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Toxicological information

Skin sensitisation

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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 19 October 2009 and 03 November 2009.
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 reliability has been changed to reflect the use of this study for read across purposes. Read across from sodium dihydrogenorthophosphate to disodium hydrogenorthophosphate is justified on the following basis: Both are ionic inorganic compounds, structurally the only difference between the two is the replacement of one hydrogen atom with an additional sodium atom to give disodium hydrogenorthophosphate. Removing one hydrogen atom from the anion will not enhance any sensitisation potential and the cation remains the same but is increased in quantity, the stimulation index results from the LLNA test with sodium dihydrogenorthophosphate do not indicate that increasing the exposure levels has an impact on the results so increasing the levels of sodium is unlikely to increase the sensitisation potential. 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 disodium hydrogenorthophosphate.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

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)
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)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sponsor's identification : Sodium dihydrogenorthophosphate
Description : white granular solid
Batch number : 444/09
Date received : 18 September 2009
Storage conditions : room temperature in the dark over silica gel

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 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:
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 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

Study design: in vivo (LLNA)

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.

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%)

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 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

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 The stimulation index (SI) results are given in Table 2.
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 2.

Any other information on results incl. tables

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/Nodea

Stimulation Indexb

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

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

Please see attachment "Appendix2Summary of Positive Control Data for the Local Lymph Node Assay”

Applicant's summary and conclusion

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. The study is considered to be reliable and acceptable for use as a key study.
Read across from sodium dihydrogenorthophosphate to disodium hydrogenorthophosphate is justified on the following basis:
Both are ionic inorganic compounds, structurally the only difference between the two is the replacement of one hydrogen atom with an additional sodium atom to give disodium hydrogenorthophosphate. Removing one hydrogen atom from the anion will not enhance any sensitisation potential and the cation remains the same but is increased in quantity, the stimulation index results from the LLNA test with sodium dihydrogenorthophosphate do not indicate that increasing the exposure levels has an impact on the results so increasing the levels of sodium is unlikely to increase the sensitisation potential.
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 disodium hydrogenorthophosphate.
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" (adopted 24 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 of10% 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.

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-sensitiserunder the conditions of the test.