Graphite, acid-treated

Acute Toxicity: dermal

Administrative data

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 October 2010 - 03 May 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was generated according to internationally accepted guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 440/2008, L 142, Annex Part B, 30 May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1200 (Acute Dermal Toxicity)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP according to German Chemikaliengesetz and Directive 2004/9/EC

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Graphite, acid-treated

EC Number:

291-367-8

EC Name:

Graphite, acid-treated

Cas Number:

90387-90-9

Molecular formula:

C(Carbon) + acid + oxidant Not available by EC, (CnX mHX)

IUPAC Name:

carbon

Details on test material:

Name of test material (as cited in study report): Graphite, acid treated
- Substance type: Inorganic
- Physical state: Solid
- Colour: Grey
- Lot/batch No.: 1706
- Storage condition of test material: Room temperature
- Density: 2.2 g/cm³
- Particle size range: 80 % < 50 mesh

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
Species/strain: healthy rats, WISTAR rats Crl: WI(Han) (full barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female - the female animals were non-pregnant and nulliparous
Number of animals: 5 male and 5 female
Age at the beginning of the study: males: 9 - 10 weeks old
females: 14 - 15 weeks old
 
Body weight on the day of administration: males: 256 – 291 g
females: 201 – 247 g
The animals were derived from a controlled full-barrier maintained breeding system.

HOUSING AND FEEDING CONDITIONS:
- Full barrier in an air-conditioned room
- Temperature: 22 +/- 3 °C
- Relative humidity: 55 +/- 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 1307)
- Free access to tap water, sulphur acidified to a pH value of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- The animals were kept individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 081110)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least five days)

Administration / exposure

Type of coverage:

semiocclusive

Vehicle:

cotton seed oil

Remarks:

Sigma, lot no. MKBB7604, expiry date: 01/06/2011

Details on dermal exposure:

PREPARATION OF THE ANIMALS:
Approximately 23 hours before the test, the fur was removed from the dorsal area of the trunk by using an electric clipper. Care was taken to avoid abrading the skin, and only animals with healthy intact skin were used.
No less than 10 % of the body surface was cleared for the application. Prior to the application a detailed clinical observation was made of all animals.

APPLICATION:
The test item was applied at a single dose, uniformly over an area which was approximately 10 % of the total body surface.
The test item was held in contact with the skin by a dressing throughout a 24-hour period. The dressing consisted of a gauze-dressing and non-irritating tape and was fixed with an additional dressing in a suitable manner.

DOSE LEVEL:
The test item was applied at a single dose of cc to each animal.

EXPOSURE PERIOD:
The test item was held in contact with the skin throughout a 24-hour period. At the end of the exposure period the residual test item was removed by using aqua ad injectionem – sterile water (Berlin Chemie, lot no. 0195A191, expiry date: 04/2013). Nevertheless there was still residual test item, which could not be removed.

Duration of exposure:

24 hours

Doses:

2000 mg/kg body weight

No. of animals per sex per dose:

5 male and 5 female

Control animals:

no

Details on study design:

- Duration of observation period following administration:
14 days

- Frequency of observations and weighing:
A careful clinical examination was made several times on the day of dosing (at least once during the first 30 minutes and with special attention given during the first 4 hours post-dose). As soon as symptoms were noticed they were recorded. Thereafter, the animals were observed for clinical signs once daily until the end of the observation period.

The animals were weighed on day 1 (prior to the application) and on days 8 and 15.

- Necropsy of survivors performed:
At the end of the observation period the animals were sacrificed with an overdosage of pentobarbital injected intraperitoneally (Narcoren®, Merial; lot no. 205100; expiry date: 10/2013) at the dosage of approximately 8 mL/kg bw. All animals were subjected to gross necropsy.

- Other examinations performed:
Cageside observations included changes in the skin and fur, eyes and mucous membranes. Also respiratory, circulatory, autonomic and central nervous systems and somatomotor activity and behaviour pattern were examined. Attention was directed to observations of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Statistics:

Not applicable since no clinical signs of irritation or mortality have been observed.

Results and discussion

Effect levels

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortality occured.

Clinical signs:

other: With regard to signs of toxicity and effects on organs no treatment-related effects have been observed. Furthermore no erythema and no oedema were observed. Scratches were observed in 3 out of 5 female and 3 out of 5 male animals. Eschar was observed in 1

Gross pathology:

With the exception of acute injection of blood vessels in the abdominal region, which is due to the euthanasia injection, no specific gross pathological changes were recorded for any animal.

Any other information on results incl. tables

Table 1:  Clinical Signs of Systemic Toxicity – Individual Data - Males

Animal No. / Sex / Dose	Time of Observation (Post-Dose)	Observations (for Signs of Dermal Irritation, see Table 3)
21/ male / 2000 mg/kg bw	during the whole observation period	no signs of toxicity
22/ male / 2000 mg/kg bw	during the whole observation period	no signs of toxicity
23/ male / 2000 mg/kg bw	during the whole observation period	alopecia on both front legs (not test-item related)
24/ male / 2000 mg/kg bw	during the whole observation period	no signs of toxicity
25/ male / 2000 mg/kg bw	during the whole observation period	no signs of toxicity

Table 2:  Clinical Signs of Systemic Toxicity – Individual Data – Females

Animal No. / Sex / Dose	Time of Observation (Post-Dose)	Observations (for Signs of Dermal Irritation, see Table 5)
26/ female / 2000 mg/kg bw	during the whole observation period	no signs of toxicity
27/ female / 2000 mg/kg bw	during the whole observation period	no signs of toxicity
28/ female / 2000 mg/kg bw	during the whole observation period	no signs of toxicity
29/ female / 2000 mg/kg bw	during the whole observation period	no signs of toxicity
30/ female / 2000 mg/kg bw	during the whole observation period	no signs of toxicity

Table 3:  Skin Irritation – Individual Data – Males

Day after Start of Application	Animal No. 21		Animal No. 22		Animal No. 23		Animal No. 24		Animal No. 25
	E/O	Comments	E/O	Comments	E/O	Comments	E/O	Comments	E/O	Comments
day 2	0/0	nsf*	0/0	s*	0/0	nsf*	0/0	nsf*	0/0	nsf*
day 3	0/0	small s*	0/0	s*	0/0	nsf*	0/0	nsf*	0/0	nsf*
day 4	0/0	small s*	0/0	s*	0/0	nsf	0/0	nsf*	0/0	nsf*
day 5	0/0	small s*	0/0	nsf*	0/0	nsf	0/0	nsf*	0/0	nsf*
day 6	0/0	nsf*	0/0	nsf*	0/0	nsf	0/0	nsf*	0/0	nsf*
day 7	0/0	nsf*	0/0	nsf*	0/0	nsf	0/0	nsf*	0/0	nsf
day 8	0/0	small s*	0/0	nsf*	0/0	nsf	0/0	nsf*	0/0	nsf
day 9	0/0	small s	0/0	nsf*	0/0	nsf	0/0	nsf	0/0	nsf
day 10	0/0	small s	0/0	nsf*	0/0	nsf	0/0	nsf	0/0	nsf
day 11	0/0	small s	0/0	nsf*	0/0	nsf	0/0	nsf*	0/0	nsf
day 12	0/0	s	0/0	nsf	0/0	nsf	0/0	nsf*	0/0	small s
day 13	0/0	s	0/0	nsf	0/0	nsf	0/0	nsf*	0/0	small s
day 14	0/0	es	0/0	nsf	0/0	nsf	0/0	nsf	0/0	es
day 15	0/0	es	0/0	nsf	0/0	nsf	0/0	nsf	0/0	es
Comments: E = erythema; O = oedema; 1, 2, 3, 4 = scoring system laid down in OECD Guideline 404 (Table 2) es = eschar; s = scratches; nsf = no specific findings, * = residual test item

Table 4:   Skin Irritation – Individual Data – Females

Day after Start of Application	Animal No. 26		Animal No. 27		Animal No. 28		Animal No. 29		Animal No. 30
	E/O	Comments	E/O	Comments	E/O	Comments	E/O	Comments	E/O	Comments
day 2	0/0	nsf*	0/0	s*	0/0	nsf*	0/0	nsf*	0/0	nsf*
day 3	0/0	nsf*	0/0	nsf*	0/0	nsf*	0/0	nsf*	0/0	nsf*
day 4	0/0	nsf*	0/0	nsf*	0/0	nsf*	0/0	nsf*	0/0	nsf
day 5	0/0	small s*	0/0	nsf*	0/0	nsf	0/0	small s*	0/0	nsf
day 6	0/0	s*	0/0	nsf	0/0	nsf	0/0	s*	0/0	nsf
day 7	0/0	s*	0/0	nsf	0/0	nsf	0/0	s*	0/0	nsf
day 8	0/0	s*	0/0	nsf	0/0	nsf	0/0	s*	0/0	nsf
day 9	0/0	s, es	0/0	nsf	0/0	nsf	0/0	nsf	0/0	nsf
day 10	0/0	s, es	0/0	nsf	0/0	nsf	0/0	nsf	0/0	nsf
day 11	0/0	s*	0/0	nsf	0/0	nsf	0/0	nsf	0/0	nsf
day 12	0/0	s, es (diameter 1 cm)	0/0	nsf	0/0	nsf	0/0	nsf	0/0	nsf
day 13	0/0	s, es (diameter 1 cm)	0/0	nsf	0/0	nsf	0/0	nsf	0/0	nsf
day 14	0/0	s, es (diameter 1 cm)	0/0	nsf	0/0	nsf	0/0	nsf	0/0	nsf
day 15	0/0	s, es (diameter 1 cm)	0/0	nsf	0/0	nsf	0/0	nsf	0/0	nsf
Comments: E = erythema; O = oedema; 1, 2, 3, 4 = scoring system laid down in OECD Guideline 404 (Table 2) es = eschar; s = scratches; nsf = no specific findings, * = residual test item

Table 5:   Absolute Body Weights in g and Body Weight Gain in %

Animal No. / Sex	g Day 1	g Day 8	g Day 15	% Day 1-15
Dose: 2000 mg/kg bw
21 / male	285	300	330	16
22 / male	291	292	320	10
23 / male	279	284	300	8
24 / male	256	255	277	8
25 / male	281	290	319	14
26 / female	247	242	246	0
27 / female	201	205	209	4
28 / female	209	215	218	4
29 / female	238	238	243	2
30 / female	220	219	230	5

Table 6:  Macroscopic Findings - Individual Data – Males and Females

Animal No. / Sex	Organ	Macroscopic Findings
Dose: 2000 mg/kg bw
21 / male	-	nsf
22 / male	-	nsf
23 / male	-	nsf
24 / male	-	nsf
25 / male	-	nsf
26 / female	-	nsf
27 / female	-	nsf
28 / female	-	nsf
29 / female	-	nsf
30 / female	-	nsf

nsf = no specific findings

Applicant's summary and conclusion

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of the present study, single dermal application of the test item Graphite, acid treated to rats at a dose of 2000 mg/kg body weight was associated with neither mortality nor signs of toxicity and did not cause erythema or oedema.
The minor findings (eschar, scratches) observed are not considered to be clearly treatment-related.
The dermal LD50 was determined to be > 2000 mg Graphite, acid treated / kg body weight.

In conformity with the criteria given in Annex VI to Commission Directive 2001/59/EC the test item Graphite, acid treated has no obligatory labelling requirement for percutaneous toxicity. According to Annex I of Regulation (EC) 1272/2008 the test item Graphite, acid treated has no obligatory labelling requirement for percutaneous toxicity and is unclassified. According to OECD-GHS (Globally Harmonised Classification System) the test item Graphite, acid treated has no obligatory labelling requirement for percutaneous toxicity.

Executive summary:

On the basis of the test results given below and in conformity with the criteria given in Annex VI to Commission Directive 2001/59/EC as well as in Annex I of Regulation (EC) 1272/2008, the substance should be not classified.

LD₅₀:                                                        > 2000 mg /kg bw

Species/strain:                                          WISTAR Crl: WI(Han) rats

Vehicle (moistening):                                cottonseed oil

Number of animals:                                   5 male and 5 female

Duration of exposure:                              24 hours

Method:                                                   OECD 402, EC 440/2008, OPPTS 870.1200

Table1:   Results per Step

Sex	Dose (mg/kg bw)	Number of Animals	Number of Intercurrent Deaths
male	2000	5	0
female	2000	5	0

Signs of toxicity related to dose level used, time of onset and duration:

No treatment-related effects were observed.

Effect on organs (related to dose level):

No treatment-related effects were observed.

Signs of irritation:

No erythema and no oedema were observed. Scratches were observed in 3 out of 5 female and 3 out of 5 male animals. Eschar was observed in 1 out of 5 female and 2 out of 5 male animals.