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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Remarks:
Study was performed before Annex VII of the REACH Regulation was updated in 2016.
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 03 April 2012 and 17 April 2012.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
See read-across justification report under Section 13 ‘Assessment Reports’.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.

The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category

The source substance and the target substance are considered to be similar enough to facilitate read-across for the following reasons:
(1) Both substances are inorganic salts containing a trivalent aluminium cation and phosphoric acid. Thus, they all share the Al3+ cation and the PO43- anion as common functional groups. The source substance also contains an Na+ ion, this is not expected to influence the toxicological profile of the substance. Therefore the toxicity of the above substances will be predominantly determined by the presence of the Al3+ cation.
(2) Both substances will ultimately dissociate into the common breakdown products of the Al3+ cations and the PO43- anion.
(3) In general, independently of the cation under consideration, the water solubility of phosphates decreases with increasing degree of phosphate condensation (orthophosphate > diphosphate > triphosphate > polyphosphate).
In accordance with the provisions set out in Annex XI, Section 1.5, the results of the studies used for assessment and read-across are adequate for the purpose of classification and labelling and/or risk assessment; have adequate and reliable coverage of the key parameters addressed in the corresponding test method; cover an exposure duration comparable to or longer than the corresponding test method; and adequate and reliable documentation of the applied method is provided in the technical dossier. Orthophosphates are not considered to be genotoxic and are essential micronutrients. As such the skin sensitisation potential of the target substance will be predominantly determined by the presence of the Al3+ cation. On this basis the standard testing requirements, as detailed in Regulation (EC) 1907/2006 (REACH) were conducted on aluminium orthophosphate as this substance contains the greatest amount of aluminium (%w/w) in comparison to the target substance. This approach is considered to be reliable and justified and no further testing for aluminium tris(dihydrogenorthophosphate) is required.


2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report under Section 13 ‘Assessment Reports’.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report under Section 13 ‘Assessment Reports’.

4. DATA MATRIX
See read-across justification report under Section 13 ‘Assessment Reports’.
Cross-reference
Reason / purpose for cross-reference:
read-across: supporting information

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2013

Materials and methods

Test guidelineopen allclose all
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 inspection: 10 July 2012. Date of signature: 30 November 2012
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Aluminium orthophosphate
EC Number:
232-056-9
EC Name:
Aluminium orthophosphate
Cas Number:
7784-30-7
Molecular formula:
Al.H3O4P
IUPAC Name:
[phosphato(3-)-kappa~3~O,O',O'']aluminum
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

In vivo test system

Test animals

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 Ltd., 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: ad libitum (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., 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

Study design: in vivo (LLNA)

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:
Preliminary Screening Test
Using available information regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 μl of the test item 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. Local skin irritation was scored daily according to the scale included as Appendix 4. Any clinical signs of toxicity, if present, were also recorded. The
bodyweight was recorded on Day 1 (prior to dosing) and on Day 6.
The thickness of each ear was measured using an Oditest micrometer (Dyer, PA), pre-dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.

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 item at concentrations of 25%, 10% or 5% w/w in propylene glycol. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. The mice were treated by daily application of 25 μl of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item 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 item or vehicle (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, ARC UK Ltd) giving a total of 20 μCi to each mouse.

OBSERVATIONS
Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the Test were recorded.
Bodyweights: The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

TERMINAL PROCEDURES
Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 ml of PBS was added to the pooled lymph nodes.

Preparation of Single Cell Suspension: A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 ml of PBS into a petri dish labelled with the project number and dose concentration. The lymph node cell suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 ml of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The pooled lymph node cells were pelleted at 1400 rpm (approximately 190 g) for ten minutes. The pellet was resuspended in 10 ml of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was resuspended in 3 ml of 5% Trichloroacetic acid (TCA).

Determination of 3HTdR Incorporation: After approximately eighteen hours incubation at approximately 4°C, the precipitates were recover ed by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, resuspended in 1 ml of TCA and transferred to 10 ml of scintillation fluid (Optiphase 'Trisafe'). 3HTdR incorporation was measured by b-scintillation counting. The "Poly QTM" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately twenty minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately twenty minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).

Positive control substance(s):
other: Phenylacetaldehyde (90%)

Results and discussion

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 Stimulation Index Result
propylene glycol

2.5 3.49 Positive

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

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: 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
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The radioactive disintegrations per minute (dpm) per lymph node and the stimulation index (SI) are given in Table 5 below.

Any other information on results incl. tables

Preliminary Screening Test

Clinical observations, bodyweight and mortality data are given in Table 2 and local skin irritation is given in Table 3. The ear thickness measurements and mean ear thickness changes are given in Table 4.

No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness 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 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 5.

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 (Table 1):

Table 1.

Concentration (%w/w) in
propylene glycol

Stimulation Index

Result

5

1.06

Negative

10

1.01

Negative

25

0.89

Negative

Clinical Observations and Mortality Data

Individual clinical observations and mortality data for test and control animals are given in Table 5.

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 6.

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.

Table 2             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

20

19

0

0

0

0

0

0

0

0

0


0=      No signs of systemic toxicity

Table 3. Local skin irritation - preliminary screening test

Concentration

(%w/w) in

propylene glycol

Animal

number

Local skin irritation

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Left

Right

Left

Right

Left

Right

Left

Right

Left

Right

Left

Right

25

S-1

0

0

0

0

0

0

0

0

0

0

0

0


Table 4. Measurement of Ear Thickness and Mean Ear Thickness Changes – Preliminary Screening Test

Concentration

(% w/w) in

propylene

glycol

Animal number

Ear thickness measurement (mm)

Day 1

Day 3

Day 6

Pre-dose

Post-dose

Left

Right

Left

Right

Left

Right

25

S-1

0.230

0.230

0.240

0.235

0.230

0.235

Overall mean (mm)

0.230

0.238

0.233

Overall mean ear thickness change (%)

n/a

3.261

1.087

Table 5. Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration
(%w/w) in
propylene glycol

dpm

dpm/Nodea

Stimulation Indexb

Result

Vehicle

6112.62

764.08

na

na

5

6450.73

806.34

1.06

Negative

10

6192.92

774.12

1.01

Negative

25

5463.95

682.99

0.89

Negative

 

dpm = Disintegrations per minute

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 6. 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 7. 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

20

19

-1

1-2

18

17

-1

1-3

23

22

-1

1-4

18

18

0

5

2-1

21

20

-1

2-2

21

22

1

2-3

20

20

0

2-4

20

21

1

10

3-1

20

21

1

3-2

19

20

1

3-3

19

19

0

3-4

21

21

0

25

4-1

20

20

0

4-2

20

21

1

4-3

18

19

1

4-4

20

20

0

Please see attachment "Appendix 1 Current Positive Control Study for the Local Lymph Node Assay”

Please see attachment "Appendix 2 Summary of Positive Control Data for the Local Lymph Node Assay”

Applicant's summary and conclusion

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 test item does not meet the criteria for classification according to the Regulation (EC) No 1272/2008 (EU CLP). The study is considered to be reliable and acceptable for use as a key study.
Executive summary:

Introduction.

A study was performed to assess the skin sensitisation potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed to be compatible with the following:

 OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted 22 July 2010)

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

Methods.

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 25% 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 item as a suspension in propylene glycol at concentrations of 25%, 10% or 5% w/w. A further group of four animals was treated with propylene glycol alone.

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

1.06

Negative

10

1.01

Negative

25

0.89

Negative

Conclusion.

The test item was considered to be a non-sensitiser under the conditions of the test. The test item does not meet the criteria for classification according to the Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.