Calcium hydrogen phosphonate

Endpoint summary

Description of key information

Two LLNA tests are available with calcium hydrogen phosphonate. The LLNA test performed with non radiolabelled material and by pooled approaches show an equivocal result (low positivity without dose response) considered not sufficent for concluding definitely about the skin sensitizing potential. The second one perforned with radiolabelled methyl thymidine show no sensitisation potential.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

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

Qualifier:

according to guideline

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

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. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

No further details specified in the study report.

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.

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.

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.

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.

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	0.9^NS
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	0.7^NS
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	0.8^NS
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	1.0^NS
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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The aim of this first LLNA with non radiolabelled material was to assess the skin sensitisation potential of the test item calcium hydrogen phosphonate in the CBA/J strain mouse following topical applications to the dorsal surface of the ear. Three groups of four animals were treated for three consecutive days (D1, D2, D3) with 50 µL (25 µL per ear) of the test item diluted at concentrations of 50%, 25% and 10% in Pluronic® L92 1%. A further group of 4 animals was treated with Pluronic® L92 1%. On D6, the proliferation of lymphocytes in the draining auricular lymph nodes was determined by cell counting. The experimental protocol was established according to the OECD test guideline no.429 (2010).

No mortality and no signs of systemic toxicity were noted in the test and control animals during the test. No significant increases in ear thickness and in ear weight was noted in animals treated at 10%, 25%, and 50%. The test item has to be considered as non-irritant at these concentrations.

The stimulation index (SI) calculated by pooled approach was 1.26, 1.48, and 1.36 for the treated groups at 10%, 25% and 50% respectively. The EC1.4 determined by linear regression was 19.55%

The result of the study is positive due to the fact that the SI is higher than 1.4 in the treated group at 25%. However, it may be considered as a borderline result (i.e. SI values around the cut-off value 1.48 versus 1.40 in the treated group at 25% and 1.36 versus 1.4 in the treated group at 50%). Futhermore no dose response was noted.

The result of the positive control substancea-Hexylcinnamaldehyde (HCA) dissolved in acetone/olive oil (4:1; v/v) was used to demonstrate the appropriate performance of the assay

In conclusion, the results of this LLNA may be considered not sufficient clear for concluding definitely about the skin sensitizing potential of the test item Calcium Hydrogen phosphonate.

Thus, a second study, LLNA with radiolabelled material was performed 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 substancea-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.

Justification for classification or non-classification

In conclusion, regarding the different data available, calcium hydrogen phosphonate, tested in a suitable vehicle, was shown to have no sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.

Thus, the following classification/labelling is triggered:

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