Graphite

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-02-24 to 2010-06-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed GLP-study according to OECD guidline

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Reference 2

Reference Type:

publication

Title:

An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: First round

Author:

Ehling, G. et al.

Year:

2005

Bibliographic source:

Toxicology (2005), 212, 60-68

Reference 3

Reference Type:

publication

Title:

An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: 2nd round

Author:

Ehling, G. et al.

Year:

2005

Bibliographic source:

Toxicology (2005), 212, 69-79

Reference 4

Reference Type:

publication

Title:

An Integrated Model for the Differentiation of Chemical-Induced Allergic and Irritant Skin Reactions

Author:

Homey, B. et al.

Year:

1998

Bibliographic source:

Toxicol. Appl. Pharmacol. (1998), 153, 83-94

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

(April 24, 2002); see principles if other than guideline

Deviations:

no

Principles of method if other than guideline:

- OECD 429, section 5 allows to use "other endpoints for assessment of proliferation may be employed provided there is
justification and appropriate scientific support, including full citations and description of the methodology." instead of radioactive labelling.
- Here the methodology according to Ehling et al. was applied (see references)

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate is attached to full study report

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

1

Reference substance name:

Graphite

EC Number:

231-955-3

EC Name:

Graphite

Cas Number:

7782-42-5

Molecular formula:

C

IUPAC Name:

carbon

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

mouse

Strain:

Balb/c

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River (Sulzfeld, Germany)
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 20g
- Housing:Makrolon cage type II, cages and adsorbing softwood materials were changed twice a week
- Diet (e.g. ad libitum): Commercial pellets (Ssniff R/M-H V1534, Ssniff-Spezialdiäten, Soest, Germany), ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: at least two weaks prior to the start of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22°C +/- 2°C
- Humidity (%): 55% +/- 15%
- Photoperiod (hrs dark / hrs light): 12hrs/12hrs

Study design: in vivo (LLNA)

Vehicle:

other: AOO: Acetone:olive oil (5:1)

Concentration:

Test item: 0.5%, 1%, 2.5%, 5%, 10% (10% Graphite in AOO was the maximum achievable dose.)
HCA: 3%, 10%, 30%

No. of animals per dose:

6 animals per dose

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: 10% was the maximum achievable dose.
- Irritation: Daily observation either for local irritation at the application site or for systemic toxicity. See "Any other information on materials and methods incl. tables"
- Lymph node proliferation response: See "Any other information on materials and methods incl. tables"

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Ehling et al. (see references)
- Criteria used to consider a positive response: See "Any other information on materials and methods incl. tables"

Positive control substance(s):

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

Statistics:

Dunnett test, differences between treatment groups and vehicle control were considered as statistically significant at the level of p<0.05

Results and discussion

Positive control results:

The differentiation index was greater than 1 for concentrations of 10 % and 30 % HCA. In conclusion, HCA was tested as skin sensitizer by reason that it showed with at least one concentration a differentiation index greater than 1. The result positively confirms the performance of the present LLNA study 18G10014. Also see "Any other information on results incl. tables".

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks:

See "Any other information on results incl. tables". Differentiation index (DI) was calculated.

Test group / Remarks:

0.5% Expanded Graphite Powder

Remarks on result:

other: See "Any other information on results incl. tables". Differentiation index: no differentiation index was calculated, since no significant changes in cell count, ear weight, or lymph node weight have been observed for the test item.

Results 2

Parameter:

SI

Remarks:

See "Any other information on results incl. tables". Differentiation index (DI) was calculated.

Test group / Remarks:

1% Expanded Graphite Powder

Remarks on result:

other: See "Any other information on results incl. tables". Differentiation index: no differentiation index was calculated, since no significant changes in cell count, ear weight, or lymph node weight have been observed for the test item.

Results 3

Parameter:

SI

Remarks:

See "Any other information on results incl. tables". Differentiation index (DI) was calculated.

Test group / Remarks:

2.5% Expanded Graphite Powder

Remarks on result:

other: See "Any other information on results incl. tables". Differentiation index: no differentiation index was calculated, since no significant changes in cell count, ear weight, or lymph node weight have been observed for the test item.

Results 4

Parameter:

SI

Remarks:

See "Any other information on results incl. tables". Differentiation index (DI) was calculated.

Test group / Remarks:

5% Expanded Graphite Powder

Remarks on result:

other: See "Any other information on results incl. tables". Differentiation index: no differentiation index was calculated, since no significant changes in cell count, ear weight, or lymph node weight have been observed for the test item.

Results 5

Parameter:

SI

Remarks:

See "Any other information on results incl. tables". Differentiation index (DI) was calculated.

Test group / Remarks:

10% Expanded Graphite Powder

Remarks on result:

other: See "Any other information on results incl. tables". Differentiation index: no differentiation index was calculated, since no significant changes in cell count, ear weight, or lymph node weight have been observed for the test item.

Any other information on results incl. tables

- OECD 429, section 5 allows to use "other endpoints for assessment of proliferation may be employed provided there is

justification and appropriate scientific support, including full citations and description of the methodology." instead of radioactive labelling.

- Here the methodology according to Ehling et al. was applied (see references)

- Differentiation idices were calculated according to Homey et al. (see references)

RESULTS FOR: Reference item: HCA

Significant changes in cell counts, ear weight, lymph node weight have been observed for the reference item HCA. Therefore, proliferation and ear weight index were calculated by dividing the means of cell counts and ear weights of the reference item treated groups by the vehicle treated ones. The differentiation index (DI) was calculated according to the equation in chapter7.6[max. index ear weight = 2.09 (internal laboratory standard), max. index proliferation = 5 (Homey et al, 1998)].

TABLE 2: Calculation of the differentiation index (DI) for HCA.

Group	treatment	DI	DI	evaluation of DI
01	3 % HCA	-37.68	< 1	non sensitizing
02	10 % HCA	4.48	> 1	sensitizing
03	30 % HCA	2.66	> 1	sensitizing

 

The differentiation index was greater than 1 for concentrations of 10 % and 30 % HCA (table 2). In conclusion, HCA was tested as skin sensitizer by reason that it showed with at least one concentration a differentiation index greater than 1. The result positively confirms the performance of the present LLNA study 18 G 10 014.

RESULTS FOR: Test item: ear weights, lymph node weights and cell counts

Ear weights, lymph node weights, and cell counts for the test item Expanded Graphite Powder are presented below in tables 4 to 7. Expanded Graphite Powder did not induce any significant changes of cell counts, lymph node weights, or ear weights.Therefore, no differentiation index (DI) was calculated. The test item has been assessed as non-sensitizing.

TABLE 3: Ear weights, lymph node (LN) weights, and cell counts for 0.5 % Expanded Graphite Powder.

Animal Number	Ear weight [mg]	LN weight [mg]	Cell counts*10^6
5201	20.74	3.85	5.7
5202	19.77	5.05	6.3
5203	21.23	5.4	7.1
5204	19.99	6.19	12
5205	19.98	4.84	6.4
5206	21.07	5.34	7.4
mean	20.48	5.11	7.48
S.D.	0.65	0.77	2.29
N	6	6	6

 

TABLE 4: Ear weights, lymph node (LN) weights, and cell counts for 1 % Expanded Graphite Powder.

Animal Number	Ear weight [mg]	LN weight [mg]	Cell counts*10^6
6201	20.33	4.56	6.4
6202	20.5	6.13	6.1
6203	21.33	5.43	9.3
6204	20.38	6.76	10.5
6205	19.92	5.96	8.2
6206	20	5.27	7.3
mean	20.41	5.69	7.97
S.D.	0.50	0.77	1.71
N	6	6	6

 

TABLE 5: Ear weights, lymph node (LN) weights, and cell counts for 2.5 % Expanded Graphite Powder.

Animal Number	Ear weight [mg]	LN weight [mg]	Cell counts*10^6
7201	19.53	5.15	7.4
7202	21.27	7.3	7.6
7203	19.65	2.89	3.4
7204	19.85	5.14	7.9
7205	19.41	5.42	8
7206	19.72	4.19	4.4
mean	19.91	5.02	6.45
S.D.	0.69	1.46	2.01
N	6	6	6

 

TABLE 6: Ear weights, lymph node (LN) weights, and cell counts for 5 % Expanded Graphite Powder.

Animal Number	Ear weight [mg]	LN weight [mg]	Cell counts*10^6
8201	19.68	3.97	6
8202	20.04	4.99	7
8203	19.32	5.19	8.9
8204	19.35	4.95	6.8
8205	20.42	4.52	6
8206	20.08	2.9	5.7
mean	19.82	4.42	6.73
S.D.	0.44	0.86	1.18
N	6	6	6

 

TABLE 7: Ear weights, lymph node (LN) weights, and cell counts for 10 % Expanded Graphite Powder.

Animal Number	Ear weight [mg]	LN weight [mg]	Cell counts*10^6
9201	21.89	5.27	8.9
9202	19.66	4.39	5.4
9203	22.05	4.37	5.6
9204	20.56	4.84	8.9
9205	20.85	7.08	5.6
9206	20.35	5.57	5
mean	20.89	5.25	6.57
S.D.	0.92	1.01	1.82
N	6	6	6

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

Expanded Graphite Powder did not induce any significant changes of cell counts, lymph node weights, and ear weights. Therefore, no differentiation index was calculated for the test item. Upon these data, Expanded Graphite Powder is not a skin sensitizer according to Regulation (EC) 1272/2008 and remains unclassified.

Executive summary:

Female mice were exposed topically on the dorsum of both ears to test item Expanded Graphite Powder for three consecutive days at concentrations of 0.5 %, 1 %, 2.5 %, 5 %, and 10 % in acetone/olive oil. Control mice were treated with HCA in acetone/olive oil (reference item), and acetone/olive oil (vehicle control). All mice were sacrificed after three days and small pieces were stamped from each ear and weighed. The draining lymph nodes were excised, weighed, and single cell suspensions were prepared. Cell counts of the LNC suspensions were measured.

HCA showed significant and very significant increases in cell counts, lymph node weights, and ear weights. Therefore, a differentiation index (DI) was calculated for the reference item. This DI describes the relation between skin-draining lymph node cell activation (lymph node cell count index) and skin inflammation (ear weight index). A DI > 1 indicates an allergic reaction (skin sensitization) whereas a DI < 1 demonstrates an irritant potency of a test chemical (Homey et al., 1998). HCA was tested at two concentrations (10 % and 30%) as skin sensitizing (DI > 1). This result positively confirmed the performance of the present LLNA study 18G10014. Upon these data, HCA revealed a sensitizing potential and would be classified according to Regulation (EC) 1272/2008.

Expanded Graphite Powder did not induce any significant changes of cell counts, lymph node weights, and ear weights. Therefore, no differentiation index was calculated for the test item. Upon these data, Expanded Graphite Powder is not a skin sensitizer according to Regulation (EC) 1272/2008 and remains unclassified.