Lead dinitrate

Endpoint summary

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:

15 January 2014 - 17 February 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

yes

Remarks:

Environmental parameters: A temperature of 17°C was registered on 20 January 2014 instead of 19°C (minimal value) as planned in the study plan. This deviation did not in any case influence the result and the development of this study.

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:

other: CBA/J (CBA/J@Rj)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Elevage Janvier Labs (F-53941 Le Genest Saint Isle, France)
- Age at study initiation: 8 weeks
- Weight at study initiation: 19.2 to 23.9 g
- Housing: Individually, in suspended solid-floor polypropylene cages furnished with softwood woodflakes. 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.
- Diet: food (A04, SAFE) was supplied freely
- Water: drinking water (tap water from public distribution system) was supplied freely
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): between 18°C and 23°C
- Humidity (%): between 38% and 60%
- Air changes (per hr): approximately thirteen changes per hour
- Photoperiod (hrs dark / hrs light): the lighting was controlled by a time switch to give twelve hours continuous light (07.00 to 19.00) and twelve hours darkness

Vehicle:

dimethylformamide

Concentration:

10%, 25%, 50%

No. of animals per dose:

4 (An additional mouse was treated in each group in case of problem which may occur during the study, in particular during the excision of lymph nodes)

Details on study design:

PRELIMINARY STUDY
- Study design: As no information was available regarding irritant potential or systemic toxicity of the test item in the mouse, a preliminary screening test was performed using one mouse. The mouse was treated by application of 25 μL of the test item at 50% in Dimethylformamide to the dorsal surface of each ear for two days (Day 1 and Day 2). Any signs of toxicity or excessive local irritation noted during this period were recorded. Ear thickness and bodyweight of the mice were recorded on day 1 (prior to dosing).
- Observations: No mortality and no signs of systemic toxicity were noted at the concentration of 50%. As no sign of excessive irritation was noted at the concentration of 50%, this concentration was chosen as the highest concentration for the main studies.

MAIN STUDIES
Two experiments were performed.
- ANIMAL ASSIGNMENT: 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.
- TREATMENT ADMINISTRATION: 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.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Positive control results:

A study was performed to assess the sensitivity of the strain of mouse to a known sensitiser. Three groups, each of four animals, were treated with 50 μL (25 μL per ear) of α-Hexylcinnamaldehyde, as a solution in acetone/olive oil (4:1; v/v) (AOO) at concentrations of 5%, 10% and 25% (v/v). A further control group of four animals was treated with acetone/olive oil (4:1; v/v) alone. The Stimulation Index (SI) expressed as the cell count for each treatment group divided by the mean cell count of the vehicle control group are as follows: - 5% test item: SI 1.07 (negative) - 10% test item: SI 1.59 (positive) - 25% test item: SI 2.46 (positive) An EC1.4 value of 6.72% could be derived.

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

Experiment 1: The Stimulation Index (SI) calculated by pooled approach was 1.25, 1.51 and 1.13 for the treated groups at 10%, 25% and 50% respectively. The EC1.4 determined by linear regression was 18.65%. Experiment 2: To confirm these results, a second test was performed at the same concentrations. The Stimulation Index (SI) calculated by pooled approach was 1.92, 2.04 and 1.72 for the treated groups at 10%, 25% and 50% respectively. The EC1.4 could not be determined in this experiment.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

According to the OECD guideline, evaluation of LLNA results is based on the measurement of 3H- thymidine incorporation into the lymph node cells in the first place. However, it is stated that “other endpoints for assessment of proliferation may be employed provided there is justification and appropriate scientific support”. Lymph node cell count is more direct measurement of cell proliferation than determination of DNA synthetis and therefore considered an appropriate parameter for evaluation of cell proliferation in the assay. The internationally validated cut-off value for this method is SI=1.4 (instead of 3).

Clinical observations and mortality

No mortality and no signs of systemic toxicity were noted in the test and control animals during the test.

Weight evolution

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.

Local irritation & Stimulation Index

1st experiment:

Slight dryness was noted on day 6 in all animals at the three concentrations. Remaining of the test item was noted on day 3 in all animals at 50%.

No significant increase in ear thickness and in ear weight was noted in animals treated at 10%, 25% and 50%. Therefore, the test item must be considered as not excessively irritant at these three concentrations.

Group	Test item	Excessive irritation (Y/N)	Cell count (x 10^6 cells/ml)	SI	result	EC1.4
1	vehicle (DMF)	N	31.44	/	/	/
2	10%	N	39.24	1.25	negative	18.65%
3	25%	N	47.43	1.51	positive	18.65%
4	50%	N	35.42	1.13	negative	18.65%

2nd experiment:

Remaining of the test item was noted on day 6 in all animals at 10% and 25%. No significant increase in ear thickness and in ear weight was noted in animals treated at 10% and 50%. Therefore, the test item must be considered as not excessively irritant at these two concentrations. Slight increase in ear thickness (+14.2%) associated with a significant increase in ear weight, was noted in animals treated at 25% (+26.4%). Therefore, the test item must be considered as excessively irritant at this concentration.

Group	Test item	Excessive irritation (Y/N)	Cell count (x 10^6cells/ml)	SI	result	EC1.4
1	vehicle (DMF)	N	20.73	/	/	/
2	10%	N	39.77	1.92	positive	/
3	25%	Y	42.39	2.04	positive	/
4	50%	N	35.58	1.72	positive	/

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: other: OECD 429

Conclusions:

In view of these results, under these experimental conditions, lead nitrate crystals must be classified as a skin sensitiser, in accordance with the criteria for classification, packaging and labelling of dangerous substances and preparations of the E.E.C. Directives 67/548, 2001/59 and 99/45. The item must be characterised by the symbol “Xi” and the warning label “Irritant” with the risk sentence R43 “May Cause Sensitisation by Skin Contact”.
In accordance with the Regulation EC No 1272/2008, the test item must be classified in category 1 “Skin sensitisation”, subcategory 1B. The signal word “Warning” and hazard statement H317 “May cause an allergic skin reaction” are required.

Executive summary:

A mouse local lymph node assay (LLNA) was performed to assess the skin sensitisation potential of lead nitrate crystals in the CBA/J mouse strain following topical applications to the dorsal surface of the ear.

Three groups of four animals were treated for three consecutive days (Day 1/2/3) with 50 μL (25 μL per ear) of the test item diluted in dimethyl formamide (DMF) at concentrations of 10%, 25% and 50% (w/w). A further group of four animals was treated with DMF.

On D6, the proliferation of lymphocytes in the draining auricular lymph nodes was determined by cell counting. The experimental protocol was established according tothe OECD Guideline No.429 dated 22 July 2010and the test method B.42 of the Council regulation No. 640/2012 of 06 July 2012.

Results of experiment 1

No mortality and no signs of systemic toxicity were noted in the test and control animals during the test. Slight dryness was noted on day 6 in all animals at the three concentrations. Remaining of the test item was noted on day 3 in all animals at 50%.

No significant increase in ear thickness and in ear weight was noted in animals treated at 10%, 25% and 50%. Therefore, the test item must be considered as not excessively irritant at these three concentrations.

The Stimulation Index (SI) calculated by pooled approach was 1.25, 1.51 and 1.13 for the treated groups at 10%, 25% and 50% respectively.
The EC1.4 determined by linear regression was 18.65%.

Results of experiment 2

To infirm or confirm this results, a second test was performed at the same concentrations. No mortality and no signs of systemic toxicity were noted in the test and control animals during the second test. Remaining of the test item was noted on day 6 in all animals at 10% and 25%.
No significant increase in ear thickness and in ear weight was noted in animals treated at 10% and 50%. Therefore, the test item must be considered as not excessively irritant at these two concentrations. Slight increase in ear thickness (+14.2%) associated with a significant increase in ear weight, was noted in animals treated at 25% (+26.4%). Therefore, the test item must be considered as excessively irritant at this concentration.

The Stimulation Index (SI) calculated by pooled approach was 1.92, 2.04 and 1.72 for the treated groups at 10%, 25% and 50% respectively.
The EC1.4 could not be determined in this experiment.

In view of these results, under these experimental conditions, lead nitrate crystals must be classified as a skin sensitiser, in accordance with the criteria for classification, packaging and labelling of dangerous substances and preparations of the E.E.C. Directives 67/548, 2001/59 and 99/45. The item must be characterised by the symbol “Xi” and the warning label “Irritant” with the risk sentence R43 “May Cause Sensitisation by Skin Contact”.

In accordance with the Regulation EC No 1272/2008, the test item must be classified in category 1 “Skin sensitisation”, subcategory 1B. The signal word “Warning” and hazard statement H317 “May cause an allergic skin reaction” are required.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Migrated from Short description of key information:
In a mouse local lymph node assay (LLNA), the skin sensitisation potential of lead nitrate crystals was investigated. The assay was repeated twice. In both experiments, a stimulation of lymphocyte proliferation was recorded at least at one concentration tested, without indications of excessive irritation at that concentration.

Justification for selection of skin sensitisation endpoint:
OECD 429 Guideline study, compliant with GLP

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

Justification for selection of respiratory sensitisation endpoint:
Respiratory exposure to lead nitrate crystals is negligible because only 0.03% of the lead dinitrate particles are within the inhalable size limits (<10 µm) and therefore an acute inhalation test is not required (see IUCLID section 4.5 for discussion of the complete dustiness test)

Justification for classification or non-classification

Lead nitrate crystals should be classified as follows:

• in accordance with EEC Directives 67/548, 2001/59 and 99/45: "May cause sensitisation by skin contact" (R43)
• in accordance with Regulation (EC) No 1272/2008: Category 1B "Skin sensitisation" with signal word "Warning" and hazard statement H317 "May cause allergic skin reaction".