Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2013, March
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study, well conducted on the similar substance Reaction mass of dipotassium phosphonate and Phosphonic acid, potassium salt (1:1). Rationale for Read Across is attached at point 13

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: potassium phosphonate KH2PO3/K2HPO3
- Substance type: inorganic
- Physical state: clear free-flowing white/colourless solution
- Storage condition of test material: ambient condition
- Stability: no assay of test item stability, nor its concentration and homogeneity in solvent were undertaken
- Expiration date of the lot/batch: 16/12/2013
- Preparation: the test item was used in the form supplied, without any further dilution. No analysis was carried out.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Animal supply and acclimatisation

Species and strain : Mice, CBA/JN
Sex : Females (nulliparous and non-pregnant)
Age and weight range : 7 to 8 weeks old, 21 to 25 grams
(at order)
Supplier : Charles River Italia S.p.A., Calco (Lecco), Italy
Breeder : Charles River France Laboratories, Iffa Credo, Domaine des Oncins B.P. 0109, F 69592 L’ARBRESLE CEDE
X, France.
Date of arrival : 07 March 2013
Weight range at arrival : 21 to 23 grams
Acclimatisation period : At least 5 days
Veterinary health check : After arrival

Caging

No. of animals/cage : 1/cage during the study; up to 5 during acclimatisation
Housing : Polysulphone solid bottomed cages measuring 35.5 x 23.5 x 19 cm with nesting material
Cage tray control : Daily inspected and changed as necessary (at least twice/week)

Water and diet

Water : Drinking water supplied to each cage via a water bottle
Water supply : Ad libitum
Diet : 4 RF 21 (Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI) Italy)
Diet supply : Ad libitum throughout the study

Records of analyses of water and diet are kept on file at RTC. Components present in the drinking water or diet are not at a level likely to interfere with the purpose or conduct of the study.

Housing conditions (parameters set)

Room lighting : Artificial (fluorescent tubes), daily light/dark cycle of 12/12 hours
Air changes : Approximately 15 to 20 air changes per hour
Temperature range : 22°C ± 2°C
Relative humidity range : 55% ± 15%
Actual conditions were monitored and recorded and records retained. No relevant deviations occurred.

Study design: in vivo (LLNA)

Vehicle:
unchanged (no vehicle)
Concentration:
Preliminary phase : 100, 50, 25, 10, 5% w/w
Main Phase : 100, 25, 50% w/w
No. of animals per dose:
Preliminary phase

Group Treatment Dose level Animal number
Number (% w/w)+ Females (odd)

1 Test item 100 1
Test item 50 3
Test item 25 5
Test item 10 7
Test item 5 9
Vehicle 0 11

+ = in terms of test item as supplied


MAIN PHASE

Group Treatment Dose level Animal number
Number (% w/w)+ Females (odd)

1 Vehicle 0 13-19
2 Test item 25 21-27
3 Test item 5029-35
4 Test item 10 37-43
5 Positive control 25 45-51

+ = in terms of test item and positive control item as supplied





Details on study design:
RANGE FINDING TESTS:
- Irritation: A preliminary irritancy test was carried out to select three concentrations to be used in a main assay, according to the criteria described in the relevant guideline for this test. A main phase was then carried out to fully evaluate lymph node cell reaction.





Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Differences between each treated group and the control group (individual BrdU labelling indices) were assessed by Dunnett's test. The homogeneity of the data was verified by Bartlett's test before Dunnett's test. If data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied.

Results and discussion

Positive control results:
In the group treated with the positive control item, a stimulation index (SI) of 2.96 was calculated. As it was greater than 2, the test system was regarded as valid.

In vivo (LLNA)

Results
Parameter:
SI
Remarks on result:
other: No increase in cell proliferation of draining lymph nodes was observed in any treatment group. The calculated stimulation indices (SI) were 0.93, 0.79 and 0.77, respectively at low, mid- and high dose levels

Any other information on results incl. tables

Preliminary test

 

Five concentrations (the undiluted test item and 50, 25, 10, 5% w/w) were selected to be used in the preliminary phase.

No signs of toxicity (clinical signs or toxicologically relevant body weight losses) were observed at any of the tested concentrations.

The evaluation of visible reactions showed no erythema at any of the concentrations investigated.

The evaluation of ear thickness indicated that the reaction was acceptable (increase of less than 25% compared to Day 1) at the concentration of 100% (undiluted test item).

The evaluation of ear punch weight indicated that the reaction was acceptable (increase of less than 25% with respect to the negative control) at all the investigated concentrations.

According to the results described above, the undiluted test item was the highest concentration selected for the main phase.

 

Main Assay - In vivo phase

 

No mortality nor significant clinical signs were recorded in animals treated at all dose levels.

Body weight decreases/reduced body weight gains observed in some animals from all groups (including controls) were considered of low entity and/or incidental and thus not toxicologically relevant.

Five concentrations (the undiluted test item and 50, 25, 10, 5% w/w) were selected to be used in the preliminary phase.

No signs of toxicity (clinical signs or toxicologically relevant body weight losses) were observed at any of the tested concentrations.

The evaluation of visible reactions showed no erythema at any of the concentrations investigated.

The evaluation of ear thickness indicated that the reaction was acceptable (increase of less than 25% compared to Day 1) at the concentration of 100% (undiluted test item).

The evaluation of ear punch weight indicated that the reaction was acceptable (increase of less than 25% with respect to the negative control) at all the investigated concentrations.

According to the results described above, the undiluted test item was the highest concentration selected for the main phase.

 

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The results obtained in this study indicate that the test item does not elicit a sensitisation response in mice following dermal exposure.
Executive summary:

Preliminary phase

The undiluted test item and four concentrations (50, 25, 10, 5% w/w) in acetone:olive oil 4:1 v/v were selected to be used in the preliminary phase, in order to identify a non toxic and minimally irritant concentration and avoid false positive results.

No signs of toxicity (clinical signs or body weight losses) were observed at the tested concentrations.

According to the results of the irritation screening, the undiluted test item was judged to be not irritant.

 

Main assay

In the main assay, the test item was topically administered at the concentrations of 100, 50 and 25% w/w, in acetone:olive oil 4:1 v/v.

No mortality or clinical signs were recorded in any animal.

Changes in body weight observed during the study were within the expected range for this strain and age of animals.

No increase in cell proliferation of draining lymph nodes was observed in any treatment group. The calculated stimulation indices (SI) were 0.93, 0.79 and 0.77, respectively at low, mid- and high dose levels.

In the group treated with the positive control item, a stimulation index (SI) of 2.96 was calculated. As it was greater than 2, the test study was regarded as valid.

The results obtained in this study indicate that the test item does not elicit any sensitisation response in mice following dermal exposure.