Reaction mass of [[(2-hydroxyethyl)imino]dimethylene]bisphosphonic acid, sodium salt and 4-(Phosphonomethyl)-2-hydroxy-2-oxo-1,4,2-oxazaphosphorinane, sodium salt

Endpoint summary

Description of key information

In the key in vivo skin sensitisation study for HEBMP-xNa, conducted according to OECD Test Guideline 429 and in compliance with GLP, the test material was concluded to be not sensitising to skin (Harlan Laboratories, 2012e).  

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:

The study was performed between 03 May 2012 and 16 May 2012.

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Harlan Laboratories UK Ltd., Oxon, UK. 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 (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) 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.

Vehicle:

other: 1% pluronic L92 in distilled water

Concentration:

Test item at concentrations of 10%, 5% or 2.5% w/w in 1% pluronic L92 in distilled water.

No. of animals per dose:

Groups of five mice were treated per concentration.

Details on study design:

PREPARATION OF TEST ITEM
For the purpose of the study, the test item was freshly prepared as a emulsion in 1% pluronic L92 in distilled water. This vehicle was chosen as it produced the highest concentration that was suitable for dosing. The concentrations used are given in the procedure section. 

The test item was formulated within two hours of being applied to the test system. It is assumed that the formulation was stable for this duration. 
No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. 

PREPARATION OF POSITIVE CONTROL:
The positive control item (α-Hexylcinnamaldehyde, tech., 85%) was freshly prepared as a 25% v/v dilution in 1% pluronic L92 in distilled water.

PROCEDURE:
PRELIMINARY SCREENING TEST:
Using available information regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µl of the test item at a maximum attainable concentration of 10% w/w in 1% pluronic L92 in distilled water, 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 in the 'any other information on materials and methods section (see Scale for Erythema). 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 Days 1 to 3 and Days 1 to 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 five mice were treated with the test item at concentrations of 10%, 5% or 2.5% w/w in 1% pluronic L92 in distilled water. 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 five mice received the vehicle alone in the same manner.

The positive control animals were similarly treated to the test animals except that 25 µl of the positive control item, α Hexylcinnamaldehyde, tech., 85%, at a concentration of 25% v/v in 1% pluronic L92 in distilled water was applied to the dorsal surface of each ear.

The thickness of each ear was measured using an Oditest micrometer (Dyer, PA), pre dose on Day 1 and on Days 2, 3, 4, 5 and 6. Any changes in the ear thickness were noted. Daily mean ear thickness changes were calculated. 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.

3H-METHYL THYMIDINE ADMINISTRATION:
Five days following the first topical application of the test item, vehicle control or positive control item (Day 6) all mice were injected via the tail vein with 250 µl of phosphate buffered saline (PBS) containing 3H methyl thymidine (3HTdR) 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.

Local Skin Irritation Observations: All animals were observed daily. Any signs of local skin irritation noted during the test were recorded.

Bodyweights: The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

TERMINAL PROCEDURES:
Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. For each individual animal of each group the draining auricular lymph nodes were excised and processed. For each individual animal 1 ml of PBS was added to the lymph nodes.

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

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

Positive control substance(s):

other: α-Hexylcinnamaldehyde, tech., 85%

Statistics:

Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships by analysis of homogeneity of variance followed by one way analysis of variance (ANOVA). In the event of a significant result from the ANOVA, pairwise comparisons were performed between control and treated groups. For homogenous datasets Dunnett’s Multiple Comparison test was used and for non homogenous datasets Dunnett’s T3 Multiple Comparison Method was used.
Probability values (p) are presented as follows:
P<0.001 ***
P<0.01 **
P<0.05 *
P>0.05 (not significant)

Positive control results:

The positive control at 25% v/v in 1% pluronic L92 in distilled water gave a Stimulation Index of 9.31, giving a positive result from skin sensitisation.

Key result

Parameter:

SI

Value:

1.54

Test group / Remarks:

2.5% w/w in 1% pluronic L92 in distilled water

Remarks on result:

other: negative

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

5% w/w in 1% pluronic L92 in distilled water

Remarks on result:

other: negative

Key result

Parameter:

SI

Value:

1.01

Test group / Remarks:

10% w/w in 1% pluronic L92 in distilled water

Remarks on result:

other: negative

Key result

Parameter:

SI

Remarks:

Positive control, α-Hexylcinnamaldehyde

Value:

9.31

Test group / Remarks:

25% w/w in 1% pluronic L92 in distilled water

Remarks on result:

other: positive

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 excessive irritation indicated by anequal to or greater than 25% increase in mean ear thicknesswere noted.

Based on this information the dose levels selected for the main test were 10%, 5% and 2.5% w/w in 1% pluronic L92 in distilled water.

See attached background material for Tables 1, 2 and 3.

Main Test

Estimation of the Proliferative Response of Lymph Node Cells

The radioactive disintegrations per minute per animal and the stimulation index are given in Table 4 (see attached background material)

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:

Treatment Group	Concentration	Stimulation Index	Result
Test Item	2.5%w/win 1% pluronicL92 in distilled water	1.54	Negative
	5%w/win 1% pluronicL92 in distilled water	1.00	Negative
	10%w/win 1% pluronicL92 in distilled water	1.01	Negative
Positive Control Item	25% v/v in 1% pluronicL92 in distilled water	9.31	Positive

Clinical Observations and Mortality Data

Individual clinical observations and mortality data for test and control animals are given in Table 5 and local skin irritation is given in Table 6. The ear thickness measurements and mean ear thickness changes are given in Table 7.

See attached background material for Tables 5, 6 and 7.

There were no deaths. No signs of systemic toxicity were noted in the test or control group animals during the test. Very slight erythema was noted on Days 3 and 4 on both ears of the animals treated with the positive control item. No visual local skin irritation was noted in the vehicle control group animals or test item group animals. No excessive irritation indicated by anequal to or greater than 25% increase in mean ear thicknesswas noted.

Bodyweight

Individual bodyweights and bodyweight changes for test and control animals are given in Table 8 (see attached background material)

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.

Interpretation of results:

GHS criteria not met

Conclusions:

In the in vivo skin sensitisation study for HEBMP-xNa, conducted according to OECD Test Guideline 429 and in compliance with GLP, the test material was concluded to be not sensitising to skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In the key in vivo skin sensitisation study for HEBMP-xNa, conducted according to OECD Test Guideline 429 and in compliance with GLP, the test material was concluded to be not sensitising to skin (Harlan Laboratories, 2012e).  

Following a preliminary screening test, in which no clinical signs of toxicity were noted at a maximum attainable concentration of 10% 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 five animals, were treated with 50 µl (25 µl per ear) of the test item as an emulsion in 1% pluronic L92 in distilled water at concentrations of 10%, 5% or 2.5% w/w. A further group of five animals was treated with 1% pluronic L92 in distilled water alone. A concurrent positive control test, using a group of five animals, was also performed with the known sensitiser, α‑hexylcinnamaldehyde tech., 85%, at a concentration of 25% v/v in 1% pluronic L92 in distilled water.

There were no deaths during the study. No signs of systemic toxicity were noted in the test or control group animals during the test. The stimulation index, which is a measure of primary proliferation of radiolabelled lymphocytes is expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group. The corresponding calculated stimulation index (SI) values were 1.54 at 2.5% test item concentration, 1.00 at 5% test item concentration and 1.01 at 10% test item concentration. These values were less than threefold compared to control values, which is the criteria for classification as a ‘non-sensitiser'. Very slight erythema was noted on Days 3 and 4 on both ears of the animals treated with the positive control item. No visual local skin irritation was noted in the vehicle control group animals or test item group animals. No excessive irritation indicated by an equal to or greater than 25% increase in mean ear thickness was noted.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available information, HMDTMP-xNa does not require classification for skin sensitisation according to Regulation (EC) No 1272/2008.