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REACH

Graphite

EC number: 231-955-3 | CAS number: 7782-42-5

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Description of key information

In a combined repeated dose toxicity study with reproduction/developmental toxicity screening test via the oral (dietary) route (OECD TG 422), there were no systemic adverse effects observed up to nominal exposure levels corresponding to the maximum recommended dose of 1000 mg/kg bw/day.

In two sub-acute repeated dose toxicity studies via the inhalation route (OECD TG 412), Synthetic Graphite (SG; w/o Quartz) and Expanded Graphite (EG; with Quartz) were tested individually.

In both studies, exposure was generally well tolerated and there were no signs of systemic toxicity. Both graphite qualities showed local responses in the respiratory tract with partly recovery after 28 days, that were to be expected for a poorly soluble dust with low toxicity. Testing of SG resulted in a NOAEC of 12 mg/m³ (LOAEC = 36 mg/m³), whereas testing of EG resulted in a NOAEC of 8 mg/m³ (LOAEC = 24 mg/m³).

An additional sub-chronic repeated dose toxicity study via the inhalation route (OECD TG 413) with expanded graphite did not show systemic effects after treatment with the test item. Local responses were observed in the respiratory tract at all doses investigated, which were in line with effects expected for poorly soluble particles with low toxicity. No NOAEL could be derived for local responses in the respiratory tract, the local LOAEC was 3.20 mg/m³.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproductive / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-02-22 to 2010-09-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

March 1996

Deviations:

GLP compliance:

yes (incl. QA statement)

Limit test:

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Strain Wistar Crl:(WI)WU
- Age at study initiation: 8 weeks
- Weight at study initiation: 292 - 293g
- Housing: Makrolon, type III cages, two rats of the same sex and dose group per cage; cages and absorbing softwood bedding material were changed twice a week or more often if necessary
- Diet for the control group: (e.g. ad libitum): Ssniff V1534, Ssniff Spezialdiäten, Soest, Germany, ad libitum
- Diet for the treatment group: test item will be sent to ssniff by Fraunhofer ITEM and added to the same commercial chow in three different concentrations, ad libitum
- Water (e.g. ad libitum): Tap water from Hannover city water supplier, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 2°C
- Humidity (%): 55% +/- 15%
- Photoperiod (hrs dark / hrs light): 12/12
- Air exchange rate: approx. 15 times per hour

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- The test item will be sent to Ssniff Spezialdiäten by Fraunhofer ITEM and added to the same commercial chow as used for the control animals (Ssniff V1534) in three different concentrations
- Food will be offered ad libitum
- Food will be changed weekly or on the dates of determination of food consumption
- For each treatment group there will be a food mixture with the corresponding concentration of the test item
- These food mixtures will be prepared and delivered once by Ssniff, Soest, Germany
- From each concentration three samples will be taken from three different sites of the first container after delivery for checking the content and homogeneity of the test item in preparation
- These samples will be investigated by repeat determination
- Based on the chemical properties of both graphite and quartz, the investigation of the stability is not regarded necessary

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Concentration and homogeneity of the test item in food will be analysed by base-digestion using 4M NaOH followed by turbidity measurement
- The content of graphite powder will be determined photometrically at a wavelength of 860nm using a calibration curve
- Determination will be according to a validated method (non-GLP) allowing an evaluation of the precision and accuracy of results
- The relative standard error for the graphite content determination is confirmed to be below +/- 20% within the operative concentrations
- Consequently, acceptance criteria for concentration and homogeneity will be set at +/- 20% of the target value

Duration of treatment / exposure:

- animals will be treated via food for two weeks before mating and during the mating period
- after successful mating, treatment of the females will be continued until day 4 post partum and subsequent sacrifice
- treatment of all males and females not successfully mated will continue until their sacrifice

Frequency of treatment:

Please see "Duration of treatment / exposure"

Remarks:

Doses / Concentrations:
0, 1200, 3600, 12000 mg/kg food
Basis:
nominal in diet

No. of animals per sex per dose:

10 males and 10 females per dose group; in a second mating trial an additional 10 males and 10 females per dose group were used (no histopathological examination, hematology and clinical chemistry, locomotor activitiy, functional observation battery, determination of organ weight in second trial)

Control animals:

yes, plain diet

Details on study design:

DOSE SELECTION RATIONALE:
- Doses selected based on a palatability study (Fraunhofer ITEM study-no: 12N100002), were food containing 10g/kg food (estimated target dose of 1000mg/kg/day) was accepted w/o any problems.
- This dose equals the recommended limit dose given in OECD 422 and was therefore chosen as the high dose.
-Other doses were determined using a stagger of approx. three

RATIONALE FOR ANIMAL ASSIGNMENT
- Randomisation to groups based on body weight using PROVANTIS 8.2.0.8

Positive control:

No positive control inculded in this study.

Observations and examinations performed and frequency:

INVESTIGATIONS IN F0

CLINICAL INVESTIGATIONS
- Animals will be inspected at least once daily.
- Cages in which animals exhibit unusual behavior or appearance will be tagged for inspection by the veterinarian or his designate.
- For those animals that continue to show a deterioration in health status, euthanasia and necropsy will be performed only if the animal's condition is severely influenced.
- Once per week, all animals will be inspected outside their home cages. The results of these examinations will be documented.

BODY WEIGHT
- Individual body weight of all animals will be recorded weekly to the nearest 0.1 g until sacrifice of the animals.
- After success¬ful mating, body weight of the females will be determined on days 0, 7, 14, and 20 p.c., as well as 0 and 4 post partum (p.p., day of birth = day 0 p.p.)

FOOD CONSUMPTION
- Individual food consumption of all animals will be recorded weekly to the nearest 0.1 g until sacrifice of the animals by determining the difference between initial and remaining food.
- After successful mating, food consumption of the females will be determined on days 0, 7, 14, and 20 p.c. as well as on days 0 and 4 p.p. The actual test item intake of the animals will be calculated based on food consumption.

HEMATOLOGY AND CLINICAL CHEMISTRY
- Blood sample will be taken w/o overnight fasting from the retrobulbar plexus of 5 males and 5 females per group towards the end of week two of treatment (shortly before start of the mating period)
- Blood will be collected under light isofluran ® anesthesia and collected in tubes with K2-EDTA (1.5-2mg/mL) and heparin lithium salt (>7.5 IU/mL).
- In the collected blood samples, the parameters given in table “Hematology and Clinical Chemistry” in section “Any other information on material and methods, incl. tables” will be analysed
- Clinical laboratory data will be checked using commercially available control samples.

LOCOMOTOR ACTIVITY
- Spontaneous locomotor activity over 60 minutes using the „Actimot“ computerized light-beam system (TSE, Homburg/Ts., Germany) will be determined shortly before sacrifice in males and towards the end of week two of treatment (shortly before the start of the mating period) in females.
- The data will be analyzed in 15-minutes intervals. In addition, the total values for distance, time in rest, time in movement, rearing time, and number of rearings will be determined.

FUNCTIONAL OBSERVATION BATTERY
- A functional observational battery (FOB) based on Gad (1982) and Moser et al. (1991) will be determined shortly before sacrifice in males and towards the end of week two of treatment (shortly before the start of the mating period) in females.
- In addition to the determination of forelimb grip strength (Meyer et al., 1979), the FOB will include the following endpoints: Righting reflex, body temperature, salivation, startle response, respiration, urination, mouth breathing, convulsions, pineal response, piloerection, diarrhea, pupil response, lacrimation, impaired gait, stereotypy, toe pinch, tail pinch, wire maneuver, hind-leg splay, tremors, extensor thrust, positive geotropism, activity and limb rotation.

MATING
- Animals will be mated beginning after two weeks of treatment for two consecutive weeks or until successful mating.
- Overnight mating will performed 1 male : 1 female of the same dose group.
- Vaginal smears will be taken the next morning.
- Mating will be considered successful if sperm and/or a vaginal plug is found.
- The day of finding sperm is considered day 0 p.c.
- Time to successful insemination (precoital time) will be determined.
- In a second mating trial an additional 10 males and 10 females per dose group were used (no histopathological examination, hematology and clinical chemistry, locomotor activitiy, functional observation battery, determination of organ weight in second trial)

POST MORTEM INVESTIGATIONS
- Males will be sacrificed after successful mating, but not before day 28 of treatment.
- Females will be sacrificed as soon as possible after day 4 p.p.
- All animals will be sacrificed by C02 overdose and subsequent exsanguination. The treatment of all animals will be continued until their sacrifice.

GROSS PATHOLOGY
- Necropsy will be performed in all F0 males and females.
- The number of corpora lutea as well as implantation sites will be determined, using ammonium sulphide staining in case of no macroscopically visible implantations
- The organs and tissues given in table “Tissues and Organs” in section “Any other information on material and methods, incl. tables” will be collected from each rat and fixed in 10% neutral buffered formalin (unless specified otherwise)

ORGAN WEIGHTS
- Weights of the following organs will be determined: liver, kidneys, adrenals, thymus, spleen, brain, heart, testes and epidydimides.

HISTOPATHOLOGY
- In 5 males and 5 females of the high dose group 4 and the control group 1, complete histopathological investigation will be performed.
- In testes, staging of the seminal epithelium will be performed.

INVESTIGATIONS IN F1

CLINICAL OBSERVATIONS
- Litters are inspected at least once daily.
- Cages in which dams or litters exhibit unusual behaviour or appearance will be tagged for inspection by the veterinarian or his designate.

SURVIVAL AND PUP WEIGHT
- Number of offspring and the occurrence of any dead pups or abnormalities will be recorded for all groups.
- Individual pup sex will be determined and individual weight of the pups will be recorded to the nearest 0.1 g on days 0 and 4 p.p.

Sacrifice and pathology:

INVESTIGATIONS IN F0

POST MORTEM INVESTIGATIONS
- Males will be sacrificed after successful mating, but not before day 28 of treatment.
- Females will be sacrificed as soon as possible after day 4 p.p.
- All animals will be sacrificed by C02 overdose and subsequent exsanguination. The treatment of all animals will be continued until their sacrifice.

GROSS PATHOLOGY
- Necropsy will be performed in all F0 males and females.
- The number of corpora lutea as well as implantation sites will be determined, using ammonium sulphide staining in case of no macroscopically visible implantations
- The organs and tissues given in table “Tissues and Organs” in section “Any other information on material and methods, incl. tables” will be collected from each rat and fixed in 10% neutral buffered formalin (unless specified otherwise)

ORGAN WEIGHTS
- Weights of the following organs will be determined: liver, kidneys, adrenals, thymus, spleen, brain, heart, testes and epidydimides.

HISTOPATHOLOGY
- In 5 males and 5 females of the high dose group 4 and the control group 1, complete histopathological investigation will be performed.
- In testes, staging of the seminal epithelium will be performed.

INVESTIGATIONS IN F1
- All pups will be humanely sacrificed as soon as possible after day 4 p.p. and carefully examined for gross abnormalities.

Other examinations:

Please see section "Observations and examinations performed and frequency" as well as section "Sacrifice and pathology".

Statistics:

- Statistical comparison of groups will be performed separately for each sex at the level of alpha=0.05.
- Body weights as well as food consumption data will be analyzed using analysis of variance.
- If the group means differ significantly with this method, the means of the treatment groups will be compared with the mean of the control group 1 using Dunnett's modification of the t-test.
- Kruskall-Wallis ANOVA and Mann-Whitney U-test will be applied in the case of non-homogeneous data.
- Qualitative data will be analyzed using the two-tailed FISHER test with Bonferroni correction or Chi-square test.
- If appropriate, other statistical tests can be applied.

Clinical signs:

no effects observed

Description (incidence and severity):

that were regarded as test item related

Mortality:

no mortality observed

Description (incidence):

that were regarded as test item related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

that were regarded as test item related

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

that were regarded as test item related

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Locomotor Activity and Functional Observation Battery

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

that were regarded as test item related

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL OBSERVATIONS IN F0 ANIMALS
- During gestation, one control animal showed red discharge from vagina on day 24 p.c. This animal did not give birth but showed one implantation site at necropsy.
- During Lactation, eye discharge was observed in one high dose animal on day 0 p.p.
- No other clinical signs were observed during the study, and none of the clinical observations were dose dependent so that they were not regarded as test item related.

BODY WEIGHT IN F0 ANIMALS
- No effects of test item exposure were observed on body weight or body weight gain of the animals at any time point of the study.

FOOD CONSUMPTION IN F0 ANIMALS
- No effects of test item exposure were observed on food consumption of the animals at any time point of the study.

HEMATOLOGY IN F0 ANIMALS
- In female rats in the low dose group, a marginal but statistically significant increase in the absolute and relative number of segmented neutrophiles (SEGC, SEGM) and accordingly a slight decrease in the relative lymphocyte count (LYM) were observed.
- A significant increase in the prothrombin time (PT) in females in the medium dose group is due to the high interindividual variance.
- Overall, hematological and clinical chemistry data were found in the ranges expected for the species, strain, sex and age.
- Therefore, the findings albeit significantly different from controls in individual cases, are considered to be of no toxicological relevance.

CLINICAL CHEMISTRY IN F0 ANIMALS
- Significantly decreased alanine aminotransferase (ALT) levels were observed in males in the high dose group.
- Slightly but significantly increased potassium (K) levels were found in males in the medium dose group.
- However, all values were within the normal range of this strain and age.

LOCOMOTOR ACITIVITY IN F0 ANIMALS
- No effects of test item exposure were observed, neither for any of the investigated time intervals, nor for the whole test period.

FUNCTIONAL OBSERVATIONAL BATTERY I F0 ANIMALS
- No effects of test item exposure were observed on any of the investigated endpoints.

GROSS EXAMINATION OF F0 ANIMALS
- The main findings consisted in reduced size of testes and epidydimides, which were found in all groups including the control group.
- Eight animals showed this observation, out of which 4 were able to produce a litter, while 4 were not.
- None of the macroscopic findings were considered test item induced.

ORGAN WEIGHTS IN F0 ANIMALS
- The only statistically significant differences from the control group consisted in an increase of the absolute and relative weight of the left, but not of the right, adrenal gland in the male medium dose group, an increase in relative heart weight in the male medium dose group, as well as a decrease in relative, but not absolute, liver weight in the male medium and high dose groups.
- No effects were observed in females, and none of the observed differences in males was considered an adverse effect of the test item exposure.

HISTOPATHOLOGY OF F0 ANIMALS
- Substance-related findings were not observed in the histologically examined organs of males and females of the control and high dose group.
- Several other findings in various organs of the examined experimental groups were noted which also occurred incidentally and were not unusual for this strain and age.

Dose descriptor:

NOAEL

Effect level:

ca. 813 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: NOAEL = highest dose tested, actually ingested dose recalculated from food consumption

Dose descriptor:

NOAEL

Remarks:

during premating period

Effect level:

ca. 1 067 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: NOAEL = highest dose tested, actually ingested dose recalculated from food consumption

Dose descriptor:

NOAEL

Remarks:

during gestation

Effect level:

ca. 930 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: NOAEL = highest dose tested, actually ingested dose recalculated from food consumption

Dose descriptor:

NOAEL

Remarks:

during lactation

Effect level:

ca. 1 159 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: NOAEL = highest dose tested, actually ingested dose recalculated from food consumption

Critical effects observed:

not specified

MATING AND PREGNANCY DATA

- Since the outcome of the first mating was insufficient, additional 10 pairs per group were added to the study for one more mating trial.

- Consequently, each group consisted of 20 pairs for mating.

- For the four groups, mating yielded 19, 19, 20, and 18 sperm positive females, in groups 1-4, respectively.

- This resulted in 14, 10, 13, and 10 pregnancies; 11, 9, 13, and 10 females with liveborn offspring in F0 dams, and 11, 9, 12, and 10 females with live litters on day 4 p.p., respectively.

- Animals no. 1102, 1107, and 2112 did not give birth, but were found pregnant at necropsy, when they showed at least one implantation site.

- Animal no. 1101 gave birth to 3 pups, which were all stillborn.

- Animal no. 3113 gave birth to only one live pup, which was missing on day 1 p.p.

- The results show that mating outcome throughout all groups including controls in both mating trials was insufficient. The reasons for this phenomenon are unclear.

- However, no effect of the test item exposure was observed on any of the observed endpoints like precoital time or fertility (number of mated females, number of pregnant females, number of implantation sites, number of liveborn pups).

LITTER SIZE, SURVIVAL AND PUP WEIGHT

- No effect of the test item exposure could be observed on litter size and pup survival as well as pup body weight.

- Pup clinical observations included sporadical bad condition, cold to touch and one finding of a damged tail.

- None of these findings were considered test item related and no abnormal pups were observed.

QUALITATIVE HISTOLOGICAL EXAMINATION OF THE TESTES/EPIDIDYMIDES TAKING INTO ACCOUNT THE TUBULAR STAGES OF THE SPERMATOGENIC CYCLE

- The randomly scanned seminiferous tubules 4/5 males of the control and 1/5 males of the high dose group showed the appropriate cell layers in their approximate normal numbers, this means that stages I-VIII contained a layer of spermatogonia, a layer of pachytene spermatocytes and several layers of round spermatids interspersed with elongated spermatids.

- Stages IX-XIV contained a layer of spermatogonia and prepachytene spermatocytes, several layers of late pachytene or dividing (stage XIV) spermatoytesand several layers of elongated spermatids.

- There was no evidence of degenerating spermatids or spermatocytes.

- Very slight (multi)focal interstitial mononuclear cell infiltration was recorded in the epididymis of 3/5 males of the control and in 2/6 males of the high dose group.

- These alterations are considered to be incidental findings.

- Aspermia was observed in one male of the low dose and high dose group each associated with severe changes in the corresponding testis.

- A unilateral slight/moderate focal spermatic granuloma was detected in one male of the mid dose and high dose group each.

- The testis of one male of the control group showed very slight degeneration/depletion of spermatocytes and spermatids associated with Sertoli-cell vacuolation and spermatid retention in one testes and a slight multifopcal tubular atrophy in the other testis.

- In one male rat of the high dose group a very severe unilateral diffuse tubular atrophy corresponding to the gross finding reduction in size was observed.

- One male of the low dose group was detected with very severe diffuse tubular atrophy associated with very severe germ cell degeneration in both testes and formation of multinucleated giant cells.

- Three (3/6) males of the high dose group were seen with very slight focal/multifocal degeneration of germ cells and 2/6 males with very slight unilateral focal/multifocal vacuolation of Sertoli cells.

- The lesions found in 1male of the control, in one male of the low dose group and in 5/6 males of the high dose group are considered to be incidental.

- The severe changes of one male of the low and high dose group each is likely to be spontaneous as single case as well as the very minimal lesions found in the 4 males of the high dose group.

ANALYSIS OF TEST ITEM CONCENTRATION IN FOOD

- The results of the analyses of the test item concentration in food of group 2-4 are summarized in Table “Food Analysis”.

- The mean substance intake calculations are summarized in Table “Substance Intake”.

- The analyses showed that preparations contained 108, 103, and 96 % of the target concentration of the three dose groups, respectively.

- Based on mean body weights and food consumption data, this resulted in the following actual substance intake: 91, 261, and 813 mg/kg body weight/day for males; 120, 343, and 1067 mg/kg body weight/day for females in the premating period; 106, 309, and 930 mg/kg body weight/day for females during gestation; and 111, 370, and 1159 mg/kg body weight/day for females during lactation, respectively.

TABLE: SUMMARY

Observations	Values
Dosage (mg/kg food)	0	1200	3600	12000

Pairs started (N)	20	20	20	20
Females showing evidence of copulation (N)	19	19	20	18
Females achieving pregnancy (N)	14	10	13	10
Conceiving days 1-5 (N)	19	18	19	18
Conceiving days 6-14 (N)	0	1	1	0
Pregnancy = 21 days (N)	0	0	0	0
Pregnancy=22 days (N)	4	3	2	3
Pregnancy => 23 days (N)	8	6	11	7
Dams with live young born (N)	11	9	13	10
Dams with live young at day 4 p.p. (N)	11	9	12	10
Corpora lutea/dam (mean)	13.2	12.0	13.2	13.5
Implants/dam (mean)	11.9	9.8	12.5	13.1
Live pups/dam at birth (mean)	10.9	9.8	10.7	11.2
Live pups/dam at day 4 (mean)	10.8	8.8	10.4	11.1
Sex ratio (m/f) at birth (mean)	61	39	62	61
Sex ratio (m/f) at day 4 (mean)	60	34	61	61
Litter weight at birth (mean)	67.9	60.6	66.8	70.2
Litter weight at day 4(mean)	115.8	92.4	115.7	109.1
Pup weight at birth (mean)	5.9	6.4	6.3	6.4
Pup weight at day 4(mean)	10.1	10.7	10.7	10.1
ABNORMAL PUPS
Dams with 0	11	9	13	10
Dams with 1	0	0	0	0
Dams with 2	0	0	0	0
Dams with 3	0	0	0	0
LOSS OF OFFSPRING
Pre-implantation (corpora lutea minus implantations)
Females with 0	8	3	5	5
Females with 1	2	3	4	4
Females with 2	1	2	3	0
Females with 3>	3	2	1	1
Pre-natal (implantations minus live births)
Females with 0	2	3	4	2
Females with 1	6	5	3	1
Females with 2	0	1	1	3
Females with 3>	3	1	5	4
Post-natal (live births minus alive at post natal day 4)
Females with 0	10	6	9	9
Females with 1	1	1	4	1
Females with 2	0	1	0	0
Females with 3>	0	1	0	0

TABLE: FOOD ANALYSIS

	Low Dose	Medium Dose	High Dose
Target concentration in food, mg/kg food	1200	3600	12000
Chemically determined concentration, mg/kg food (% of target value)	1300 (108%)	3700 (103%)	11500(96%)

TABLE: SUBSTANCE INTAKE

Week no.	Group 2 (Low dose)			Group 3 (mid dose)			Group 4 (high dose)
	Mean BW [g]	Mean FC [g/animal/d]	SI [mg/kg BW/day]	Mean BW [g]	Mean FC [g/animal/d]	SI [mg/kg BW/day]	Mean BW [g]	Mean FC [g/animal/d]	SI [mg/kg BW/day]
0	293			292			292
1	325	26	104	323	26	298	323	26	926
2	351	26	96	347	26	277	347	26	862
3	365	22	78	361	22	225	360	22	703
4	384	25	85	378	25	245	378	25	761

Mean		25	91		25	261		25	813
SD		2	12		2	32		2	100
N		4	4		4	4		4	4

Analytical Concentration [mg/kg food]			1300			3700			11500

% target dose			91			87			81

Conclusions:

Based on the results described above, the NOAEL for this study was determined as the high (limit) dose level of 11.500 mg/kg food. This corresponds to the following actual substance intake: 813 mg/kg body weight/day for males; 1067 mg/kg body weight/day for females in the premating period, 930 mg/kg body weight/day for females during gestation and 1159 mg/kg body weight/day for females during lactation, respectively.

Executive summary:

SUMMARY AND CONCLUSION

- The aim of this study was to evaluate possible adverse effects of Expanded Graphite Powder after repeated exposure especially on male and female reproductive performance, such as gonadal function, mating behaviour, conception, development of the conceptus and parturition after exposure via food.

- The study was conducted following the OECD Guideline 422 and in compliance with the Principles of Good Laboratory Practice as well as with the German Animal Protection Law.

- The dose levels were based on a palatability study (Fraunhofer ITEM study no. 12N10002), in which the animals accepted food containing 10 g/kg food (estimated target dose of 1000mg/kg/day) without any problems.

- The dose of 1000 mg/kg/day is also recommended as limit dose by OECD Guideline 422.

- Consequently, this dose was chosen as the high dose in the present study, and the actual concentration in food was determined based on actual body weights and food consumption data, which resulted in a value of 12 g/kg food.

- The other doses were determined using a stagger of approx. 3. The analyses showed that preparations contained 1300, 3700, and 11.500 mg/kg food (108, 103, and 96 % of the target concentration) of the three dose groups, respectively.

- Based on mean body weights and food consumption data, this resulted in the following actual substance intake: 91, 261, and 813 mg/kg body weight/day for males; 120, 343, and 1067mg/kg body weight/day for females in the premating period; 106, 309, and 930 mg/kg body weight/day for females during gestation; and 111, 370, and 1159 mg/kg body weight/day for females during lactation, respectively.

- Wistar rats (Crl:WU) were used in this study.

- The study commenced with 40 male and 40 female rats which were randomly assigned to the control or one of the test item exposed groups.

- Since the outcome of the first pairing was insufficient, a second subset of 40 female and 40 male animals was randomly assigned to the four groups for one more mating trial.

- In the control group, a closed formula in pellet (ssniff Spezialdiäten) form was used as the diet for this study.

- For the treatment groups, the test item was sent to ssniff by Fraunhofer ITEM and added to the same commercial chow in three different concentrations.

- The animals were treated via food for 2 weeks before mating and during the mating period.

- After successful mating (day of finding sperm in vaginal smears = day 0 post conceptionem [p.c.]), treatment of the females was continued until day 4 post partum (p.p., day of birth = day 0 p.p.) and subsequent sacrifice.

- Treatment of all males and females not successfully mated was continued until their sacrifice.

- After successful mating, body weight and food consumption of the females was determined on days 0, 7, 14, and 20 p.c., as well as 0 and 4 p.p.

- A blood sample was taken without overnight fasting from the retrobulbar plexus of 5 males and 5 females per group towards the end of week two of treatment (shortly before the start of the mating period).

- In the collected blood samples hematology and clinical chemistry was investigated.

- Spontaneous locomotor activity over 60 minutes was determined shortly before sacrifice in males and towards the end of week two of treatment (shortly before the start of the mating period) in females.

- A functional observational battery (FOB) was applied shortly before sacrifice in males and towards the end of week two of treatment (shortly before the start of the mating period) in females.

- Animals were mated beginning after two weeks of treatment for two consecutive weeks or until successful mating.

- Overnight mating was performed 1 male: 1 female of the same dose group.

- Vaginal smears were taken the next morning.

- Mating was considered successful if sperm and/or a vaginal plug was found.

- The day of finding sperm was considered day 0 p.c.

- Time to successful insemination (precoital time) was determined.

- Litters were inspected at least once daily. Number of offspring and the occurrence of any dead pups or abnormalities was recorded for all groups. Individual pup sex was determined and individual weight of the pups was recorded on days 0 and 4 p.p. All pups were humanely sacrificed as soon as possible after day 4 p.p. and carefully examined for gross abnormalities.

- Males were sacrificed after successful mating, but not before day 28 of treatment.

- Females were sacrificed as soon as possible after day 4 p.p. All animals were sacrificed by C02 overdose and subsequent exsanguination. The treatment of all animals was continued until their sacrifice.

- Necropsy was performed in all F0 males and females.

- The number of corpora lutea as well as implantation sites was determined, using ammonium sulphide staining in case of no macroscopically visible implantations.

- Organ weight was determined for specified organs.

- In 5 males and 5 females of the high dose group 4 and the control group 1, complete histopathological investigation according to OECD Guideline 422 was performed.

- In testes, staging of the seminal epithelium was performed.

RESULTS

- Please see “Summary Table”

- None of the sporadically observed clinical findings regarded as test item related.

- No effects of test item exposure were observed on body weight, body weight gain or food consumption of the animals at any time point of the study.

- Overall, haematological and clinical chemistry data were found in the ranges expected for the species, strain, sex and age. Therefore, sporadical findings, albeit significantly different from controls in individual cases, are considered to be of no toxicological relevance.

- No effects of test item exposure were observed on locomotor activity, neither for any of the investigated time intervals, nor for the whole test period.

- No effects of test item exposure were observed on any of the investigated endpoints of the Functional Observational Battery.

- Since the outcome of the first mating was insufficient, additional 10 pairs per group were added to the study for one more mating trial. Consequently, each group consisted of 20 pairs for mating. For the four groups, mating yielded 19, 19, 20, and 18 sperm positive females, in groups 1-4, respectively. This resulted in 14, 10, 13, and 10 pregnancies; 11, 9, 13, and 10 females with liveborn offspring in F0 dams, and 11, 9, 12, and 10 females with live litters on day 4 p.p., respectively. The results show that mating outcome throughout all groups was insufficient. The reasons for this phenomenon are unclear.

- No effect of the test item exposure could be observed on litter size and pup survival as well as pup body weight.

- None of the sporadically observed clinical findings in pups were considered test item related and no abnormal pups were observed. The main necropsy findings consisted in reduced size of testes and epidydimides, which were found in all groups including the control group. Sporadically observed statistically significant differences in organ weights were not considered an adverse effect of the test item exposure.

- Substance-related findings were not observed in the histologically examined organs of males and females of the control and high dose group.

CONCLUSION

Based on the results described above, the NOAEL for this study (parental, reproductive and developmental) was determined as the high (limit) dose level of 11.500 mg/kg food. This corresponds to the following actual substance intake: 813 mg/kg body weight/day for males; 1067 mg/kg body weight/day for females in the premating period, 930 mg/kg body weight/day for females during gestation and 1159 mg/kg body weight/day for females during lactation, respectively.

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEL
: 813 mg/kg bw/day
Study duration:: subacute
Species:: rat
Quality of whole database:: Guideline-compliant study (OECD TG 422), conducted under GLP.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-03-09 to 2010-08-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed GLP study according to OECD technical guidelines

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

adopted on Sept 7, 2009

Deviations:

Qualifier:

according to guideline

Guideline:

EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate attached to full study report.

Limit test:

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Strain Wistar Crl:(WI)WU
- Age at study initiation: 8 weeks
- Weight at study initiation: males ca. 250g, females ca. 175g
- Housing: Makrolon, type III cages, two rats of the same sex and dose group per cage; cages and absorbing softwood bedding material were changed twice a week or more often if necessary
- Diet (e.g. ad libitum): Ssniff V1534, Ssniff Spezialdiäten, Soest, Germany, ad libitum
- Water (e.g. ad libitum): Tap water from Hannover city water supplier, ad libitum
- Acclimation period: 2-3 weeks (to be trained to be accustomed to nose-only tubes)

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

Route of administration:

inhalation

Type of inhalation exposure:

nose only

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: Low dose (MMAD/GSD) = 2.31µm/2.04
Mid dose (MMAD/GSD) = 2.36µm/2.11
High doese (MMAD/GSD) = 2.44µm/2.22

Details on inhalation exposure:

- Generation of particulate aerosols by dispersing the dry powder
- Dispersion is achieved by a feeding system and a high-pressure, high-velocity pressurized air dispersion nozzle developed by Fraunhofer ITEM.
- An MMAD in the range recommended by the guideline, i.e. approx. 1-3 µm will be generated.
- For each nose-only exposure unit, the aerosol will be generated by a high-pressure pneumatic disperser. The disperser will be fed with the test item under computerized control, i.e. with a feedback loop to the actual aerosol concentrations measured by an aerosol photometer.
- The photometer gives a scattering light signal which is proportional to the particle concentration, if the particle size distribution is constant.
- The ratio between photometer signal and concentration will be determined throughout the study by comparing to gravimetric concentrations.
- Filter samples are taken at a free port that is not used for animal exposure. The frequency of filter sampling will be chosen as needed to assure a stable aerosol generation.
- In this system, aerosols will be supplied to each rat individually, and exhaled air is immediately exhausted.
- The airflow to each rat will be approximately 1 l/min which is calculated to be laminar. Therefore measurement of the oxygen concentration is not necessary.
- Prior to the 28-day exposure of rats, technical trials to adjust particle size distributions and exposure levels will be conducted.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Please also see details on inhalation exposure.
- Air flow, temperature and relative humidity will be measured continuously and recorded by 20-minute means.
- The limits will be set at 22° C + 2° C for temperature and 55 % + 15 % for relative humidity.
- Additionally, the MMAD will be determined at least two times for each exposure unit (3 units) by a cascade impactor
- The airflow, the temperature and the relative humidity will be monitored continuously and recorded as 10 min mean values.

Duration of treatment / exposure:

6hrs per day, 5 consecutive days per week, 4 weeks

Frequency of treatment:

6hrs per day, 5 consecutive days per week, 4 weeks

Remarks:

Doses / Concentrations:
8 mg/m3 (low), 24 mg/m3 (mid), 72 mg/m3 (high)
Basis:
nominal conc.

No. of animals per sex per dose:

28d exposure: 5 males / 5 females per dose
28d recovery in control and high dose, with additional 5 males / 5 females per dose group

Control animals:

yes

Details on study design:

- Dose selection rationale: Based on dose range finding study conducted prior to this study (Fraunhofer ITEM Study-No: 09G0548)
- According to the MPDD Model (v 2.0), in the low, mid and high dose groups depositited masses of approx. 0.3, 0.7 and 1.8 mg/lung can be expected for an aerosol with an MMAD of approx. 2.5µm (3.3% deposition rate)
- Based on this a particle overload situation is induced in the high dose group
- Rationale for selecting satellite groups: To demonstrate reversibility of effects
- Post-exposure recovery period in satellite groups: 28d

Positive control:

- According to the MPDD Model (v 2.0), in the low, mid and high dose groups depositited masses of approx. 0.3, 0.7 and 1.8 mg/lung can be expected for an aerosol with an MMAD of approx. 2.5µm (3.3% deposition rate)
- Based on this, a particle overload situation is induced in the high dose group

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day (with the exception of weekends and public holidays: once daily), i.e. before and after exposure
- Cage side observations checked in table "Scheduled Investigations in the 28d-study" (Section: Any other information on materials and methods incl. tables) were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Once a week: careful examination for abnormalities concerning observation of the general condition, fur, grooming activity and visible mucous membranes

BODY WEIGHT: Yes
- Time schedule for examinations: twice a week to the nearest 0.1g throughout the study for all animals

FOOD CONSUMPTION:
- Food consumption will be recorded weekly during the study period, including post-exposure observation period

GROSS PATHOLOGY/NECROPSY - ORGAN WEIGHT
- All animals will be subjected to a complete necropsy including careful examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents.
- For anesthesia an overdose of carbon dioxide will be used.
- The abdominal cavity will be opened and the diaphragm will be cut carefully allowing the lungs to collapse.
- Heart, esophagus, upper half of trachea, thymus and lung associated lymph nodes (LALN) will be removed from the lung convolution.
- The lung will be inflated under a pressure of about 20 cm water with formalin and will be fixed by immersion for a minimum of 2 hours, and used for histopathology.
- Thereafter the weight of the lower part of the trachea will be recorded and the weight of the lung will be calculated.
- The following organs will be trimmed and wet weights will be recorded: liver, kidneys, adrenals, testes, epididymides, ovaries, uterus, thymus, spleen, brain, and heart.
- All tissues listed in OECD Guideline no. 412, table 2 will be prepared for histopathology.

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- The hematological and clinico-chemical investigations will be done after the end of exposure following a 16-hour fasting period
- Tap water ad libitum
- 5m/5f animals per dose group; see table "Scheduled Investigations in the 28d-study" (Section: Any other information on materials and methods incl. tables).
- Blood will be taken under slight halothane anesthesia by puncture of the retroorbital plexus.
- Parameters checked in "Hematology and Clinical Chemistry" (Section: Any other information on materials and methods incl. tables) will be examined

URINALYSIS: Yes
- Appearance, volume, specific gravity, pH, protein, glucose, ketones, bilirubin, blood, nitrite, urobilinogen, and leukocytes will be measured semi-quantitatively (5m/5f animals per dose group; see table "Scheduled Investigations in the 28d-study" (Section: Any other information on materials and methods incl. tables).

Sacrifice and pathology:

HISTOPATHOLOGY
- In 5m/5f animals per group after end of exposure.
- For the recovery group animals, all tissues will be preserved but only those showing changes on day 29 will be examined histopathologically.
- full histopathology on the respiratory tract and other organs and tissues, as listed in OECD 412 of all animals in the clean air control group and the Expanded Graphite Powder high dose group and all animals that died or were killed during the study.
- histopathology of lung lobes, including bronchi and the lung-associated lymph nodes (LALN), trachea, larynx, pharynx and the nasal cavities in all animals of all groups.
- Lungs will be fixed in buffered formalin (10%), embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H & E).
- A special stain will be applied for diagnosis of fibrotic changes: Masson trichrome.

Statistics:

- Differences between groups will be considered statistically significant at p < 0.05.
- Data will be analyzed using analysis of variance. If the group means differ significantly by the analysis of variance the means of the treated groups will be compared with the means of the control groups using Dunnett's test.
- The statistical evaluation of the histopathological findings will be done with the two-tailed Fisher test by the P.L.A.C.E.S. system.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

See section "Details on results"

Mortality:

mortality observed, treatment-related

Description (incidence):

See section "Details on results"

Body weight and weight changes:

no effects observed

Description (incidence and severity):

See section "Details on results"

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

See section "Details on results"

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

See section "Details on results"

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

See section "Details on results"

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See section "Details on results"

Histopathological findings: neoplastic:

no effects observed

Details on results:

SURVIVAL OF ANIMALS AND CLINICAL OBSERVATIONS
- One rat was found dead (before a high body weight loss had been measured)
- Upon necropsy, a highly dilatated left kidney space and ureter were observed, with the right kidney being highly enlarged and purative.
- This severe kidney finding is incidentally observed in this rat strain and probably caused by a bacterial infection.
- All other animals survived the study and, generally, tolerated well the exposure at all concentrations.
- The only relevant clinical observation was an increased rate of eye irritation in some rats of the high dose groups.
- Eye lids appeared slightly red-brownish discoloured. This effect is considered as particle-related, however, it disappeared until the next morning.
- The macroscopical observations made upon sacrifice of animals reflect some test item-related effects such as the test/reference item deposition in the deep lung areas (discoloration), however, severe test item-related adverse effects were not observed macroscopically.

BODY WEIGHT DATA
- Statistically significant changes were not observed in the treatment groups as compared to controls.

FOOD CONSUMPTION
- Upon necropsy, test item- or dose-related macroscopical findings were not observed. Statistically significant values are considered as incidental findings.

HEMATOLOGY, CLINICAL CHEMISTRY AND URINALYSIS
- Overall, hematological and clinical chemistry data were found in the ranges expected for the species, strain, sex and age.
- Therefore, those findings, albeit significantly different from controls, are considered to represent incidental findings.

HEMATOLOGY
- For red blood cells, hemoglobin and hematocrit statistically significant decreases were observed in the male groups and partially in the female groups.
- Although these are considered as test-item related alterations the alterations are very slight and the values are still in the range usually observed for rats of this species, strain, sex and age (historical data).
- No treatment related adverse effects were detected in the other hematology parameters.

CLINICAL CHEMISTRY
- All values are in the range expected for this species, strain, sex and age.
- Significant differences are considered to be due to biological variance.

URINALYSIS
- All values are in the range expected for this species, strain, sex and age.
- Statistically significant increases were not observed.

GROSS PATHOLOGY/NECROPSY - ORGAN WEIGHTS
- Upon necropsy, test item- or dose-related macroscopical findings were not observed.
- The absolute and relative organ wet weights did show statistically significant changes as compared to controls only in the target organ lungs, i.e. absolute and relative lung weights (males: high dose groups only; females: mid and high dose group).

HISTOPATHOLOGY
- Test substance-related findings were observed in the nasal cavity, trachea, lung and lung-associated lymph nodes (LALN).
- Deposition of particle-laden macrophages was observed in lung, trachea and LALN.
- In the lung, the majority of the particle-laden macrophages were located within the alveoli with a minor portion lodged within the interstitium (perivascular, peribronchiolar and intraseptal).
- At the end of the recovery study, the interstitial portion of particle-laden macrophages was more prominent than after the end of the main study indicating a time-dependent translocation of particles from the alveoli into the interstitium. This is also reflected by a higher burden of particle-laden macrophages in the lung-associated lymph nodes in the recovery animals.
- In the trachea, it was difficult to distinguish whether the subepithelial particle deposits at the carina of the bifurcation were cell-associated or lying freely within the interstitium.
- Although this site is known as a common site for lesions, presumably because this area receives a higher dose from direct aerosol impaction, only minimal or mild adaptive mucous cell hyperplasia could be observed in this region in a few rats from the Graphite high-dose group of the main study.
- In the nasal cavity, the observed changes were mainly located in the anterio-ventral compartment which is mostly exposed to the airflow.
- Dose-dependent changes were eosinophilic hyaline cytoplasmic inclusions within respiratory epithelial cells (all Graphite exposure groups), (adaptive) mucous (goblet) cell hyperplasia (Graphite mid- and high-dose group), subepithelial inflammatory cell infiltration (Graphite mid- and high-dose group), respiratory epithelial hyperplasia (Graphite high-dose group) and squamous cell metaplasia (Graphite high-dose group).
- Eosinophilic inclusions were also observed in a female control rat, and are known as common spontaneous age-related
- The increased incidence of eosinophilic globules in the Graphite low-dose group of the present study is interpreted as an adaptive rather than adverse effect, because no association with inflammatory cell infiltration and mucous cell hyperplasia was observed at this dose level.
- Thus the Graphite mid-dose group is considered to represent the adverse-effect level in the nasal cavity. There was only a weak decrease of the induced nasal cavity lesions in the animals of the recovery study as compared to the main study.
- In the lung, clearly adverse effects such as markedly increased incidence of interstitial mononuclear cell infiltration and interstitial fibrosis, were seen in the Graphite high-dose group.
- However, due to its high incidence in the Graphite mid- and high-dose group, bronchiolo-alveolar hyperplasia of the bronchiolar type (alveolar bronchiolization) is already interpreted as a mild adverse rather than adaptive effect.
- This type of hyperplasia is considered to be non-preneoplastic and to represent an “attempt of the lung” to facilitate a more efficient removal of inhaled materials via the mucociliary escalator by extension of bronchiolar epithelium into the alveolar ducts.
- In contrast, bronchial/bronchiolar mucous cell hyperplasia seen in the Graphite high-dose group only is considered as an adaptive non-adverse change. With the exception of mucous cell hyperplasia, all induced changes in the lung as well as in the LALN persisted until the end of the recovery study.
- In all other organs, including larynx and nasopharynx, no effects of the Graphite exposure could be observed.

Dose descriptor:

NOAEC

Effect level:

8 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Effects in the respiratory tract

Critical effects observed:

not specified

Macroscopical Findings Upon Sacrifice

- Macroscopical observations made upon sacrifice of animals are summarized below (lungs, lung-associated lymph nodes and other organs have been under inspection).

- They reflect some test item-related effects such as the test/reference item deposition in the deep lung areas (discoloration), however, severe test item-related adverse effects were not observed macroscopically.

TABLE: Macroscopical Findings Upon Sacrifice on Day 29 and Day 56

Treatment	Clean Air	Expanded Graphite low	Expanded Graphite mid	Expanded Graphite high
Aerosol concentration (mg/m³)	-	8	24	72
Group #	1	2	3	4
Number of Rats(M+F each)	5 5	5* 0	5 0	5 5
Day	29 56	29 56	29 56	29 56
MALES
Within normal limits	4 5	0 -	0 -	0 0
LUNGS
Multiple grey discoloration	0 0	4 -	5 -	5 2
Multiple grey-black discoloration	0 0	0 -	0 -	5 3
LALN
slightly enlarged	0 0	0 -	1 -	3 3
Grey discolored	0 0	0 -	5 -	5 0
Black discolored	0 0	0 -	0 -	0 5
Testes and Epididymides
Left: Slightly decreased in size	1 0	0 -	0 -	0 0
Right testis: Slightly enlarged	- -	1 -	0 -	0 0
Left testis: Slightly decreased	- -	- -	1 -	0 0
FEMALES
Within normal limits	5 4	0 -	0 -	0 0
LUNGS
Multiple grey discoloration	0 0	5 -	4 -	5 1
Multiple grey-black discoloration	0 0	0 0	0 -	0 4
LALN
slightly enlarged	0 0	- -	0 -	0 3
Grey discolored	0 0	0 -	5 -	0 0
Black discolored	0 0	0 -	0 -	2 5
Uterus Horns
Slightly cystic enlargements	0 1	1 -	3 -	3 1
Ovaries
Slightly decreased	0 0	0 -	0 -	2 0

* One rat found dead on day 22 (not included in this table) - LALN: lung-associated lymph nodes

Histopathology: Non-Item Substance-Related Noteworthy Findings in other Organs

- One rat which died before the terminal sacrifice date showed a spontaneous bilateral very severe purulent pyelonephritis (= cause of death) associated with inflammation of a ureter, atrophy of the coagulating glands and seminal vesicles, epithelial erosion of the glandular stomach and lymphoid depletion of the thymus and spleen.

- Common spontaneous findings in groups 1 and 4 from which all protocol organs were examined included estrus cycle-dependent luminal dilatation of the uterus, degeneration of the seminiferous tubules in the testis, microgranulomas in the liver, basophilic tubules (tubular basophilia) in the kidneys and subepithelial mononuclear cell infiltration in the larynx.

- These findings occurred at incidences between 2/5 and 5/5 per sex and group and all were considered to be spontaneous and unrelated to Graphite exposure.

- Common spontaneous findings were basophilic tubules (tubular basophilia) in the kidneys (males only) and microgranulomas in the liver occurring at incidences of up to 4/5 rats per group.

Conclusions:

Based on this 28-day nose-only inhalation study an NOAEL of 8 mg/m3 was derived based on the histopathological examination of the respiratory tract. Expanded Graphite Powder showed effects that were to be expected for a poorly soluble dust with low toxicity.

Executive summary:

- A 28-day nose-only inhalation in the rat according to Commission Regulation (EC) No 440/2008, Part B.8. and OECD Guideline No. 412 was conducted to assess and evaluate potential toxic effects of respirable fractions of Expanded Graphite Powder

-Thirty male and 30 female Wistar rats [strain Crl:WI(WU)] were used for this study and allocated to 4 groups each: Clean Air Control, Expanded Graphite Powder low (8 mg/m³), Expanded Graphite Powder mid (24 mg/m³), and Expanded Graphite Powder high (72 mg/m³). The target aerosol concentrations were achieved satisfactorily, i.e. to 103% - 102% - 99%, respectively.

- One rat was found dead (death due to a severe kidney finding that is incidentally observed in this rat strain).

- All other male and female test animals survived treatment and were euthanized at scheduled dates.

- Effects indicating systemic toxicity were not observed. Sex-specific differences were not detected.

- Body weight development did not show any statistically significant changes as compared to concurrent controls.

- Food consumption did not show treatment-related significant changes as compared to concurrent controls. Statistically significant values are considered as incidental findings.

- Hematology and clinical chemistry data were all in the ranges expected for the species, strain, sex and age. Some statistically significant data are considered as incidental findings.

- Absolute and relative lung weights were statistically significantly increased in the high dose group (males) or the mid and high dose group (females). After a 28-day recovery these values were still significantly increased.

- All other organ weights did not show any statistically significant changes as compared to concurrent controls.

- The histopathological examination in the nasal cavity showed as dose-dependent changes eosinophilic hyaline cytoplasmic inclusions within respiratory epithelial cells (all Expanded Graphite groups), (adaptive) mucous (goblet) cell hyperplasia (Graphite mid- and high-dose group), subepithelial inflammatory cell infiltration (Graphite mid- and high-dose group), respiratory epithelial hyperplasia (Graphite high-dose group) and squamous cell metaplasia (Graphite high-dose group). Thus the Graphite mid-dose group is considered to represent the adverse-effect level in the nasal cavity.

- In the lung, clearly adverse effects such as markedly increased incidence of interstitial mononuclear cell infiltration and interstitial fibrosis, were seen in the Graphite high-dose group.

- Based on this 28-day nose-only inhalation study an NOAEL of 8 mg/m3 was derived based on the histopathological examination of the respiratory tract. Expanded Graphite Powder showed effects that were to be expected for a poorly soluble dust with low toxicity.

Reference 2

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-04-21 to 2010-08-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed GLP study according to OECD technical guidelines

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

adopted on Sept 7, 2009

Deviations:

Qualifier:

according to guideline

Guideline:

EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate attached to full study report.

Limit test:

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Strain Wistar Crl:(WI)WU
- Age at study initiation: 11-12 weeks
- Weight at study initiation: males ca 320g, females ca. 190g
- Housing: Makrolon, type III cages, two rats of the same sex and dose group per cage; cages and absorbing softwood bedding material were changed twice a week or more often if necessary
- Diet (e.g. ad libitum): Ssniff V1534, Ssniff Spezialdiäten, Soest, Germany, ad libitum
- Water (e.g. ad libitum): Tap water from Hannover city water supplier, ad libitum
- Acclimation period: 5-6 weeks (to be trained to be accustomed to nose-only tubes)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 2°C
- Humidity (%): 55% +/- 15%
- Photoperiod (hrs dark / hrs light): 12/12
- Air exchange rate: min 10-fold air change per hour

Route of administration:

inhalation

Type of inhalation exposure:

nose only

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: Low dose (MMAD/GSD) = 2.16µm/1.63
Mid dose (MMAD/GSD) = 2.33µm/1.59
High dose (MMAD/GSD) = 2.34µm/1.66

Details on inhalation exposure:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Duration of treatment / exposure:

6hrs per day, 5 consecutive days per week, 4 weeks

Frequency of treatment:

6hrs per day, 5 consecutive days per week, 4 weeks

Remarks:

Doses / Concentrations:
12 mg/m3 (low), 36 mg/m3 (mid), 108 mg/m3 (high)
Basis:
nominal conc.

No. of animals per sex per dose:

28d exposure: 5 males / 5 females per dose
28d recovery in control and high dose, with additional 5 males / 5 females per dose group

Control animals:

yes

Details on study design:

- Dose selection rationale: Based on dose range finding study conducted prior to this study (Fraunhofer ITEM Study-No: 09G0548)
- According to the MPDD Model (v 2.11), in the low, mid and high dose groups depositited masses of approx. 0.45, 1.0 and 2.7 mg/lung can be expected for an aerosol with an MMAD of approx. 2.5µm (3.3% deposition rate)
- Based on this a particle overload situation is induced in the high dose group
- Rationale for selecting satellite groups: To demonstrate reversibility of effects
- Post-exposure recovery period in satellite groups: 28d

Positive control:

- According to the MPDD Model (v 2.11), in the low, mid and high dose groups depositited masses of approx. 0.45, 1.0 and 2.7 mg/lung can be expected for an aerosol with an MMAD of approx. 2.5µm (3.3% deposition rate)
- Based on this a particle overload situation is induced in the high dose group

Observations and examinations performed and frequency:

Sacrifice and pathology:

Statistics:

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

(See section "Details on results")

Mortality:

mortality observed, treatment-related

Description (incidence):

(See section "Details on results")

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

(See section "Details on results")

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

(See section "Details on results")

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

(See section "Details on results")

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

(See section "Details on results")

Histopathological findings: neoplastic:

no effects observed

Details on results:

SURVIVAL OF ANIMALS AND CLINICAL OBSERVATIONS
- All animals survived the study and, generally, tolerated well the exposure at all concentrations.
- The only relevant clinical observation was an increased rate of eye irritation in some rats of the high dose groups.
- Eye lids appeared slightly red-brownish discoloured. This effect is considered as particle-related, however, it disappeared until the next morning.
- The macroscopical observations made upon sacrifice of animals reflect some test item-related effects such as the test/reference item deposition in the deep lung areas (discoloration), however, severe test item-related adverse effects were not observed macroscopically.

BODY WEIGHT DATA
- Statistically significant changes were observed in the male treatment group during the recovery phase (not in the female groups).
- During the exposure period no statistical significance was found.
- Therefore, this finding is considered as incidental due to a higher body weight in the control group accompanied by a small standard deviation.
- A treatment-related effect cannot be derived from this data.

FOOD CONSUMPTION
- Upon necropsy, test item- or dose-related macroscopical findings were not observed.
- Statistically significant values are considered as incidental findings.

HEMATOLOGY, CLINICAL CHEMISTRY AND URINALYSIS
- Overall, hematological and clinical chemistry data were found in the ranges expected for the species, strain, sex and age.
- Therefore, those findings, albeit significantly different from controls, are considered to represent incidental findings.

HEMATOLOGY
- For red blood cells, hemoglobin and hematocrit statistically significant decreases were observed in the male groups and partially in the female groups.
- Although these are considered as test-item related alterations the alterations are very slight and the values are still in the range usually observed for rats of this species, strain, sex and age (historical data).
- No treatment related adverse effects were detected in the other hematology parameters.

CLINICAL CHEMISTRY
- All values are in the range expected for this species, strain, sex and age.
- Significant differences marked in the tables are considered to be due to biological variance.

URINALYSIS
- All values are in the range expected for this species, strain, sex and age.
- Statistically significant increases were not observed.

GROSS PATHOLOGY/NECROPSY – ORGAN WEIGHTS
- Upon necropsy, test item- or dose-related macroscopical findings were not observed.
- The absolute and relative organ wet weights did show statistically significant changes as compared to controls only in the target organ lungs, i.e. absolute and relative lung weights (males: high dose groups only; females: mid and high dose group).

HISTOPATHOLOGY
- Test particle-related findings were observed in the nasal cavity, trachea, lung and lung-associated lymph nodes (LALN).
- Deposition of particle-laden macrophages was observed in lung, trachea and LALN.
- In the lung, the majority of the particle-laden macrophages were located within the alveoli with a minor portion lodged within the interstitium (perivascular, peribronchiolar and intraseptal).
- At the end of the recovery study, the interstitial portion of particle-laden macrophages was more prominent than after the end of the main study indicating a time-dependent translocation of particles from the alveoli into the interstitium. This is also reflected by a higher burden of particle-laden macrophages in the lung-associated lymph nodes in the recovery animals.
- In the trachea, it was difficult to distinguish whether the subepithelial particle deposits at the carina of the bifurcation were cell-associated or lying freely within the interstitium.
- Although this site is known as a common site for lesions, presumably because this area receives a higher dose from direct aerosol impaction, only minimal adaptive mucous cell hyperplasia could be observed in this region in a single rat from the Graphite mid-dose group.
- In the nasal cavity, the observed changes were all located in the anterio-ventral compartment which is mostly exposed to the airflow.
- Dose-dependent changes were eosinophilic hyaline cytoplasmic inclusions within respiratory epithelial cells (all exposure groups), (adaptive) mucous (goblet) cell hyperplasia (all exposure dose groups) and subepithelial inflammatory cell infiltration (mid- and high-dose group).
- Eosinophilic cytoplasmic inclusions are known as common spontaneous age-related change.
- The significant incidence of eosinophilic globules in the Graphite low-dose group of the present study is interpreted as an adaptive rather than adverse effect, because no association with inflammatory cell infiltration was observed at this dose level.
- In contrast to another study with Expanded Graphite, no respiratory epithelial hyperplasia and no squamous cell metaplasia could be observed in the nasal cavities of the present study.
- In addition, the severity scores for the eosinophilic inclusions were minimally lower than in the previous study.
- The mid-dose group is considered to represent the adverse-effect level in the nasal cavity.
- The same is true for the lung where adverse effects such as significantly increased incidence of interstitial mononuclear cell infiltration, interstitial fibrosis and bronchiolo-alveolar hyperplasia of the bronchiolar type (alveolar bronchiolization) were seen in the mid- and high-dose group.
- The bronchiolar type of bronchiolo-alveolar hyperplasia is considered to be non-preneoplastic and to represent an “attempt of the lung” to facilitate a more efficient removal of inhaled materials via the mucociliary escalator by extension of bronchiolar epithelium into the alveolar ducts.
- Although in contrast to the previous study, bronchial/bronchiolar mucous cell hyperplasia in the lungs was not observed, the adverse lung effects were slightly more pronounced in the present study than in the previous one, probably due to the higher dosage of Graphite used in the present study.
- Although some of the exposure-related findings slightly decreased in incidence and severity like mucous cell hyperplasia in the nasal cavity, they persisted until the end of the recovery time.
- In all other organs, including larynx and nasopharynx, no effects of the test item exposure could be observed.

Dose descriptor:

NOAEC

Effect level:

12 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Effects in the respiratory tract

Critical effects observed:

not specified

-Macroscopical observations made upon sacrifice of animals are summarized below (lungs, lung-associated lymph nodes and other organs have been under inspection).

-They reflect some test item-related effects such as the test/reference item deposition in the deep lung areas (discoloration), however, severe test item-related adverse effects were not observed macroscopically.

TABLE: Macroscopical Findings at Sacrifice on Day 31 and Day 58

Treatment	Clean Air	Synthetic Graphite low	Synthetic Graphite mid	Synthetic Graphite high
Aerosol concentration (mg/m³)	-	12	36	108
Group #	1	2	3	4
Number of Rats (M+F each)	5 5	5 0	5 0	5 5
Day	31 58	31 58	31 58	31 58
MALES
Within normal limits	2 5	0 -	0 -	0 0
LUNGS
Multiple grey-black discoloured areas	0 0	5 -	5 -	5 5
LALN
Slight enlargement	0 0	0 -	0 -	1 0
Grey-black discolouration	0 0	0 -	2 -	5 5
Testes and Epididymides
Slight reduction in size	3 0	1 -	2 -	2 0
FEMALES
Within normal limits	4 4	0 -	0 -	0 0
LUNGS
Multiple grey-black discoloured areas	0 0	5 -	5 -	5 5
LALN
Slight enlargement	0 0	0 -	0 -	3 0
Grey-black discolouration	0 0	0 -	5 -	5 5
Kidneys
Yellow-brownish raised area	1 0	0 -	0 -	0 0
Unilateral cyste	0 1	0 -	0 -	1 0
Sligh unilateral dilatation	0 0	1 -	0 -	0 0
Uterus Horns
Slightly cystic enlargement	0 0	4 -	1 -	1 0

LALN: lung-associated lymph nodes

Histopathology: Non-Item Substance-Related Noteworthy Findings in other Organs

- Common spontaneous findings in groups 1 and 4 from which all protocol organs were examined included estrus cycle-dependent luminal dilatation of the uterus, degeneration of the seminiferous tubules in the testis associated with aspermia, oligospermia and atrophy of the epididymides, myocardial degeneration and mononuclear cell infiltration of the heart, microgranulomas in the liver, basophilic tubules (tubular basophilia), tubular dilatation and transitional cell hyperplasia in the kidneys and epithelial hyperplasia of the thymus.
- These findings occurred at incidences between 2/5 and 5/5 per sex and group and all were considered to be spontaneous and unrelated to Synthetic Graphite exposure.
- Common spontaneous findings were basophilic tubules (tubular basophilia) in the kidneys, microgranulomas and perivascular mononuclear cell infiltration in the liver, degeneration of the seminiferous tubules in the testis and epithelial hyperplasia in the thymus.
- All these findings occurred at incidences between 2/5 and 4/5 per sex and group.

Conclusions:

Based on this 28-day nose-only inhalation study an NOAEL of 12 mg/m3 was derived based on the histopathological examination of the respiratory tract. Synthetic Graphite Powder showed effects that were to be expected for a poorly soluble dust with low toxicity.

Executive summary:

- A 28-day nose-only inhalation in the rat according to Commission Regulation (EC) No 440/2008, Part B.8. and OECD Guideline No. 412 to evaluate potential toxic effects of Synthetic Graphite Powder

- Thirty male and 30 femaleWistar rats [strain Crl:WI(WU)] were used for this study and allocated to 4 groups each: Clean Air Control, Synthetic Graphite Powder low (12 mg/m³), Synthetic Graphite Powder mid (36 mg/m³), and Synthetic Graphite Powder high (108 mg/m³). The target aerosol concentrations of 12, 36 and 108 mg Synthetic Graphite Powder/m³were achieved satisfactorily, i.e. to 103% - 101% - 101%, respectively.

- All test animals survived treatment and were euthanized at scheduled dates.

- Effects indicating systemic toxicity were not observed. Sex-specific differences were not detected.

- Body weight development did not show treatment-related statistically significant changes as compared to concurrent controls.

- Food consumption did not show treatment-related significant changes as compared to concurrent controls. Statistically significant values are considered as incidental findings.

- Hematology and clinical chemistry data were all in the ranges expected for the species, strain, sex and age. Some statistically significant data are considered as incidental findings.

- Wet lung weights were statistically significantly increased dose-dependently in the mid and high dose groups of both sexes (absolute data: males, in addition, also in the low dose group - relative data: females, in addition, also in the low dose group). After a 28-day recovery these values were still significantly increased.

- All other organ weights did not show any treatment-related statistically significant changes as compared to concurrent controls.

- The histopathological examination in thenasal cavityshowed as dose-dependent changes eosinophilic hyaline cytoplasmic inclusions within respiratory epithelial cells (all Synthetic Graphite groups),(adaptive) mucous (goblet) cell hyperplasia (Graphite low, mid- and high-dose group) and subepithelial inflammatory cell infiltration (Graphite mid- and high-dose group). Overall, the Graphite mid-dose group is considered to represent the adverse-effect level in the nasal cavity.

- In the lung, clearly adverse effects such as markedly increased incidence of interstitial mononuclear cell infiltration and interstitial fibrosis were seen in the Graphite mid and high dose group.

- In larynx and nasopharynx and all other organs, no treatment-related effects were observed.

- Based on this 28-day nose-only inhalation study an NOAEL of 12 mg/m3 was derived based on the histopathological examination of the respiratory tract. Synthetic Graphite Powder showed effects that were to be expected for a poorly soluble dust with low toxicity.

Reference 3

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-08-06 to 2022-09-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Version / remarks:

25 June 2018

Qualifier:

according to guideline

Guideline:

EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: SGL CARBON GmbH (86400 Meitingen, Germany), batch number: 2205/09
- Purity: 96.45%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature (15 to 25°C)
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: The crystal structure of graphite consists of planar hexagonal layers of carbon atoms. Graphite is the most stable form of carbon under standard conditions. A transformation into any other modification or form of carbon could only be achieved with extremely high energy expenditure (temperature, pressure). For this reason, graphite with a purity of >95% shows no physical aging effect. The test item was therefore considered acceptable foruse for the duration of the study.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- The test item was used as supplied

Species:

rat

Strain:

Wistar

Remarks:

CRL:WI(Han)

Details on species / strain selection:

The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The Crl:WI(Han) strain was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 46 to 52 days.
- Weight at study initiation: Males: 170 to 230 g, Females: 128 to 194 g
- Fasting period before study: no
- Housing: Five animals of the same sex were housed together (main and lung sampling animals). For validation animals, three of the same sex were group-housed in polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals. Wood based bedding was used which was changed at appropriate intervals each week. For environmental enrichment, aspen gnawing material and plastic shelter was provided to each cage throughout the study and replaced when necessary. In addition, diamond twists were provided to each cage from Week 4 and replaced when necessary.
- Diet: Teklad 2014C Diet, ad libitum
- Water: Potable water from the public supply was provided ad libitum via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
- Acclimation period: 11 days before commencement of treatment. Additionally, the animals on study were acclimated to the method of restraint, over a three-day period preceding the first test item exposure.

DETAILS OF FOOD AND WATER QUALITY:
- Certificates of analysis for the diet were scrutinized and approved before any batch of diet was released for use. Certificates of analysis are routinely provided by the water supplier. Certificates of analysis were also received from the suppliers of the wood based bedding and Aspen gnawing material. No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 40-70%
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light: 12 hours dark with simulated periods of dawn and dusk.

IN-LIFE DATES: From: 22 November 2021 To: 21 to 23 February 2022 (06 December 2021 for validation animals)

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

snout only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

> 2.4 - < 2.5 µm

Geometric standard deviation (GSD):

2.95

Remarks on MMAD:

During preliminary generation trials for this study, it was found that the particle size of the material is slightly above (mass median aerodynamic diameter (MMAD) 2.3-2.6 µm; σg 1.92-2.61) the range stipulated in the OECD TG 413 (MMAD ≤2 µm with σg or GSD of 1-3).
As a consequence, several adjustments within the generation equipment and settings were made in order to reduce the particle size. Measures taken and resulting MMADs are listed below:
- Increase in airflow: MMAD: 2.5 µm; GSD:2.23
- Jet mill and elutriator added: MMAD: 2.5 µm; GSD:2.74
- Jet mill and elutriator removed, airflow further increased: MMAD: 2.1 µm; GSD 2.50
- Repeat of higher airflow: MMAD: 2.2 µm; GSD 2.54
- Repeat of higher airflow: MMAD: 1.9 µm; GSD 2.44
The result of these trials showed that increasing the airflow was the most successful method of reducing the particle size to a minimum MMAD range of 1.9-2.2 µm, which was within the maximum range set out in OECD TG 413.
These parameters were therefore applied for aerosol generation in the main study.
Under the conditions of the main study, the MMAD of individual particle size distribution samples and the overall mean MMAD for all groups exposed to Expanded Graphite Powder was slightly above the guideline recommended size. Actions taken during preliminary characterization trials to reduce the MMAD included using an in-line micronizer and processing (mechanically milling) the test item, however, these did not offer any improvement in the MMAD. The test item was therefore used as supplied for animal exposures. It is considered that the obtained MMAD (mean of 2.4 to 2.5 μm (mean GSD of 2.71 to 2.95)) was the smallest possible at the target concentrations. Under the conditions of this study the aerosols were considered respirable to the rat.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Flow through nose-only chamber. Aluminum alloy construction comprising a base unit, three animal exposure sections, a top section, and a pre-chamber. An elutriator was incorporated into the Expanded Graphite Powder exposure systems, in between the aerosol generator and the prechamber. The elutriator connected to the pre-chamber by aerosol tubing.
- Method of holding animals in test chamber: Plastic nose-only restraint tube
- Source and rate of air: Inlet airflow was provided from in-house compressed air system with breathing quality. Inlet flow: 49 L/minute. Extract flow: 50 L/minute
- Method of conditioning air: Aerosol conditioning pre-chamber was used.
- System of generating particulates/aerosols: Wright Dust Feed Mechanism. The WDF mechanism is designed to produce and maintain test atmospheres containing dust by suspending material scraped from the surface of a compressed powder in a stream of dry air.
- Temperature, humidity, pressure in air chamber: Chamber air temperature was measured throughout exposure using an electronic thermometer probe placed in the breathing zone of the animals via an unused exposure port. Chamber relative humidity was measured throughout exposure using an electronic hygrometer probe placed in the breathing zone of the animals via an unused exposure port. Chamber air temperature and relative humidity were monitored continuously and recorded from 30 minutes into exposure, then hourly thereafter and at the end of exposure.
- Air flow rate: Inlet flow: 49 L/minute.
- Air change rate: Not specified.
- Method of particle size determination: Determined by cascade impaction with Marple 290 Series (298 configuration) combined with gravimetric analysis.
- Treatment of exhaust air: Extract airflow was drawn by in-house vacuum system and filtered locally.

TEST ATMOSPHERE
- Brief description of analytical method used: Aerosol samples were collected by a glass fiber filter, held in an open face filter holder at a sample flow of 2 L/minute (Group 4) and 5 L/minute (Group 2 and Group 3). Sample volume was measured by wet-type gas meter. A minimum of 3 samples/group/day were taken at approximately 60, 180 and 300 minutes during exposure. Samples were analysed gravimetrically.
- Samples taken from breathing zone: yes. Samples taken from animal exposure port.

VEHICLE (if applicable)
- Justification for use and choice of vehicle: Not specified.
- Composition of vehicle: Compressed air only
- Concentration of test material in vehicle: 0, 3.20, 9.94 and 31.10 µg/l
- Purity of vehicle: breathing quality

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Calculation of MMAD and GSD was performed by linear regression of the probit of the cumulative percentage, by mass, of particles smaller than the Effective cut-off diameter (ECD) of each stage versus the logarithm of the ECD of each stage.
For more details on analytical verification of doses or concentrations, please refer to section "Details on inhalation exposure".

Duration of treatment / exposure:

6 hours/day

Frequency of treatment:

Week 1 to 12: Day 1 to 5 of each study week.
Week 13: 6 days (Days 1 to 5 and 7).
Additional exposures were conducted for main study animals to cover the period of necropsy.

Dose / conc.:

3.2 mg/m³ air

Remarks:

Achieved concentration

Dose / conc.:

9.94 mg/m³ air

Remarks:

Achieved concentration

Dose / conc.:

31.1 mg/m³ air

Remarks:

Achieved concentration

No. of animals per sex per dose:

10/sex/dose plus 5 males/dose for lung sampling

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The target exposure levels were selected based on results from a 4-week inhalation study in the rat (Study No. 02 G 10 003). In that study concentrations of 8.21, 24.4 or 71.5 μg/L were well tolerated, there were no systemic effects.
Microscopically, test item-related findings were observed in the nasal cavity, trachea, lung and lung associated lymph nodes. In the nasal cavity, mucous cell hyperplasia and subepithelial inflammatory cell infiltration were seen at ≥24.4 μg/L with additional findings of respiratory epithelial hyperplasia and squamous cell metaplasia at 71.5 μg/L. In the lung, multifocal alveolar accumulation of particle-laden macrophages and bronchioloalveolar hyperplasia were observed at all concentrations. Additional observations of increased incidence of interstitial mononuclear cell infiltration and interstitial fibrosis were seen at 71.5 μg/L. In the trachea, subepithelial deposits of particles were observed at ≥24.4 μg/L with accumulation of particle-laden macrophages in the lymph nodes at all concentrations.
Pathological changes in the respiratory tract at 24.4 μg/L were generally of lower incidence and/or severity than those seen at 71.5 μg/L. Therefore for this study, a high exposure level targeted at 30 μg/L is anticipated to induce test-item related changes similar to those previously seen but is expected to be tolerated for 13 weeks. Target exposure levels of 9.5 and 3.0 μg/L have been selected for the intermediate and low groups respectively to identify a no-observed adverse effect concentration, if possible, and to explore any possible concentration relationship.
- Rationale for animal assignment:: Randomly allocated on arrival. Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals. Each sex was allocated separately.
- Fasting period before blood sampling for clinical biochemistry: Blood samples were collected after overnight withdrawal of food and prior to dosing.
- Post-exposure recovery period in satellite groups: no
- Section schedule rationale:: To allow satisfactory inter-group comparison.

Positive control:

Not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment.
Cages were inspected daily for evidence of animal ill-health amongst the occupants. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization periods, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed weekly physical examination was performed on each animal to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded twice weekly on Weeks -1 to 4 (on Day 1 and 5 of each study week), then weekly thereafter, and before necropsy.
More frequent weighings were instituted, when appropriate, for animals displaying ill-health, so that the progress of the observed condition could be monitored. These data are retained in the study data but are not reported.

FOOD CONSUMPTION:
- The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started and for each week throughout the study.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes . Prior to each examination, the pupils of each animal were dilated using tropicamide ophthalmic solution (Mydriacyl). The adnexae, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and fundus were examined.
- Time schedule for examinations: The eyes of the animals were examined by means of a binocular indirect ophthalmoscope before treatment and in week 13
- Dose groups that were examined: All main study and spare animals at pretreatment. Main study animals of Groups 1 and 4 in week 13. As no test item-related changes were observed, the examination was not extended to Groups 2 and 3 in Week 13.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected after overnight withdrawal of food and prior to dosing in week 13
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: All main study animals
- Parameters checked: Hematocrit (Hct)*, Hemoglobin concentration (Hb), Erythrocyte count (RBC), Absolute reticulocyte count (Retic), Mean cell hemoglobin (MCH)*, Mean cell hemoglobin concentration (MCHC)*, Mean cell volume (MCV), Red cell distribution width (RDW), Total leucocyte count (WBC), Differential leucocyte count (Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC)), Platelet count (Plt).
* Derived values calculated in ClinAxys.
Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written description from the blood film was made where appropriate. In the presence of platelet clumping a manual count of the differential white blood cell parameters was performed. Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using a Stago STA Compact Max analyzer and appropriate reagent in respect of: Prothrombin time (PT) - using IL PT Fibrinogen reagent, Activated partial thromboplastin time (APTT) - using IL APTT reagent.
- Week 13 coagulation samples for Group 1 to 4 males were analyzed outside of validated stability, the samples were analyzed 3 hours and 10 minutes after separation following storage at ambient temperature, 10 minutes outside of validated criteria (room temperature stability is established as up to 3 hours). Published data on coagulation parameters from rat citrate plasma suggests that PT and APTT can be analyzed without significant effect for up to 6 hours after collection following room temperature storage (Goyal et al, 2015). Considering the published data the minor time deviation is considered negligible and is unlikely to have any impact on the reported data. However, as samples were analyzed outside of validated criteria, they are reported for information only. As all male coagulation samples were subject to the same deviation a comparison between the groups can be made. Samples for females were collected on a different day and were not subject to the same deviation.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected after overnight withdrawal of food and prior to dosing in week 13
- Animals fasted: Yes
- How many animals: All main study animals
- Parameters checked: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Total bilirubin (Bili), Urea, Blood urea nitrogen (BUN), Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Total protein (Total Prot), Albumin (Alb).
Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: At termination, within 24 hours of final exposure.
- Dose groups that were examined: All dose groups
- Number of animals: All main study animals
- Parameters checked: Total and differential cell count (neutrophils, eosinophils, mononuclear cells (includes monocytes and macrophages) and lymphocytes) of BAL cells, Lactate dehydrogenase (LDHL), Total protein (Prot)
- Dehydrogenase (LDH) and total protein (TP) results for Group 4 males 31 to 35 and Group 4 females 131-135 were analyzed out of stability and are reported for information. In error the samples were stored frozen at -70°C for approximately 16 hours and analyzed the day after collection. As no stability is available for LDH and TP in frozen BAL fluid the results of these analytes from these samples are reported for information only and excluded from the calculation of study means. LDH and TP results are available from Group 4 males 36 to 40 and Group 4 females 136 to 140 from Week/Day 14.2 and 14.3, respectively (Day 2 and 3 of terminal procedures). There are sufficient data from Group 4 (N=5 from each sex) to assess for an effect of exposure to the test item on this endpoint and therefore this deviation has no impact on the interpretation of the study data.

LUNG BURDEN: Yes
- Time schedule for analysis: At termination (Day 92)
- Dose groups that were examined: All lung sampling animals of Groups 1 to 4
- Number of animals: 5 males per group
- Parameters checked: Gravimetric determination of test material concentration in lung samples.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. All main study animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

HISTOPATHOLOGY: Yes.
- Full List of organs investigated for main study animals of Groups 1 and 4 killed at a scheduled interval.
- Lungs including bronchi, tracheobronchial lymph node, nasal turbinates including nasopharynx, trachea including bifurcation and abnormalities investigated in all main study animals of Groups 2 and 3 killed at a scheduled interval.

For more information on organs and tissues selceted for pathology, please refer to Table 1 in section "Any other information on materials and methods incl. tables".

Statistics:

For more details on statistical methods used, please refer to section "Any other information on materials and methods incl. tables".

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical signs that could be related to the treatment with test item. Clinical signs associated with the dosing procedure included wet fur and red staining for all groups (including control) and powder/black powder on nose/muzzle/head/eyes for test groups. These signs are considered to be associated with the method of restraint/delivery (for more information, please refer to attached detailed tabular summary in section “Overall remarks, attachments”).

Mortality:

no mortality observed

Description (incidence):

There was no mortality observed throughout the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no effect on body weight gain. All differences from control were consistent with normal variation and considered incidental (for more information, please refer to attached detailed tabular summary in section “Overall remarks, attachments”).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no effect on food consumption.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no findings that could be related to treatment with the test item.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Mean neutrophil counts were higher than control for both sexes exposed to 9.94 or 31.1 μg/L in Week 13, up to 2.0X and 1.4X control, for males and females respectively.
Mean eosinophil counts were also higher than control for males exposed to 31.1 μg/L (1.8X control). Consequently mean total white blood cell counts were slightly higher than control for both sexes exposed to 9.94 or 31.1 μg/L, up to 1.2X and 1.3X control, for males and females respectively.
There was a large degree of variability in the individual data for white cell populations, however the majority of individual counts for males were above the control range. In females, individual counts were typically within the range seen for control but were consistently towards the higher end of the range. All other differences from control were consistent with normal variation and considered incidental (for more information, please refer to attached detailed tabular summary in section “Overall remarks, attachments”).

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There was no test item related effect on blood chemistry parameters. All differences from control were consistent with normal variation and considered incidental (for more information, please refer to attached detailed tabular summary in section “Overall remarks, attachments”).

Endocrine findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

effects observed, treatment-related

Description (incidence and severity):

During bronchoalveolar lavage assessment, an exposure concentration related higher cellular infiltration into the lung was observed for all groups exposed to Expanded Graphite Powder. This was predominantly related to an influx of neutrophil and mononuclear (monocytes and macrophages) cells (for more information, please refer to attached detailed tabular summary in section “Overall remarks, attachments”).

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute (up to 1.74x of control for males and 1.90x for females of the HD) and body weight adjusted (up to 1.81x of control for males and 1.86x of control for females) mean lung and bronchi weights were higher than control in all exposure groups. Differences were concentration-related and adjusted weight was statistically significantly increased for all groups exposed to the test item (for more information, please refer to attached detailed tabular summary in section “Overall remarks, attachments”).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Abnormal color of the lungs, and dark and/or enlarged tracheobronchial and mediastinal lymph nodes were noted in both sexes in all exposure groups. All other macroscopic findings were considered not test item-related (for more information, please refer to attached detailed tabular summary in section “Overall remarks, attachments”).

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related findings were noted in the lungs, tracheobronchial and mediastinal lymph nodes, trachea, tracheal bifurcation and nasal cavity.
Particle laden alveolar macrophages accompanied by bronchioloalveolar hyperplasia and increased incidence of perivascular inflammatory cell infiltrates were noted in the lungs in males and females in all treatment groups. In the tracheobronchial and mediastinal lymph nodes, particle laden macrophages and increased generalized cellularity were noted in males and females in all treatment groups.
Subepithelial particle deposits were noted in the trachea and tracheal bifurcation of occasional males and females at 9.94 or 31.1 μg/L, and in females at 3.20 μg/L.
In the nasal cavity, eosinophilic globules of the olfactory and respiratory epithelia were noted in the nasal turbinates and nasal pharynx of males and females in all treatment groups. Additionally, mucous cell hyperplasia, subepithelial inflammatory cell infiltrates of the olfactory and respiratory epithelia were noted in males and females at 9.94 or 31.1 μg/L. Squamous cell metaplasia of the olfactory epithelium in one male at 31.1 μg/L and subepithelial inflammatory cell infiltrates of the respiratory epithelium in one female at 3.20 μg/L, were also noted.
All other microscopic findings were considered not test item-related (for more information, please refer to attached detailed tabular summary in section “Overall remarks, attachments”).

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

The analysis of the bronchoalveolar lavage fluid (BALF) supernatant demonstrated an exposure concentration-related increase in mean lactate dehydrogenase values up to 6.0X and 8.7X of control, for males and females, respectively. There was a high degree of variability in the individual data however, the majority of values were above the concurrent control range (6/10, 7/10 and 4/5 concentrations above the control range for males exposed to 3.20, 9.94 or 31.1 μg/L; 5/10, 8/10 and 5/5 concentrations above the control range for females exposed to 3.20, 9.94 or 31.1 μg/L). In additon, mean total protein was up to 3.7X and 3.4X higher than control (for males and females, respectively) in BALF supernatants for all groups exposed to the test item (for more information, please refer to attached detailed tabular summary in section “Overall remarks, attachments”).

The amount of expanded graphite powder was quantified in right lung tissue. Due to the low exposure concentrations, it was only possible to quantify the test item in the group exposed to 31.1 μg/L, which yielded test item contents of 3.32 mg/g lung. Although results for the other dose groups were below the LOQ for the method, a tendency towards a concentration-related progression of residual test item in the lungs was observed (0.40 mg/g lung for the 3.20 µg/l group, 1.52 mg/g lung for the 9.94 µg/l group and 3.32 mg/g lung for the 31.1 µg/l group) (for more information, please refer to attached detailed tabular summary in section “Overall remarks, attachments”).

Key result

Dose descriptor:

LOAEC

Effect level:

3.2 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

3.2 mg/m³ air

System:

respiratory system: lower respiratory tract

Organ:

alveolar duct

Treatment related:

yes

Dose response relationship:

yes

Conclusions:

In a subchronic inhalation toxicity study according to OECD TG 413, expanded natural graphite did not elicit systemic test item-related effects at concentrations of 3.20, 9.94 or 31.10 mg/m³. Local responses were observed in respiratory tract tissues and regional lymph nodes at all concentrations with a clear exposure concentration relationship.
Based on these findings, a no observed adverse effect concentration (NOAEC) for local effects could not be established, the lowest observed adverse effect concentration (LOAEC) for local effects was 3.20 mg/m³.
The NOAEC for systemic effects is considered to be 31.10 mg/m³.

The nature of these effects correlated with effects observed with other poorly soluble particles.

Executive summary:

In a subchronic inhalation toxicity study according to OECD TG 413, expanded natural graphite (96.45% purity) was administered to 10 CRL:WI(Han) rats/sex/concentration (plus 5 male rats/concentration for lung burden analysis) by dynamic nose only exposure at concentrations of 3.20, 9.94 and 31.10 mg/m³ in air for 6 hours/day, 5 days/week for a total of 13 weeks (92 days).

There were no compound-related effects in mortality, body weight, food consumption and clinical chemistry. In general, no systemic test item-related effects were noted.

Local responses were observed in respiratory tract tissues and regional lymph nodes at all concentrations with a clear exposure concentration relationship. These consisted of microscopic findings of particle laden macrophages accompanied by bronchioloalveolar hyperplasia and increased incidence of inflammatory cell infiltrates in the lung, particle laden macrophages and increased generalized cellularity in tracheobronchial and mediastinal lymph nodes, particle deposits in the trachea and tracheal bifurcation; and mucous cell hyperplasia, eosinophilic globules and inflammatory cell infiltrates of the olfactory and respiratory epithelia, and/or squamous metaplasia of the olfactory epithelium in the nasal cavity. These effects correlated with changes in bronchoalveolar lavage fluid, circulating white cell counts, organ weights and macroscopic observations in the respiratory tract.

These effects resemble the general particle effect of biopersistent granular dusts on the lungs.

Based on the results of this study, the no observed adverse effect concentration (NOAEC) for systemic effects is considered to be at least 31.10 mg/m³, i.e. the highest dose tested. Due to the local effects in the lungs, a NOAEC for local effects could not be established; therefore, the lowest observed adverse effect concentration (LOAEC) for local effects was 3.20 mg/m³.

This subchronic inhalation toxicity study in the rat is acceptable and satisfies the guideline requirement for a subchronic inhalation study according to OECD TG 413.

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed
Study duration:: subchronic
Species:: rat
Quality of whole database:: GLP-compliant studies conducted according to the relevant OECD TG 412/413.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-03-09 to 2010-08-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed GLP study according to OECD technical guidelines

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

adopted on Sept 7, 2009

Deviations:

Qualifier:

according to guideline

Guideline:

EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate attached to full study report.

Limit test:

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Route of administration:

inhalation

Type of inhalation exposure:

nose only

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: Low dose (MMAD/GSD) = 2.31µm/2.04
Mid dose (MMAD/GSD) = 2.36µm/2.11
High doese (MMAD/GSD) = 2.44µm/2.22

Details on inhalation exposure:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Duration of treatment / exposure:

6hrs per day, 5 consecutive days per week, 4 weeks

Frequency of treatment:

6hrs per day, 5 consecutive days per week, 4 weeks

Remarks:

Doses / Concentrations:
8 mg/m3 (low), 24 mg/m3 (mid), 72 mg/m3 (high)
Basis:
nominal conc.

No. of animals per sex per dose:

28d exposure: 5 males / 5 females per dose
28d recovery in control and high dose, with additional 5 males / 5 females per dose group

Control animals:

yes

Details on study design:

Positive control:

Observations and examinations performed and frequency:

Sacrifice and pathology:

Statistics:

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

See section "Details on results"

Mortality:

mortality observed, treatment-related

Description (incidence):

See section "Details on results"

Body weight and weight changes:

no effects observed

Description (incidence and severity):

See section "Details on results"

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

See section "Details on results"

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

See section "Details on results"

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

See section "Details on results"

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See section "Details on results"

Histopathological findings: neoplastic:

no effects observed

Details on results:

Dose descriptor:

NOAEC

Effect level:

8 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Effects in the respiratory tract

Critical effects observed:

not specified

Macroscopical Findings Upon Sacrifice

- Macroscopical observations made upon sacrifice of animals are summarized below (lungs, lung-associated lymph nodes and other organs have been under inspection).

TABLE: Macroscopical Findings Upon Sacrifice on Day 29 and Day 56

Treatment	Clean Air	Expanded Graphite low	Expanded Graphite mid	Expanded Graphite high
Aerosol concentration (mg/m³)	-	8	24	72
Group #	1	2	3	4
Number of Rats(M+F each)	5 5	5* 0	5 0	5 5
Day	29 56	29 56	29 56	29 56
MALES
Within normal limits	4 5	0 -	0 -	0 0
LUNGS
Multiple grey discoloration	0 0	4 -	5 -	5 2
Multiple grey-black discoloration	0 0	0 -	0 -	5 3
LALN
slightly enlarged	0 0	0 -	1 -	3 3
Grey discolored	0 0	0 -	5 -	5 0
Black discolored	0 0	0 -	0 -	0 5
Testes and Epididymides
Left: Slightly decreased in size	1 0	0 -	0 -	0 0
Right testis: Slightly enlarged	- -	1 -	0 -	0 0
Left testis: Slightly decreased	- -	- -	1 -	0 0
FEMALES
Within normal limits	5 4	0 -	0 -	0 0
LUNGS
Multiple grey discoloration	0 0	5 -	4 -	5 1
Multiple grey-black discoloration	0 0	0 0	0 -	0 4
LALN
slightly enlarged	0 0	- -	0 -	0 3
Grey discolored	0 0	0 -	5 -	0 0
Black discolored	0 0	0 -	0 -	2 5
Uterus Horns
Slightly cystic enlargements	0 1	1 -	3 -	3 1
Ovaries
Slightly decreased	0 0	0 -	0 -	2 0