Ashes (residues), nonhazardous municipal solid waste

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3.1.2008-17.1.2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

The deviation has no impact on the study results.

Principles of method if other than guideline:

non-radioactive method

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Ashes (residues)

EC Number:

268-627-4

EC Name:

Ashes (residues)

Cas Number:

68131-74-8

Test material form:

solid: bulk

Details on test material:

- Name of test material (as cited in study report): Ashes (residues)
- Substance type: technical product
- Physical state: solid
- Main components: SiO2 (42.12%), Al2O3 (32.16%), Fe2O3 (10.88%), TiO2, MnO, MgO, CaO, Na2O, K2O, SO3 - constituents with content less than 10%
- Expiration date of the lot/batch: unlimited
- Appearance: greyish black powder

In vivo test system

Test animals

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 to 10 weeks (at start of dosing)
- Weight at study initiation: 21 to 24.6 g (at start of dosing)
- Housing: Animals in groups of maximum six in macrolon cages with sterilized softwood shavings.
- Diet: Pelleted standard diet for experimental animals ad libitum. Microbiological control and content of nutrients will be performed according SOP No. 75 and 83.
- Water: Drinking tap water ad libitum. Water quality corresponded to Regulation No. 252/2004 Czech Coll. of Law, Health Ministry
- Acclimation period: 5 days
- Prophylactic arrangement: Cleaning and disinfection of animal room was regularly performed as it is described in appropriate SOP No.10.
- Cage identification: By cage number, study number and group specific colour.
- Animal identification: By felt tip marking (from 1 to 6 per each group and group specific colour).
- Random selection: According to SOP No.42.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): Room temperature 22 +/- 3°C, permanently monitored
- Humidity (%): Relative humidity 30 – 70 %, permanently monitored
- Air changes (per hr): approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): Light: 12 hours light/dark cycle: 6am-6pm/6pm-6am

STUDY TIME SCHEDULE
Pilot experiment: 7.-10.1.2008
Animal arrival/ start of acclimatization: 2.1.2008
Experimental starting date: 3.1.2008
First day of administration: 14.1.2008
End of treatment period: 16.1.2008
Necropsy: 17.1.2008

Study design: in vivo (LLNA)

Vehicle:

other: 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. Concentration in formulation:
30% (w/v) 300 mg/mL
3% (w/v) 30 mg/mL
0.3% (w/v) 3 mg/mL

No. of animals per dose:

Exposed groups - 18 females (6 animals per concentration)

Details on study design:

PILOT EXPERIMENT
The highest concentration 30% was administered to three animals to assess a possible systemic toxicity. The route of administration was same as in the main study. During pilot experiment no clinical symptoms of systemic toxicity and no macroscopic changes (after necropsy) were found out in all three animals.


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
Animals were subjected to a clinical examination (health check) shortly after arrival.
Study animals were randomly allocated to the dose groups manually and assigned animal numbers.

- Criteria used to consider a positive response:
The results of the LLNA were evaluated according to the following criteria.
The following thresholds were determined by Ulrich (2007) from analysis of historical data:
Ear weight index: 1.05
LN weight: 1.2
LN cell count: 1.3

1. Values which exceed these thresholds were considered positive
- when a statistically significant increase in one of the parameters occurs and a clear concentration-dependence can be derived
- or with no statistical significance, but a clear concentration-dependence.

2. Values which are below these thresholds were considered positive
- when a statistical significant occurs in one of the parameters together with a clear concentration dependence.

3. Values were considered negative:
- in case of being below the thresholds and without a statistical significance
- in case of being below the thresholds, with a statistical significance, but without a clear concentration-dependence
- in case of being above the thresholds, without statistical significance and without a clear concentration-dependence.


TREATMENT PREPARATION AND ADMINISTRATION:
- Dosage volume: 25μL / ear / animal
- Preparation for administration: All solutions were prepared by dissolving an appropriate amount of Ashes (residues) in the vehicle to obtain a concentration of 30%, 3% or 0.3% (w/v). Before start of application the suspension was mixed for 5 minutes with a magnetic stirrer.
- Frequency of preparation: each day of administration.
- Application: The volume of the application form was constant at all groups of animals - 25 microliter of the appropriate dilution to the dorsum of each ear once a day morning for 3 consecutive days. The application was performed very slowly by micropipette. Losses caused by draining from the ear must be minimized. This route of administration is listed in guideline and it is similar to expected exposure conditions at the workplace. The application form of test substance was prepared immediately before administration.
- Post mortem investigations:
- Organ weights: Immediately after death, the both ears were cut off and circular pieces from the apical area of each ear with a diameter of 8mm (=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 of the lymph nodes were prepared by gentle mechanical disaggregation through 100 m-mesh nylon gauze with pooling of 1 mL PSB (Phosphate Buffered Saline). The cell concentrations in resulting suspension were measured by Counter Celltac-α (Nihon Kohden) with veterinary software.

Positive control substance(s):

other: dinitrochlorbenzene

Statistics:

For statistical calculations the software Statgraphic ® Centurion (version XV, USA) was used. At first the global comparison of all three values of the concentration groups with 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.

Results and discussion

Positive control results:

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.
In the positive control group, the cell concentration in suspension and weight of lymph nodes was statistically significantly increased against negative control group – test LLNA was efficient.
In the positive control group, the weight of ear target was statistically significantly increased against negative control group – test LLNA was efficient.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

other: LN weight

Value:

>= 5.62 - <= 6.43

Test group / Remarks:

0,3%, 3%, 30%

Results 2

Key result

Parameter:

other: LN cell count

Value:

>= 9.93 - <= 12.22

Test group / Remarks:

0,3%, 3%, 30%

Results 3

Key result

Parameter:

other: Ear weight

Value:

>= 24.83 - <= 26.25

Test group / Remarks:

0,3%, 3%, 30%

Any other information on results incl. tables

Table No. 3.  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

Figures with asterisk = values statistically significant on probability level 0.05 (Mann-Whitney test)

Figures with cross = values exceeding thresholds

NC-Negative control group

PC-Positive control group

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

Under the given test conditions, the test substance, Ashes (residues), elicited negative result in LLNA test.

Executive summary:

The Local Lymph Node Assay (LLNA) was used. This method was performed with endpoint different from that described in original guideline (non radioactive measuring of cell proliferation). This testing was conducted according to EU method B.42 with modifications as described in publications of Ulrich P, Streich J, Suter W, 2001; Ehling et al., 2005; Ehling et al., 2005A.

In this study the contact allergenic potential of Ashes (residues) was evaluated after topical application to female BALB/c mice. Six mice per group were exposed by test and control substances on the dorsum of both ears once a day during 3 consecutive days. Draining lymph nodes were taken off at 24 hours after the last application. Concentrations: positive control DNCB (dinitrochlorobenzene): 0.5% (w/v) andAshes (residues): 30%, 3%, 0.3% (w/v). Endpoints: ear weight, auricular (ear-draining) lymph node weights and cell counts = lymph node (LN) hyperplasia.

The animals exposed to test substance showed no pathological skin reactions and no other negative clinical symptoms of intoxication throughout the experiment. There was no difference in body weight increment of all groups in comparison to the vehicle control. There were no clinical observations attributable to the treatment with test substance. The positive control substance DNCB elicited a reaction pattern with statistically significant increase LN hyperplasia, which was in congruence with his expected mode of action as a contact allergen.

The test substance did not show a tendency to increase ear weight in anyone of dose levels tested. These results confirmed existing dermal irritation data (acute dermal irritation study was performed in CETA with negative results, see study No. 46/07/4).

            

Comparison of values between treated groups and control group revealed that the test substance, Ashes (residues), did not cause statistically significant increase in LN cell count or in LN weight. Also index of LN weight was not exceeded in any dose level.The index for LN cell count exceeded the threshold in groups of animals treated by the test substance in the dose levels 0.3% and 30% but without a clear concentration-dependence.

In conclusion, at the given experimental conditionsthe test substance, Ashes (residues), elicited negative result in LLNA test.