Graphite

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a combined repeated dose toxicity study with the reproductive/developmental toxicity screening test (OECD TG 422), graphite was tested up to the limit dose given in OECD 422 (nominal 1000mg/kg bw/day) via oral administration via food (incl. analytical verification).

There were no signs of systemic toxicity and no signs of any effects on development, reproduction, or fertility observed. Since no hazard was identified, the NOAEL based on nominal food intake is therefore at least 1000 mg/kg bw/day.

Additional sub-acute and sub-chronic repeated dose toxicity studies via the inhalation route (for more information, please refer to IUCLID section 7.5.2) and acute toxicity studies via the oral and inhalation route (for more information, please refer to IUCLID section 7.2.1 and 7.2.2) likewise did not indicate any systemic adverse effects.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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:

other: diet

Details on exposure:

DIET PREPARATION
- 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, the investigation of the stability is not regarded necessary

Details on mating procedure:

- M/F ratio per cage: Overnight mating will performed 1 male : 1 female of the same dose group. Vaginal smears will be taken the next morning.
- Length of cohabitation: Animals will be mated beginning after two weeks of treatment for two consecutive weeks or until successful mating.
- Proof of pregnancy: 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.
- Any other deviations from standard protocol: 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)

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:

continuous via diet

Dose / conc.:

91 mg/kg bw/day (actual dose received)

Remarks:

males LD (based on 1300 mg/kg food)

Dose / conc.:

261 mg/kg bw/day (actual dose received)

Remarks:

males MD (based on 3700 mg/kg food)

Dose / conc.:

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

Remarks:

males HD (based on 11500 mg/kg food)

Dose / conc.:

120 mg/kg bw/day (actual dose received)

Remarks:

females LD during premating (based on 1300 mg/kg food)

Dose / conc.:

343 mg/kg bw/day (actual dose received)

Remarks:

females MD during premating (based on 3700 mg/kg food)

Dose / conc.:

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

Remarks:

females HD during premating (based on 11500 mg/kg food)

Dose / conc.:

106 mg/kg bw/day (actual dose received)

Remarks:

females LD during gestation (based on 1300 mg/kg food)

Dose / conc.:

309 mg/kg bw/day (actual dose received)

Remarks:

females MD during gestation (based on 3700 mg/kg food)

Dose / conc.:

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

Remarks:

females HD during gestation (based on 11500 mg/kg food)

Dose / conc.:

111 mg/kg bw/day (actual dose received)

Remarks:

females LD during lactation (based on 1300 mg/kg food)

Dose / conc.:

370 mg/kg bw/day (actual dose received)

Remarks:

females MD during lactation (based on 3700 mg/kg food)

Dose / conc.:

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

Remarks:

females HD during lactation (based on 11500 mg/kg food)

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), where food containing 10g/kg food (estimated target dose of 1000mg/kg/day) was accepted without 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 (if not random): Randomisation to groups based on body weight using PROVANTIS 8.2.0.8
- Fasting period before blood sampling for clinical biochemistry: no

Positive control:

No positive control included in this study.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 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.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once per week, all animals will be inspected outside their home cages. The results of these examinations will be documented.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weight of all animals will be recorded weekly to the nearest 0.1 g until sacrifice of the animals. After successful 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 AND COMPOUND INTAKE (if feeding study): Yes
- 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, 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. The actual concentration in food was determined based on actual body weights and food consumption data.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:

OTHER:

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.

Sperm parameters (parental animals):

In testes, staging of the seminal epithelium was performed.

Litter observations:

- Litters were inspected at least once daily. Cages in which dams or litters exhibited unusual behaviour or appearance were tagged for inspection by the veterinarian or his designate. 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.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: Males were sacrificed after successful mating, but not before day 28 of treatment.
- Maternal animals: Females were sacrificed as soon as possible after day 4 p.p.
All animals were sacrificed by CO2 overdose and subsequent exsanguination. The treatment of all animals was continued until their sacrifice.

GROSS NECROPSY
- Necropsy was be performed in all F0 males and females.
- The number of corpora lutea as well as implantation sites were 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” were collected from each rat and fixed in 10% neutral buffered formalin (unless specified otherwise)

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

Postmortem examinations (offspring):

SACRIFICE
- All pups were humanely sacrificed as soon as possible after day 4 p.p. and carefully examined for gross abnormalities.

Statistics:

- Statistical comparison of groups were performed separately for each sex at the level of alpha=0.05.
- Body weights as well as food consumption data were analyzed using analysis of variance.
- If the group means differed significantly with this method, the means of the treatment groups were 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 were applied in the case of non-homogeneous data.
- Qualitative data were analyzed using the two-tailed FISHER test with Bonferroni correction or Chi-square test.
- If appropriate, other statistical tests were applied.

Clinical signs:

no effects observed

Description (incidence and severity):

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

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

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 and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No effects of test item exposure were observed on food consumption of the animals at any time point of the study.

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):

Haematological data were found to be 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. 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.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Clinical chemistry data were found to be 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. 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.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

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.

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

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

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

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

Other effects:

not specified

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

There was no evidence of degenerating spermatids or spermatocytes.
All findings and lesions were considered spontaneous and incidental.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

The results show that mating outcome throughout all groups including control groups in both mating subsets was insufficient. The reasons for this phenomenon are unclear. However, no effect of the test item exposure was observed on any of the investigated endpoints like precoital time or fertility (number of mated females, number of pregnant females, number of implantation sites, number of liveborn pups). No effect of the test item exposure could be observed on litter size.

- Reproductive function: sperm measures:
The randomly scanned seminiferous tubules from 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) spermatocytes and 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 multifocal 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 one male 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.

- Reproductive performance:
For investigation of mating data, each group consisted of 20 pairs for mating. For the four groups, mating yielded 19 (control), 19 (LD), 20 (MD), and 18 (HD) 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 control groups in both mating subsets was insufficient. The reasons for this phenomenon are unclear. However, no effect of the test item exposure was observed on any of the investigated endpoints like precoital time or fertility (number of mated females, number of pregnant females, number of implantation sites, number of liveborn pups).

Key result

Dose descriptor:

NOAEL

Effect level:

> 813 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No adverse effects at highest dose tested.

Key result

Dose descriptor:

NOAEL

Remarks:

females in premating period

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effects at highest dose tested.

Key result

Dose descriptor:

NOAEL

Remarks:

females during gestation

Effect level:

> 930 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effects at highest dose tested.

Key result

Dose descriptor:

NOAEL

Remarks:

females during lactation

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effects at highest dose tested.

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

None of the sporadically observed clinical findings in pups were considered test item related. Pup clinical observations included sporadical bad condition, cold to touch and one finding of a damaged tail.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

No effect of the test item exposure could be observed on litter size and pup survival. One dam from the mid dose group (#3113) gave birth to one live pup which was missing on day 1 p.p.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No effect of the test item exposure could be observed on pup body weight.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not specified

Nipple retention in male pups:

not specified

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No abnormal pups were observed.

Histopathological findings:

not specified

Other effects:

not specified

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

exposure of dams during gestation

Generation:

F1

Effect level:

> 930 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related adverse effects observed in offspring.

Key result

Dose descriptor:

NOAEL

Remarks:

exposure of dams during lactation

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related adverse effects observed in offspring.

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Table: Summary of pregnancy data

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

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: 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 from this combined repeated dose toxicity study with the reproduction/developmental toxicity screening test, the NOAEL for this study was determined as the high (limit) dose level of 11500 mg/kg food. This corresponds to the following actual substance intakes: 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:

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422), Expanded Graphite Powder was administered to 10 Wistar rats (Crl:WU)/sex/dose in diet at dose levels of 1300, 3700 and 11500 ppm food, corresponding to actual substance intake levels of 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.

The animals were treated for 2 weeks before mating and during the mating period. Successful mating was confirmed by detection of sperm in vaginal smears and/or a vaginal plug (day 0 post conceptionem) and treatment was continued through postnatal day 4. Males were treated for at least 28 days.

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. For the four dose groups, mating yielded 19 (control), 19 (LD), 20 (MD), and 18 (HD) 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.

There were no compound-related adverse clinical signs, effects on body weight, weight gain, food consumption or functional observations in any dose group. In addition, haematological and clinical chemistry data were within expected ranges for the rat species, strain and age. Occasionally, statistically significant differences in organ weights were observed, which were not considered test item-related.

The main necropsy findings consisted of reduced size of testes and epidydimides, which were found in all groups including the control group.

There were no treatment-related effects observed for reproductive parameters, including precoital time or fertility (number of mated females, number of pregnant females, number of implantation sites, number of liveborn pups).

In addition, no effect could be observed on litter size, pup survival and pup body weight.

Based on the results of this study, the NOAEL is considered to be >11500 ppm food, corresponding to actual substance intake levels of 813 mg/kg bw/day (males), 1067 mg/kg bw/day (females in premating period), 930 mg/kg bw/day (females during gestation) and 1159 mg/kg bw/day (females during lactation), i.e. the highest dose tested.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Two qualities of Graphite were considered for the present registration dossier: Synthetic Graphite (SG) and Expanded Graphite (EG). Both are high purity graphite qualities with SG having the higher purity (>99%) and no amounts of quartz or any other crystalline impurities. EG however may contain certain (but low) amounts of quartz (typical quantity: 0.9%) and muscovite (typical quantity: 1.2%), hence making EG a worst case scenario for some endpoints (e.g. inhalation and irritation). For this reason, EG was chosen as test item for the combined repeated dose toxicity study with the reproductive/developmental toxicity screening test (OECD TG 422) as well. Since neither by inhalation nor oral administration any sign of systemic toxicity was found in all other studies and the fact that SG does not contain any impurities known to be problematic for developmental/reproductive toxicity, results obtained from testing EG in the combined repeated dose toxicity study with the reproduction/developmental toxicity screening test are valid for SG, too.

Since the present reproductive toxicity study was conducted as a combined repeated dose toxicity study with the reproductive/developmental toxicity screening test, several NOAELs were obtained, all representing the nominal dose of 1000 mg/kg bw/day, as there were no systemic treatment-related adverse effects and no effects for reproductive parameters, including precoital time or fertility (number of mated females, number of pregnant females, number of implantation sites, number of liveborn pups) observed. In addition, no effect could be observed on litter size, pup survival and pup body weight (for more information, please refer to IUCLID section 7.8.1).

However, the actual substance intake varied from about 813 mg/kg bw/day up to 1159 mg/kg bw/day.

According to ECHA’s decision on a compliance check (CCH-D-2114527073-58-01/F) published on the public activities coordination tool, "The information requirement under Section 8.7.3. of Annex X to REACH (Extended one-generation reproductive toxicity study, EOGRTS) is not addressed in this decision, because the information from the Sub-chronic toxicity study (90-day), requested in the present decision, is relevant for the design of the EOGRTS."

Graphite is an inorganic substance that consists of crystalline carbon. It is insoluble in water (IUCLID section 4.8) and can be considered to be largely inert.
In a specific reproductive/developmental toxicity study combined with a repeated dose toxicity in rats conducted via the oral route (according to the relevant OECD TG 422), graphite did not show any systemic treatment-related effects up to the recommended maximum dose level of 1000 mg/kg bw/day. In particular, no effects on reproductive parameters, including precoital time or fertility (number of mated females, number of pregnant females, number of implantation sites, number of liveborn pups) were noted. Furthermore, no effect could be observed on the development of offspring, as determined by litter size, pup survival and pup body weight (for more information, please refer to IUCLID section 7.8.1). Based on this study, graphite is not expected to be a reproductive or developmental toxicant.
The observed lack of any systemic effects in this study is substantiated by a wealth of additional in vivo toxicity data for the substance. The results of the abovementioned sub-chronic (90-day) repeated dose toxicity study performed with graphite via the inhalation route did not show test item-related systemic effects in rats. Adverse outcomes were limited to local effects resembling general effects of poorly soluble particles with low toxicity (for more information, please refer to IUCLID section 7.5.2).
Additional sub-acute repeated dose toxicity studies via the inhalation route (for more information, please refer to IUCLID section 7.5.2) and acute toxicity studies via the oral and inhalation route (for more information, please refer to IUCLID section 7.2.1 and 7.2.2) likewise did not indicate any systemic adverse effects.

Overall, based on the physicochemical properties of graphite, such as insolubility in water and the lack of systemic effects in all available in vivo studies, the current data show no indication of systemic availability and no systemic effects that could lead to an impairment of fertility and reproductive performance in vivo. Most importantly, graphite did not exert any effects on reproductive or developmental parameters when tested in vivo in a reproductive/developmental test according to OECD TG 422 in rats.
Consequently, the substance is not considered to be a reproductive toxicant and the conduct of an extended one-generation reproductive toxicity study according to Annex X, section 8.7.3, is not considered to provide additional knowledge on the reproductive toxicity of graphite.
Therefore, further experimental testing is not considered scientifically justified.

 

Effects on developmental toxicity

Description of key information

In a combined repeated dose toxicity study with the reproductive/developmental toxicity screening test (OECD TG 422), graphite was tested up to the limit dose given in OECD 422 (nominal 1000mg/kg bw/day) via oral administration via food (incl. analytical verification).

There were no signs of systemic toxicity and no signs of any effects on development, reproduction, or fertility observed. Since no hazard was identified, the NOAEL based on nominal food intake is therefore at least 1000 mg/kg bw/day.

Additional sub-acute and sub-chronic repeated dose toxicity studies via the inhalation route (for more information, please refer to IUCLID section 7.5.2) and acute toxicity studies via the oral and inhalation route (for more information, please refer to IUCLID section 7.2.1 and 7.2.2) likewise did not indicate any systemic adverse effects.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Two qualities of Graphite were considered for the present registration dossier: Synthetic Graphite (SG) and Expanded Graphite (EG). Both are high purity graphite qualities with SG having the higher purity (>99%) and no amounts of quartz or any other crystalline impurities. EG however may contain certain (but low) amounts of quartz (typical quantity: 0.9%) and muscovite (typical quantity: 1.2%), hence making EG a worst case scenario for some endpoints (e.g. inhalation and irritation). For this reason, EG was chosen as test item for the combined repeated dose toxicity study with the reproductive/developmental toxicity screening test (OECD TG 422) as well. Since neither by inhalation nor oral administration any sign of systemic toxicity was found in all other studies and the fact that SG does not contain any impurities known to be problematic for developmental/reproductive toxicity, results obtained from testing EG in the combined repeated dose toxicity study with the reproduction/developmental toxicity screening test are valid for SG, too.

Since the present reproductive toxicity study was conducted as a combined repeated dose toxicity study with the reproductive/developmental toxicity screening test, several NOAELs were obtained, all representing the nominal dose of 1000 mg/kg bw/day, as there were no systemic treatment-related adverse effects and no effects for reproductive parameters, including precoital time or fertility (number of mated females, number of pregnant females, number of implantation sites, number of liveborn pups) observed. In addition, no effect could be observed on litter size, pup survival and pup body weight (for more information, please refer to IUCLID section 7.8.1).

However, the actual substance intake varied from about 813 mg/kg bw/day up to 1159 mg/kg bw/day.

Graphite is an inorganic substance that consists of crystalline carbon. It is insoluble in water (IUCLID section 4.8) and can be considered to be largely inert.
In a specific reproductive/developmental toxicity study combined with a repeated dose toxicity in rats conducted via the oral route (according to the relevant OECD TG 422), graphite did not show any systemic treatment-related effects up to the recommended maximum dose level of 1000 mg/kg bw/day. In particular, no effects on reproductive parameters, including precoital time or fertility (number of mated females, number of pregnant females, number of implantation sites, number of liveborn pups) were noted. Furthermore, no effect could be observed on the development of offspring, as determined by litter size, pup survival and pup body weight (for more information, please refer to IUCLID section 7.8.1). Based on this study, graphite is not expected to be a reproductive or developmental toxicant.
The observed lack of any systemic effects in this study is substantiated by a wealth of additional in vivo toxicity data for the substance. The results of the abovementioned sub-chronic (90-day) repeated dose toxicity study performed with graphite via the inhalation route did not show test item-related systemic effects in rats. Adverse outcomes were limited to local effects resembling general effects of poorly soluble particles with low toxicity (for more information, please refer to IUCLID section 7.5.2).
Additional sub-acute repeated dose toxicity studies via the inhalation route (for more information, please refer to IUCLID section 7.5.2) and acute toxicity studies via the oral and inhalation route (for more information, please refer to IUCLID section 7.2.1 and 7.2.2) likewise did not indicate any systemic adverse effects.

Overall, based on the physicochemical properties of graphite, such as insolubility in water and the lack of systemic effects in all available in vivo studies, the current data show no indication of systemic availability and no systemic effects that could lead to an impairment of offspring development in vivo. Most importantly, graphite did not exert any effects on reproductive or developmental parameters when tested in vivo in a reproductive/developmental test according to OECD TG 422 in rats.
Consequently, the substance is not considered to be a developmental toxicant and the conduct of a pre-natal developmental toxicity study (OECD TG 414) in a first species according to Annex IX, section 8.7.2 and a second species according to Annex X, section 8.7.2, is not considered to provide additional knowledge on the developmental toxicity of graphite.
Therefore, further experimental testing is not considered scientifically justified.

Toxicity to reproduction: other studies

Additional information

See discussion for effects on fertility and developmental toxicity.

Justification for classification or non-classification

Based on the results of the present study and according to criteria laid down in Regulation (EC) No 1272/2008, Graphite is not regarded neither as being toxic to reproduction nor as being toxic to development or fertility.

Therefore, classification for toxicity to reproduction is not warranted.