge control group). The positive control group was challenged with DNCB. Observations for skin reactions were recorded at initiation and termination. All animals remained healthy and gained weight during the study. No skin reactions were noted on any of the test or challenge control animals at any time during the study. Animals in the positive control group had slight erythema and edema following the initial induction application and all but one had well defined to moderate erythema and slight to mild edema following challenge. Under the conditions of this study, the test material is non-sensitising when topically applied to Hartley guinea pigs.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Sensitisation:

A sensitisation study with lithium phosphate was not available. Consequently read-across was applied using a characteristically similar compound, sodium dihydrogenorthophosphate.

Read-across with Sodium dihydrogenorthophosphate (Harlan Laboratories, 2920-0003, 2010)

A study according to OECD Guideline 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. A stimulation index of less than 3 was recorded for the test material at concentrations of 10 %, 5 % and 2.5 % w/w in propylene glycol. Thus, the test material was considered to be a non‑sensitiser under the conditions of the test.

Read-across with lithium carbonate (FMC, RL, 1993)

A skin sensitisation test in Hartley guinea pigs was performed according to OECD 406 and EU method B.6 (Buehler test). Lithium carbonate Pharmaceutical Grade (0.30 g) was applied undiluted topically to the left shoulders (previously clipped free of hair) of 10 male and 10 female Hartley guinea pigs. The test material was left in contact with the skin for approximately six hours. The animals received three induction treatments one week apart. A concurrent positive control group of 10 animals was treated in a similar manner with DNCB (0.15 % weight/volume). 14 days after the third induction treatment, the animals were challenged with the test material at a virgin skin site. An additional 5 male and 5 female naive animals received 0.30 g of the undiluted test material (challenge control group). The positive control group was challenged with DNCB. Observations for skin reactions were recorded at initiation and termination. All animals remained healthy and gained weight during the study. No skin reactions were noted on any of the test or challenge control animals at any time during the study. Animals in the positive control group had slight erythema and edema following the initial induction application and all but one had well defined to moderate erythema and slight to mild edema following challenge. Under the conditions of this study, the test material is non-sensitising when topically applied to Hartley guinea pigs.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008:
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on skin sensitisation, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.