Ashes (residues), cenospheres

Endpoint summary

Description of key information

Based on read-across following an analogue approach

EU Method B.42; GLP; mice; 0.3, 3, 30%; not sensitizing

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

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:

03 Jan 2008 - 17 Jan 2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study. In accordance to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Test conducted with modifications (nonradioactive method) described in Ulrich et al. (2001. Arch Toxicol 74:733-744), Ehling et al. (2005. Toxicology 212: 1-11 and 60-68). Threshold values for evaluation based on a nonpublished personal communication.

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

Balb/c

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Breeding farm BioTest s.r.o., Konárovice, 281 25 Czech Republic, RČH CZ 21760152
- Age at study initiation: 8-10 weeks
- Weight at study initiation: 21-24.6 g
- Housing: groups of six animals (or less) in macrolon cages with sterilized softwood shavings
- Diet (e.g. ad libitum): Pelleted standard diet for experimental animals ad libitu
- Water (e.g. ad libitum): Drinking tap water ad libitum
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): ca. 15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 14 Jan 2008 To: 17 Jan 2008

Vehicle:

other: DAE 433 mixture of 40% DAE 433-mixture of 40%, dimethylacetamide, 30% acetone and 30% ethanol

Concentration:

The test substance was administered in the form of suspension in DAE 433. Concentrations in formulations were:
30% (w/v) 300 mg/mL
3% (w/v) 30 mg/mL
0.3% (w/v) 3 mg/mL

No. of animals per dose:

Pilot experiment: 3 females
Exposed groups: 18 females (6 animals in 3 groups)
Positive control group: 6 females
Negative control group: 6 females
Reserve group: 4 females

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The test material is not soluble. Therefore, suspensions of the test material in vehicle were prepared and tested.
- Irritation: No irritation, no signs of systemic toxicity and no macroscopic changes (after necropsy) were observed in a pilot experiment conducted with 3 animals treated with a 30% suspension of the test material.
- Lymph node proliferation response: Proliferation (cell concentration in the lymph nodes) was measured by cell counting, which served as parameter for comparison between test and control groups.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay (LLNA)
- Criteria used to consider a positive response:
The results of the LLNA were evaluated according to the following criteria.
According to the authors, the following threshold values had been determined from analysis of historical data and were reported as a personal communication:
Ear weight index: 1.05
LN weight: 1.2
LN cell count: 1.3

1. Values exceeding these thresholds were considered as a positive result
- if a statistically significant increase in one of the parameters compared to control occurs and a clear concentration dependence can be derived
- or, in the absence of statistical significance, a clear concentration dependence is observed.

2. Values below these thresholds were considered as a positive result
- if one of the parameters showed a statistically significant increase compared to control, along with a clear concentration dependence.

3. Values were considered as a negative result
- in case of being below the threshold values and without a statistically significant increase compared to control
- in case of being below the threshold values, with a statistically significant increase compared to control, but without showing a clear concentration dependence
- in case of being above the thresholds, without a statistically significant increase compared to control, and without showing a clear concentration-dependence.


TREATMENT PREPARATION AND ADMINISTRATION:
- Dosage volume: 25μL/ear/animal
- Preparation of suspensions: All suspensions were prepared by mixing an appropriate amount of the test material in the vehicle in order to obtain a concentration of 30%, 3% or 0.3% (w/v). Prior to application, the suspensions were mixed for 5 minutes with a magnetic stirrer.
- Frequency of preparation: each day of application
- Application: 25 µL of the appropriate dilution was applied to the dorsum of each ear once a day for 3 consecutive days.
- Other examinations:

Ear and lymph node weights: Animals were sacrificed 24 h after the last application. Immediately after sacrifice both ears were sectioned and circular pieces from the apical area of each ear with a diameter of 8 mm (= 0.5 cm²) were excised using a disposable punch and weighed together on analytical balances. Pairs of auricular LN were excised and weighed on analytical balances.

Lymph node cell counts: Both lymph nodes were prepared by gentle mechanical disaggregation through a 100-µm-mesh nylon gauze with pooling of 1 mL PSB (Phosphate Buffered Saline). The cell concentration in the resulting suspension was measured with a Counter Celltac-α (Nihon Kohden) and analysed with veterinary software.

Positive control substance(s):

other: dinitrochlorbenzene (DNCB, 0.5% w/v in vehicle)

Statistics:

For statistical calculations, the software Statgraphic ® Centurion (version XV, USA) was used. At first, the global comparison of all three values of the test groups to vehicle control was performed by applying the non-parametric Kruskal-Wallis test, and then the non-parametric two-group Mann-Whitney rank test (probability level 0.05) was applied to all two-group comparisons.

Positive control results:

In the positive control group, the cell concentration in suspension and weight of lymph nodes was statistically significantly increased compared to the negative control group.
In the positive control group, the weight of the excised ear discs was statistically significantly increased compared to the negative control group.
The positive control substance DNCB elicited a reaction pattern with statistically significant increase in ear weight and LN hyperplasia, which was in congruence with the expected mode of action of a contact allergen.

Parameter:

other: Lymph node weight

Value:

>= 5.62 - <= 6.43

Test group / Remarks:

0.3, 3, 30%

Parameter:

other: Lymph node cell count

Value:

>= 9.93 - <= 12.22

Test group / Remarks:

0.3, 3, 30%

Parameter:

other: Ear weight

Value:

>= 24.83 - <= 26.25

Test group / Remarks:

0.3, 3, 30%

Parameter:

SI

Test group / Remarks:

0.3%

Remarks on result:

not measured/tested

Parameter:

SI

Test group / Remarks:

3%

Remarks on result:

not measured/tested

Parameter:

SI

Test group / Remarks:

30%

Remarks on result:

not measured/tested

No statistically significant increases in lymph node cell count as well as ear and lymph node weights were observed in any of the test groups. The index for LN cell count exceeded the threshold value in the 0.3 and 30% test groups without a clear dose response.

Summary of results

Group	LN weight		LN cell count		Ear weight
	Mean (mg)	Index	Mean (10^6/mL)	Index	Mean (mg)	Index
NC	5.62	1.00	8.55	1.00	26.40	1.00
PC	13.52*	2.41+	26.00*	3.37+	32.37*	1.23+
0.3%	5.62	1.00	12.22	1.43+	24.83	0.94
3%	6.32	1.12	9.93	1.16	25.18	0.95
30%	6.43	1.15	11.78	1.38+	26.25	0.99

* = values statistically significant on probability level 0.05 (Mann-Whitney test)

+ = values exceeding threshold values

NC: Negative control group

PC: Positive control group

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008

Conclusions:

Under the conditions described in this study, the test substance elicited negative results in the LLNA.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

There are no substance specific data available on the skin sensitising potential of ashes (residues), cenospheres.

Ashes (residues), cenospheres and ashes (residues), coal share a common production process as substances derived from coal combustion. Ashes (residues), cenospheres represent a fraction of ashes (residues), coal separated by physical means. Both substances exhibit similarities in physicochemical properties and chemical composition. The main differences consist in a much lower content of water soluble matter and the particle size distribution of ashes (residues), cenospheres.

In terms of hazard assessment, studies available for ashes (residues), coal are therefore taken into account by read-across following an analogue approach, the results of these studies being considered a worst case for ashes (residues), cenospheres.

Ashes (residues) were tested for skin sensitisation potential in a mouse local lymph node assay (LLNA) conducted under GLP conditions. The method was performed with an endpoint different from that described in the original guideline (non radioactive measuring of cell proliferation). The assay was conducted according to EU method B.42 with modifications as described in publications (Ulrich et al., 2001; Ehling et al., 2005a, b).

The contact allergenic potential of Ashes (residues) was evaluated after topical application to female BALB/c mice. Six mice per group were exposed to test and control substances on the dorsum of both ears once a day for 3 consecutive days. Draining lymph nodes were removed 24 hours after the last application. Ashes (residues) were tested as suspensions in DAE 433 (40% dimethylacetamide, 30% acetone, 30% ethanol) at 0.3, 3 and 30% (w/v). The positive control substance dinitrochlorobenzene (DNCB) was tested at 0.5% (w/v) in DAE 433. Ear weight, auricular (ear-draining) lymph node weights and lymph node cell counts were assessed as parameters for lymph node (LN) hyperplasia.

Animals exposed to the test substance showed no pathological skin reactions and no clinical symptoms of intoxication throughout the experiment. There was no difference in body weight gain in all groups compared to the vehicle control. Ashes (residues) did not cause statistically significant increases in LN cell count, LN weight or ear weight at the dose levels tested. No Stimulation index was calculated.

The positive control substance DNCB elicited a reaction pattern with statistically significant increase in LN hyperplasia, which was in congruence with its expected mode of action as a contact allergen.

It was concluded that, under the experimental conditions of the study, Ashes (residues) yielded negative results in the LLNA (Plodíková, 2008).

Based on their physicochemical properties (solid inorganic particles of mostly water insoluble compounds), ashes (residues), cenospheres are not considered to be biologically reactive on the skin leading to the induction of a sensitising reaction. Furthermore, none of the components of ashes (residues), cenospheres is a known skin sensitiser.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on read-across following an analogue approach, the available data on the skin sensitising potential of ashes (residues), cenospheres is conclusive but not sufficient for classification according to GHS (CLP, 1272/2008/EC) criteria for classification and labelling.

There are no data available on the respiratory sensitisation potential. Thus, classification is not possible due to data lacking.