Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
The study was performed between 21 June 2011 and 13 July 2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions. The reliability has been amended in accordance with 'practical guide 6: How to report read-across and categories' which states that the maximum reliability for a read-across study is 2. The study is considered to be adequate and reliable for the purpose of registration under REACH (Regulation (EC) No. 1907/2006) and for classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP). Read-across is justified on the basis that the sensitisation potential of tetrairon tris(pyrophosphate) will be determined by the Fe cation. Pyrophosphate itself is not considered to be a sensitiser, in addition, the breakdown product of pyrophosphate (orthophosphate) is a natural component of blood and cellular fluids. As, tetrairon tris(pyrophosphate) has a lower water solubility than iron orthophosphate it is considered to be less bioavailable and therefore iron orthophosphate is considered to be a worst case for sensitisation potential of the Fe cation.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Sponsor's identification: IP 27 Iron orthophosphate
Description: off white solid
Batch number: MV3395
Purity: see CoA (attached)
Date received: 11 May 2011
Expiry date : 03 March 2014
Storage conditions: room temperature in the dark

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/Ca (CBA/CaOlaHsd)
Sex:
female
Details on test animals and environmental conditions:
Animals and Animal Husbandry
Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by by a reputable supplier. For full details please see Harlan Laboratories Ltd Study number 41101364. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non pregnant. After an acclimatisation period of at least five days the 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. At the start of the study the animals were in the weight range of 15 to 23 g, and were eight to twelve weeks old.
The animals were individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes. Free access to mains tap water and food was allowed throughout the study.
The temperature and relative humidity were controlled to remain within target ranges of 19 to 25°C and 30 to 70%, respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Remarks:
Please see below for Vehicle Determination Record
Concentration:
Groups of mice were treated with the test item at concentrations of 50%, 25% or 10% w/w in dimethyl formamide.
No. of animals per dose:
Groups of four mice were treated
Details on study design:
Preliminary Screening Test
As no toxicological information was available 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 50% w/w in dimethyl formamide, 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 irritation was scored daily according to the scale included as Appendix 5. 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 Test
Test Item Administration
Groups of four mice were treated with the test item at concentrations of 50%, 25% or 10% w/w in dimethyl formamide. The preliminary screening test suggested that the test item 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 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).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
None provided.

Results and discussion

Positive control results:
One group of five animals was treated with 50 µl (25 µl per ear) of alpha-Hexylcinnamaldehyde, Tech, 85% as a solution in dimethyl formamide at a concentration of 15% v/v. A further group of five animals was treated with dimethyl formamide 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 4.77 Positive

Alpha-Hexylcinnamaldehyde, Tech 85% 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 test material at concentrations of 50% , 25% and 10% v/v in dimethyl formamide. The stimulation index (SI) results are given in Table 4.
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 4.

Any other information on results incl. tables

Preliminary Screening Test

Clinical observations, bodyweight and mortality data are given in Table 1 and local skin irritation is given in Table 2. The ear thickness measurements and mean ear thickness changes are given in Table 3.

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. Off white residual test item on the ears was noted on Days 2 to 6.

Based on this information the dose levels selected for the main test were 50%, 25% and 10% w/w in dimethyl formamide.

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

Concentration (%w/w) in
dimethyl formamide

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

50

S-1

17

18

0

0

0

0Rt

0Rt

0Rt

0Rt

0Rt

0Rt


0=      No signs of systemic toxicity

Rt =     Off white residual test item on the ears

Table 2              Local Skin Irritation – Preliminary Screening Test

Concentration
(%
w/w) in
dimethyl formamide

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

50

S-1

0

0

0

0

0

0

0

0

0

0

0

0

Table 3       Measurement of Ear Thicknessand Mean Ear Thickness Changes – Preliminary Screening Test

Concentration
(%
w/w) in
dimethyl formamide

Animal Number

Ear Thickness Measurement (mm)

Day 1

Day 3

Day 6

pre‑dose

post dose

left

right

left

right

left

right

50

S-1

0.240

0.250

0.265

0.270

0.260

0.270

overall mean (mm)

0.245

0.268

0.265

overall mean ear thickness change (%)

na

9.184

8.163


na=     Not applicable

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 4.

Table 4              Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration
(%w/w) in
dimethyl formamide

dpm

dpm/Nodea

Stimulation Indexb

Result

Vehicle

10037.10

1254.64

na

na

10

14799.04

1849.88

1.47

Negative

25

14840.58

1855.07

1.48

Negative

50

20102.10

2512.76

2.00

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 appl

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. Off white residual test item on the ears was noted in animals treated with the test item at concentrations of 50% or 25% w/w in dimethyl formamide.

Table 5              Individual Clinical Observations and Mortality Data

Concentration
(% w/w) in
dimethyl formamide

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

10

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

25

3-1

0

0

0

0

0

0Rt

0Rt

0Rt

0

3-2

0

0

0

0

0

0Rt

0Rt

0Rt

0

3-3

0

0

0

0

0

0Rt

0Rt

0Rt

0

3-4

0

0

0

0

0

0Rt

0Rt

0Rt

0

50

4-1

0

0

0

0

0

0Rt

0Rt

0Rt

0Rt

4-2

0

0

0

0

0

0Rt

0Rt

0Rt

0Rt

4-3

0

0

0

0

0

0Rt

0Rt

0Rt

0Rt

4-4

0

0

0

0

0

0Rt

0Rt

0Rt

0Rt


0=      No signs of systemic toxicity

Rt =     Off white residual test item on the ears

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 6              Individual Bodyweights and Bodyweight Changes

Concentration
(% w/w) in
dimethyl formamide

Animal Number

Bodyweight (g)

Bodyweight Change (g)

Day 1

Day 6

Vehicle

1-1

18

19

1

1-2

18

20

2

1-3

19

19

0

1-4

22

21

-1

10

2-1

20

22

2

2-2

18

18

0

2-3

19

20

1

2-4

18

19

1

25

3-1

18

18

0

3-2

19

20

1

3-3

20

20

0

3-4

18

20

2

50

4-1

19

19

0

4-2

17

19

2

4-3

19

19

0

4-4

18

19

1

Appendix4      Scale for Erythema

Observation

Score

No erythema.................................................................................................................

0

Very slight erythema (barely perceptible)..................................................................

1

Well-defined erythema.................................................................................................

2

Moderate to severe erythema.....................................................................................

3

Severe erythema (beef redness) to eschar formation preventing grading of erythema.......................................................................................................................

4

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test material was considered to be a non-sensitiser under the conditions of the test. This study is considered to be scientifically justified for use as a key study under Regulation (EC) No. 1907/2006 (REACH) and the results are appropriate for the purposes of classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP).