Registration Dossier

Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 June 2016 to 14 June 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
OECD Guidelines for Testing of Chemicals No. 429 “Skin Sensitisation: Local Lymph Node Assay”(22 July 2010)
Deviations:
yes
Remarks:
See "Any other information" for details
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
Commission Regulation (EC) No 440/2008 of 30 May 2008, B.42. “Skin Sensitisation: Local Lymph Node Assay” (Official Journal L 142, 31/05/2008) amended by Commission Regulation (EU) No 640/2012 of 6 July 2012
Deviations:
yes
Remarks:
see "Any other information" for details
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
Purity : 99%
Batch number: PCAF151028 Rs
CAS number : 21056-98-4
Chemical name : Phosphonic acid, calcium salt (1:1), Calcium hydrogen phosphonate
Manufacturing date : 28/10/2015
Shelf life or Expiry date : 28/10/2018
Date of analysis : 27/11/2015
Specific details on test material used for the study:
No further details specified in the study report.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
Species and strain: CBA/CaOlaHsd mice
Source: Envigo, San Pietro al Natisone (UD), Zona Industriale Azzida, 57, 33049 Italy
Hygienic level: SPF at arrival; standard housing conditions during the study
Justification of strain: On the basis of OECD Guideline, mice of CBA/Ca or CBA/J strain can be used. Females are used because the existing database is predominantly based on females.
Number of animals: 4 animals / group
Sex: Female, nulliparous, non-pregnant
Age of animals at starting: 10 weeks old (age-matched, within one week)
Body weight range at starting: 19.1 – 21.5 grams (The weight variation in animals in the study did not exceed ± 20 % of the mean weight.)
Acclimatization time: 21 days
Note: In the Preliminary Experiment, mice of 11 weeks of age (23.2-24.1 grams) were used.
HusbandryAnimal health: Only healthy animals were used for the study. Health status was certified by the veterinarian.
Housing / Enrichment: Group caging / mice were provided with glass tunnel tubes
Cage type: Type II. polypropylene / polycarbonate
Bedding: Bedding was available to animals during the study
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 18.5 – 25.6°C
Relative humidity: 29 - 80 %
Ventilation: 15-20 air exchanges/hour
The temperature and relative humidity were recorded twice every day during the acclimatisation and experimental phases.
Room/Cabinet (preliminary experiment): 244/6
Room/Cabinet (non-radioactive phase): 244/5
Room/Cabinet (radioactive phase): 139 – 140
Food and feeding
Animals received ssniff® SM Rat/Mouse – “Breeding & Maintenance, 15 mm, autoclavable Complete diet for rats/mice” (Batch number: 540 5117, Expiry date: 31 July 2016 and Batch number: 278 5652, Expiry date: 30 November 2016) produced by ssniff Spezialdiäten GmbH (Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany), ad libitum.
Water supply
Animals received tap water from the municipal supply from 500 mL bottles, ad libitum

.Bedding
Bedding of certified wood chips especially designed to keep animals in the best natural environment was provided for animals during the study. Lignocel 3/4-S Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH + Co.KG (D-73494 Rosenberg, Germany) was available to animals during the study. Certified nest building material was also provided for animals (ARBOCEL crinklets natural produced by J. Rettenmaier & Söhne GmbH + Co.KG).Identification and randomisationA unique number written on the tail with a permanent marker identified each animal. The animal number was assigned on the basis of CiToxLAB Hungary Ltd.’s master file. The cages were marked with identity cards with information including study code, cage number, and dose group, sex and individual animal number. The animals were randomised and allocated to the experimental groups. The randomisation was checked by computer software using the body weight to verify the homogeneity and variability between the groups.

Study design: in vivo (LLNA)

Vehicle:
other: 1% aqueous Pluronic® PE9200
Concentration:
The Preliminary Irritation/Toxicity Test was using two doses at test item concentrations of 50 % (w/v) and 25 % (w/v) in 1% Pluronic.Based on the high variability and/or equivocal results in the body weight values of the preliminary experiment, 50% (w/v) dose group is selected as top dose for the main test. The test concentrations for the main test were 50% (w/v), 25% (w/v), 10 % (w/v) and 5% (w/v).
No. of animals per dose:
Preliminary Irritation/Toxicity Test: 2 animals/doseMain Test: 4 animal/dose
Details on study design:
Formulation
The solubility of the test item was examined in a short Preliminary Compatibility Test. The following standard OECD vehicles were assessed: AOO (acetone:olive oil 4:1 (v:v) mixture), N,N-dimethylformamide (DMF), Methyl ethyl ketone (MEK), Propylene glycol (PG), Dimethyl sulfoxide (DMSO) and 1% aqueous Pluronic® PE9200. The best vehicle taking into account the test item characteristics, its usage and the requirements of the relevant OECD guideline was considered to be 1% Pluronic. The formulation at 50 % (w/v) using 1% Pluronic as vehicle was suitable for the test.The 50% (w/v), 25% (w/v), 10 % (w/v) and 5% (w/v) formulations appeared to be suspension by visual examination.The test item was weighed and formulations prepared daily on a weight:volume basis (as % (w/v)) in the Pharmacy of CiToxLAB Hungary Ltd.Analytical determination of the test item concentration, stability and homogeneity was not performed because of the character and the short period of study.

Dose Selection and Justification of Dose Selection
The Preliminary Irritation/Toxicity Test was started according to the Study Plan on CBA/CaOlaHsd mice using two doses (2 animals/dose) at test item concentrations of 50 % (w/v) and 25 % (w/v) in 1% Pluronic. The preliminary experiment was conducted in a similar experimental manner to the main study, but it was terminated on Day 6 and the radioactive proliferation assay was not performed.The maximum concentration of test item in an acceptable solvent was established according to OECD guideline 429. Based on the observation of the solubility test, the maximum available concentration was 50 % (w/v).In the Preliminary Irritation / Toxicity Test, all mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Both ears of each mouse were observed for erythema and scored. Ear thickness was also measured using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals.During the Preliminary Irritation / Toxicity Test, no mortality or signs of systemic toxicity were observed. Test item precipitate or minimal amount of test item precipitate was observed on the ears of the animals for both dose groups on Day 3. Marked body weight loss (>5% reduction of body weight) was detected for both animals of the 50% (w/v) dose group. Thus the mean body weight loss of this group was more than 5 %. For one animal of the 25 % (w/v) dose group, the body weight loss was also more than 5 %. Consequently, the mean body weight loss of this group was slightly more than 5 %.The ear thickness values and ear punch weights were within the acceptable range. There were no indications of any irritancy at the site of application on the experimental animals.The draining auricular lymph nodes of the animals were visually examined: they were normal in both dose groups (subjective judgement by analogy with observations of former experiments).Based on the high variability and/or equivocal results in the body weight values of the preliminary experiment, 50% (w/v) dose group is selected as top dose for the main test and an additional fourth dose group was also included to ensure that there were three analysable concentrations in the main test.

Topical application
During the study, animals were topically dosed with 25 µL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.PROLIFERATION ASSAYInjection of Tritiated Thymidine (3HTdR)On Day 6, animals were taken to the radioactive suite and each mouse was intravenously injected via the tail vein with 250 µL of sterile PBS (phosphate buffered saline) containing approximately 20 µCi of 3HTdR using a gauge 25G x 1" hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).

Removal and Preparation of Draining Auricular Lymph Nodes
Five hours (± 30 minutes) after intravenous injection the mice were euthanized by asphyxiation with ascending doses of carbon dioxide (deep anaesthesia was confirmed before making incision, death was confirmed before discarding carcasses).The draining auricular lymph nodes were excised by making a small incision on the skin between the jaw and sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. The nodes were then removed using forceps. The carcasses were discarded after cervical dislocation or after cutting through major cervical blood vessels.Once removed, the nodes of each mouse were collected in separate Petri dishes containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing. The nodes of each animal were processed individually.

Preparation of Single Cell Suspension of Lymph Node Cells
A single cell suspension (SCS) of lymph node cells (LNCs) was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). LNCs were pelleted with a relative centrifugal force (RCF) of 190 x g (approximately) for 10 minutes at 4 °C.After centrifugation, supernatants were discarded. Pellets were gently resuspended and 10 mL of PBS was added to the tubes. The washing step was repeated twice. This procedure was repeated for the lymph nodes of each individual animal.Determination of Incorporated 3HTdRAfter the final washing step, supernatants were removed. Pellets were gently agitated resuspended and 3 mL of 5 % (w/v) TCA solution was added to the tubes for precipitation of macromolecules.After overnight (approximately 18 hours) incubation at 2-8 °C, precipitates were centrifuged (approximately 190 x g for 10 minutes at 4 °C), and supernatants were removed. Pellets were resuspended in 1 mL of 5 % (w/v) TCA solution and dispersed by using an ultrasonic bath. Samples were transferred into a suitable sized scintillation vial filled with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded into a ß-scintillation counter and 3HTdR incorporation was measured (10-minute measurement).The ß-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM). Background level was also measured in duplicates by adding 1 mL of 5 % (w/v) TCA solution into a scintillation vial filled with 10 mL of scintillation liquid.

OBSERVATIONS
Clinical Observations
During the study (Day 1 to Day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Individual records were maintained.

Measurement of Body Weight
Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.

EVALUATION OF THE RESULTS
The proliferative response of lymph node cells from the lymph nodes of each individual animal is expressed as radioactive disintegrations per minute (DPM) per animal. The average of the two measured DPM values of 5 % (w/v) TCA solutions was used as background DPM value.The results were expressed as disintegrations per node (DPN = DPM divided by the number of lymph nodes) for each animal following the industry standard for data presentation.The stimulation index (SI = mean DPN of treated group divided by mean DPN of the appropriate control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result.Interpretation of ResultsThe test item is regarded as a sensitizer if both of the following criteria are fulfilled:- That exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.- The data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.Acceptability of the testThe Local Lymph Node Assay is considered valid if it meets the following criteria:- the DPN value of the negative (vehicle) control group falls within the range of historical laboratory control data,- the positive control substance produces a significant lymphoproliferative response increases (SI>3),- each treated and control group includes at least 4 animals,- the test item does not cause serious systemic or local toxicity.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The use of the individual approach to calculate the SI made the use of a statistical analysis available. The statistical analysis was performed using the SPSS/PC+ (4.0.1) software package. The heterogeneity of variance between groups was checked by Bartlett's test for the measured DPM values.Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the result was positive, then Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by the Kolmogorow-Smirnow test. In the case of no normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using the Mann-Whitney U-test.

Results and discussion

Positive control results:
The result of the positive control substance a-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. The positive control substance was examined at a concentration of 25 % in the relevant vehicle (1% Pluronic) using CBA/CaOlaHsd mice.No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A lymphoproliferative response in line with historic positive control data (stimulation index value of 10.0) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.Furthermore, the DPN values observed for the vehicle and positive control substance in this experiment were in within the historical control range. Each treated and control group included 4 animals.

In vivo (LLNA)

Results
Parameter:
SI
Value:
>= 0.7 - <= 1
Test group / Remarks:
Overall
Cellular proliferation data / Observations:
CLINICAL OBSERVATION
No mortality or signs of systemic toxicity were observed during the main study. There were no indications of any irritancy at the site of application on the experimental animals.Test item precipitate or minimal amount of test item precipitate was observed for all animals of the 50% (w/v) dose group on Days 2-3 and for all animals of the 25% (w/v) dose group on Day 3.

BODY WEIGHT MEASUREMENT
Marked body weight loss (=5%) was observed for one animal of the 25% (w/v) dose group and for one animal of the 10% (w/v) dose group, however these changes were considered as individual variability.

PROLIFERATION ASSAY
The appearance of the lymph nodes was normal in the negative control group and in all examined dose groups. Larger than normal lymph nodes were observed in the positive control group.The stimulation index values were 0.9, 0.7, 0.8 and 1.0 at concentrations of 50% (w/v), 25% (w/v), 10 % (w/v) and 5% (w/v), respectively.

INTERPRETATION OF OBSERVATIONS
The test item was solid, which was formulated in 1% Pluronic. Since there were no confounding effects of irritation or systemic toxicity at the applied concentrations, the proliferation values obtained are considered to reflect the real potential of the test item to cause lymphoproliferation in the Local Lymph Node Assay. The resulting stimulation indices observed under these exaggerated test conditions was considered to be good evidence that Calcium hydrogen phosphonate is a non-sensitizer. The size of lymph nodes was in good correlation with this conclusion. Based on the observed results, the test item does not need classification according to the GHS or CLP.

Any other information on results incl. tables

Individual Body Weights for all Animals with Group Means

Identity Number

Test Group Name

Initial Body Weight (g)

Terminal Body Weight*(g)

Change#(%)

2004

1992

1975

1997

Negative (vehicle) control

1% Pluronic

 

 

Mean

21.5

19.9

19.4

19.2

20.0

21.8

19.9

20.5

19.5

20.4

1.4

0.0

5.7

1.6

2.2

1986

2000

1978

1990

Calcium hydrogen phosphonate

50 (w/v)% in 1% Pluronic

 

 

Mean

21.5

19.6

20.0

19.3

20.1

21.1

19.6

19.5

19.8

20.0

-1.9

0.0

-2.5

2.6

-0.4

1996

2002

1995

2011

Calcium hydrogen phosphonate

25 (w/v)% in 1% Pluronic

 

 

Mean

21.5

19.7

19.3

19.3

20.0

20.1

19.6

19.9

18.7

19.6

-6.5

-0.5

3.1

-3.1

-1.8

1991

2003

1994

1998

Calcium hydrogen phosphonate

10 (w/v)% in 1% Pluronic

 

 

Mean

21.1

20.6

19.9

19.4

20.3

19.8

20.9

19.7

19.2

19.9

-6.2

1.5

-1.0

-1.0

-1.7

1993

2009

2006

2001

Calcium hydrogen phosphonate

5 (w/v)% in 1% Pluronic

 

 

Mean

21.4

20.6

19.1

19.3

20.1

21.3

20.5

19.3

19.2

20.1

-0.5

-0.5

1.0

-0.5

-0.1

2019

2005

1999

2007

Positive control

25 (w/v)% HCA in 1% Pluronic

 

 

Mean

20.6

20.3

20.3

19.1

20.1

20.6

19.8

20.2

19.7

20.1

0.0

-2.5

-0.5

3.1

0.0

Notes:

*: Terminal body weights were measured on Day 6.

#: = (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100

 

DPM, DPN and Stimulation Index Values for all Groups

Test Group Name

Animal Number

Measured DPM

DPM

Number of lymph nodes

DPN

Group DPN

Stimulation Index

Background

(5% (w/v) TCA)

-

34

34

-

-

-

-

-

Negative control

(1% Pluronic)

1

2

3

4

590

468

867

606

556.0

434.0

833.0

572.0

2

2

2

2

278.0

217.0

416.5

286.0

299.4

1.0

Calcium hydrogen phosphonate

50 (w/v)% in 1% Pluronic

9

10

11

12

655

491

601

467

621.0

457.0

567.0

433.0

2

2

2

2

310.5

228.5

283.5

216.5

259.8

0.9NS

Calcium hydrogen phosphonate

25 (w/v)% in 1% Pluronic

13

14

15

16

233

429

468

674

199.0

395.0

434.0

640.0

2

2

2

2

99.5

197.5

217.0

320.0

208.5

0.7NS

Calcium hydrogen phosphonate

10 (w/v)% in 1% Pluronic

17

18

19

20

720

522

297

399

686.0

488.0

263.0

365.0

2

2

2

2

343.0

244.0

131.5

182.5

225.3

0.8NS

Calcium hydrogen phosphonate

5 (w/v)% in 1% Pluronic

21

22

23

24

637

519

697

685

603.0

485.0

663.0

651.0

2

2

2

2

301.5

242.5

331.5

325.5

300.3

1.0NS

Positive control

(25% HCA) in 1% Pluronic

25

26

27

28

6165

6046

6403

5514

6131.0

6012.0

6369.0

5480.0

2

2

2

2

3065.5

3006.0

3184.5

2740.0

2999.0

10.0**

Notes:

1. NS = Not Significant (p<0.05, Duncan Test versus negative control)

2. * = Significant (p<0.05, Duncan Test versus negative control)

3. ** = Significant (p<0.01, Duncan Test versus negative control)

 

Summarized Clinical Observations

Group

Animal No.

CLINICAL OBSERVATIONS

DAY 1

DAY 2

DAY 3

DAY 4

DAY 5

DAY 6

Negative control

(1% Pluronic)

1

 

2

 

3

 

4

 

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Calcium hydrogen phosphonate

50 (w/v)% in 1% Pluronic

5

 

6

 

7

 

8

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free*

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Calcium hydrogen phosphonate

25 (w/v)% in 1% Pluroinc

9

 

10

 

11

 

12

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

BT: symptom-free

AT: symptom-free**

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Calcium hydrogen phosphonate

10 (w/v)% in 1% Pluronic

13

 

14

 

15

 

16

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Calcium hydrogen phosphonate

5 (w/v)% in 1% Pluronic

17

 

18

 

19

 

20

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Positive control

(25% (w/v) HCA in 1% Pluronic)

21

 

22

 

23

 

24

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

BT: symptom-free

AT: symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Symptom-free

 

Symptom-free

 

Symptom-free

 

Symptom-free

Note:

1. BT: Before treatment, AT: After treatment

2. *: test item precipitate. **: minimal amount of test item precipitate

 

 

 

Results of the Preliminary Irritation / Toxicity Test

 

Individual Body Weights for all Animals with Group Means

Identity Number

Animal Number

Test Group Name

Initial Body Weight (g)

Terminal Body Weight*(g)

Change#(%)

1883

1885

1

2

50%

50%

Mean

23.6

24.1

23.9

21.8

22.3

22.1

-7.6

-7.5

-7.5

1891

1890

3

4

25%

25%

Mean

23.2

24.1

23.7

21.0

23.8

22.4

-9.5

-1.2

-5.4

Notes:

1. *: Terminal body weights were measured on Day 6

2. #: = (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100

 

Individual Ear Thickness for all Animals

Identity Number

Animal Number

Test Group Name

Ear Thickness on Day 1 (mm)

Ear Thickness on Day 3 (mm)

Ear Thickness on Day 6 (mm)

Biopsy weight*on Day 6 (mg)

Right

Left

Right

Left

Right

Left

1883

1885

1891

1890

1

2

3

4

50%

50%

25%

25%

0.22

0.21

0.21

0.21

0.21

0.22

0.21

0.21

0.22

0.22

0.21

0.22

0.22

0.22

0.21

0.21

0.22

0.21

0.21

0.22

0.23

0.22

0.21

0.21

15.97

16.54

16.19

15.55

Note:

1. *: Historical control range: 11.92-22.53 mg. Positive response is over 28.16 mg (=25%)

 

Summarized Clinical Observations

Period

Group

Animal No.

Identity No.

Clinical observations

DAY 1

50% (w/v)

1

1883

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

2

1885

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

25% (w/v)

3

1891

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

4

1980

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

DAY 2

50% (w/v)

1

1883

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

2

1885

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

25% (w/v)

3

1891

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

4

1890

Before treatment: symptom-free, ES: 0

After treatment: symptom-free, ES: 0

DAY 3

50% (w/v)

1

1883

Before treatment: symptom-free, ES: 0

After treatment: symptom-free*, ES: 0

2

1885

Before treatment: symptom-free, ES: 0

After treatment: symptom-free*, ES: 0

25% (w/v)

3

1891

Before treatment: symptom-free, ES: 0

After treatment: symptom-free**, ES: 0

4

1890

Before treatment: symptom-free, ES: 0

After treatment: symptom-free**, ES: 0

DAY 4

50% (w/v)

1

1883

Symptom-free, ES: 0

2

1885

Symptom-free, ES: 0

25% (w/v)

3

1891

Symptom-free, ES: 0

4

1890

Symptom-free, ES: 0

DAY 5

50% (w/v)

1

1883

Symptom-free, ES: 0

2

1885

Symptom-free, ES: 0

25% (w/v)

3

1891

Symptom-free, ES: 0

4

1890

Symptom-free, ES: 0

DAY 6

50% (w/v)

1

1883

Symptom-free, ES: 0

2

1885

Symptom-free, ES: 0

25% (w/v)

3

1891

Symptom-free, ES: 0

4

1890

Symptom-free, ES: 0

Notes:

1. The clinical observation of animals on the first day was performed simultaneously with the body weight measurements.

2. ES: Erythema score

3. *: test item precipitate, **: minimal amount of test item precipitate

 

Historical Control Data

 

Historical Control Data of the Positive and Negative Controls for CBA/J Rj mice (2008-2014)

CBA/J Rj mice

 

Vehicles

Acetone : Olive oil 4:1 (AOO)

1% Pluronic PE9200 in water (1%Plu)

DPN values

SI value

DPN values

SI value

 

Control

HCA 25%

HCA 25%

Control

HCA 25%

HCA 25%

Average

162.3

1627.0

11.7

117.0

931.2

8.6

Range:  min

               max

36.4

586.9

304.9

4602.1

3.2

52.7

21.4

469.6

146.3

6258.1

1.8

55.1

Number of cases

105

81

81

127

84

84

 

 

Vehicles

N,N-Dimethylformamide (DMF)

Dimethyl sulfoxide (DMSO)

DPN values

SI value

DPN values

SI value

 

Control

HCA 25%

HCA 25%

Control

HCA 25%

HCA 25%

Average

165.6

2484.6

14.7

210.0

2278.2

11.2

Range:  min

               max

20.8

1843.0

350.9

28287.0

3.8

75.7

80.6

424.3

848.2

5441.2

4.8

24.1

Number of cases

90

56

56

28

20

20

 

 

Vehicles

Propylene glycol (PG)

Methyl ethyl ketone (MEK)

DPN values

SI value

DPN values

SI value

 

Control

HCA 25%

HCA 25%

Control

HCA 25%

HCA 25%

Average

120.7

1488.1

13.4

203.9

2525.5

14.0

Range:  min

               max

38.4

288.8

510.4

3231.3

3.7

27.9

72.2

572.2

412.6

4056.4

4.4

26.5

Number of cases

22

21

21

11

9

9

HCA 25% = alpha-Hexylcinnamaldehyde 25% (w/v)

SI (Stimulation Index) = DPN of a treated group divided by DPN of the appropriate control group.

DPN (Disintegrations Per Node) = DPM (Disinitgrations Per Minute) divided by the number of lymph nodes.

In case of individual approach, SI values were calculated from the mean DPN values of the group.

 

Historical Control Data of the Positive and Negative Controls for CBA/CaOlaHsd mice (2014-2015)

CBA/J Rj mice

 

Vehicles

Acetone : Olive oil 4:1 (AOO)

1% Pluronic PE9200 in water (1%Plu)

DPN values

SI value

DPN values

SI value

 

Control

HCA 25%

HCA 25%

Control

HCA 25%

HCA 25%

Average

415.2

2922.6

7.5

197.7

1825.3

10.0

Range:  min

               max

111.3

847.8

890.3

7674.5

3.3

15.5

23.0

680.8

154.0

6755.8

3.0

33.1

Number of cases

32

32

30

134

134

128

 

 

Vehicles

N,N-Dimethylformamide (DMF)

Dimethyl sulfoxide (DMSO)

DPN values

SI value

DPN values

SI value

 

Control

HCA 25%

HCA 25%

Control

HCA 25%

HCA 25%

Average

244.6

2522.6

10.8

488.7

3212.1

7.8

Range:  min

               max

140.8

505.8

1201.3

4804.6

6.3

21.3

238.5

934.6

2017.2

4877.5

3.1

14.5

Number of cases

21

21

21

13

13

12

 

 

Vehicles

Propylene glycol (PG)

Methyl ethyl ketone (MEK)

DPN values

SI value

DPN values

SI value

 

Control

HCA 25%

HCA 25%

Control

HCA 25%

HCA 25%

Average

235.4

2371.8

10.0

260.2

4888.8

19.5

Range:  min

               max

63.3

506.0

817.3

4978.0

6.5

14.4

183.5

383.3

2356.3

8682.5

8.9

36.3

Number of cases

14

14

14

9

10

10

HCA 25% = alpha-Hexylcinnamaldehyde 25% (w/v)

SI (Stimulation Index) = DPN of a treated group divided by DPN of the appropriate control group.

DPN (Disintegrations Per Node) = DPM (Disinitgrations Per Minute) divided by the number of lymph nodes.

In case of individual approach, SI values were calculated from the mean DPN values of the group.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the assay, Calcium hydrogen phosphonate, tested in a suitable vehicle, was shown to have no sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.
Executive summary:

The aim of this study was to determine the skin sensitisation potential of Calcium hydrogen phosphonate following dermal exposure. The study was performed with vertebrate animals as no full regulatory in vitro alternative is available. The minimum number of animals was used, corresponding with the specific requirements of the Sponsor.

 

Based on the results of the Preliminary Compatibility Test, the test item characteristics, its usage and on the recommendations of the OECD Guideline, the test item was tested for formulation compatibility in 1% aqueous Pluronic® PE9200 solution (abbreviated as 1% Pluronic). The highest achievable concentration based on the regulatory requirements of the OECD guideline and the physical characteristics of the test item was 50 % (w/v).

 

The Preliminary Irritation/Toxicity Test was performed in CBA/CaOlaHsd mice using two doses (2 animals/dose): 50 and 25 % (w/v) in 1% Pluronic. Based on the observations recorded in the preliminary test, the 50 % (w/v) was selected as top dose for the main test.

 

In the main assay, twenty four female CBA/CaOlaHsd mice were allocated to six groups of four animals each:

- four groups received Calcium hydrogen phosphonate (formulated in 1% Pluronic) at 50% (w/v), 25 % (w/v), 10% (w/v) and 5% (w/v) concentrations,

- the negative control group received the vehicle (1% Pluronic),

- the positive control group received 25 % (w/v) HCA (dissolved in 1% Pluronic).

 

The test item solutions were applied on the dorsal surface of ears of experimental animals (25 µL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. The cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI).

 

No mortality or signs of systemic toxicity were observed during the study. Test item precipitate or minimal amount of test item precipitate was observed for all animals of the 50% (w/v) dose group on Days 2-3 and for all animals of the 25% (w/v) dose group on Day 3. There were no indications of any irritancy at the site of application on the experimental animals. Marked body weight loss (=5%) was observed for one animal of the 25% (w/v) dose group and for one animal of the 10% (w/v) dose group.

 

The stimulation index values were 0.9, 0.7, 0.8 and 1.0 at concentrations of 50% (w/v), 25% (w/v), 10 % (w/v) and 5% (w/v), respectively.

 

The result of the positive control substance a-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. A lymphoproliferative response in line with historic positive control data was noted for the positive control chemical, this result confirmed the validity of the assay.

 

In conclusion, under the conditions of the present assay, Calcium hydrogen phosphonate, tested in a suitable vehicle, was shown to have no sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.

 

The following classification/labelling is triggered:

Regulation (EC) No 1272/2008 (CLP) / GHS (rev. 6) 2015: none.