Non-graphitic carbon fibre

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Based on the results from an OECD 422 guideline study, the NOAEL (parental, reproductive and developmental) was determined as the high (limit) target dose level of 12.000 mg/kg food (equals 1000 mg/kg bw/day (nominal). This corresponds to the following actual substance intake: 859 mg/kg body weight/day for males; 1051 mg/kg body weight/day for females in the premating period; 994 mg/kg body weight/day for females during gestation; and 1521 mg/kg body weight/day for females during lactation, respectively.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-08-10 to 2013-01-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same 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:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Germany
- Age at study initiation: 8 weeks at delivery
- Housing: individually in polycarbonate (Makrolon) cages type III in the conventional area of the animal house, except during mating or dams with their litters
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 2 °C
- Humidity: 55 +/- 15 %
- Air changes: 15 per hour
- Photoperiod: 12 hrs dark/ 12 hrs light

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS

DIET PREPARATION
- Rate of preparation of diet (frequency): food mixtures are prepared and delivered once by ssniff Spezialdiaeten GmbH, Soest, Germany
- Mixing appropriate amounts with (type of food): V1324 (closed formula) from ssniff Spezialdiaeten GmbH, Soest, Germany. The concentration is documented by ssniff Spezialdiaeten GmbH under the supervision of the contract research organisation's quality assurance unit.

Details on mating procedure:

- animals are first mated after two weeks of treatment for two consecutive weeks or until successful mating
- overnight mating is performed with 1 male : 1 female of the same dose group
- vaginal smears are taken the next morning
- mating is considered successful if sperm and/or a vaginal plug is found

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- food mixtures are prepared and delivered once by ssniff Spezialdiaeten GmbH, Soest, Germany
- concentration is documented by ssniff under supervision of the contract research organisation's quality assurance unit
- from each concentration 3 samples are taken from 3 different sites of the 1st container after delivery for checking the homogeneity
- these samples are investigated by repeated determination
- based on the chemical properties of the test item an investigation of its stability is not regarded necessary
- homogeneity of the test item in food is analysed by base-digestion (4M NaOH) followed by turbidity measurement in aqueous media
- the content of test item is determined photometrically at a wavelength of 860 nm using a calibration curve
- the determination is performed as a non-GLP analysis
- the method is validated to allow an evaluation of the precision and accuracy of the results
- the relative standard error of the carbon fibre homogeneity determination is confirmed to be below ± 20% within the operative concentrations
- consequently, acceptance criteria for homogeneity are set at ± 20 % of the target value

Duration of treatment / exposure:

- animals are 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 is continued until day 4 post partum (p.p.) and subsequent sacrifice
- males are sacrificed after successful mating, but not before day 28 of treatment; females are sacrificed as soon as possible after day 4 p.p.
- exposure of all males and females not successfully mated is continued until their sacrifice

Frequency of treatment:

see "duration of treatment/exposure"

Details on study schedule:

see "duration of treatment/exposure"

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

= 1200 mg/kg food

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

= 3600 mg/kg food

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

= 12000 mg/kg food

No. of animals per sex per dose:

10 males and 10 females per dose and in the control group

Control animals:

yes, plain diet

Details on study design:

- dose selection rationale: Dose levels are based on a palatability study (CRO's study number: 12N12507) in which the animals accepted a target dose of 1000 mg/kg bw/day (12 g/kg food) without any problems. This dose is also recommended as limit dose in the OECD Guideline 422. Consequently, this dose was chosen as the high dose in the present study. The other doses were determined using a stagger of approx. 3.
- rationale for animal assignment (if not random): not applicable, randomised assignment
- rationale for selecting satellite groups: no satellite groups
- post-exposure recovery period in satellite groups: not applicable

Positive control:

no positive control included

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: yes
- Time schedule: clinical observations at least once daily

DETAILED CLINICAL OBSERVATIONS: yes
- Time schedule: once a week all animals are inspected outside their home cages and carefully examined for clinical symptoms

BODY WEIGHT: yes
- Time schedule for examinations: weekly to the nearest 0.1 g until sacrifice of the animals
- After successful mating, body weight of the females is determined on days 0, 7, 14, and 20 p.c., as well as on days 0 and 4 p.p.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: yes
- Actual test item intake of the animals will be calculated based on food consumption: yes

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

HAEMATOLOGY: yes
- Time schedule for collection of blood: towards the end of week two of treatment (shortly before the start of the mating period in females and shortly before sacrifice in males)
- Anaesthetic used for blood collection: yes (light isofluran anesthesia)
- Animals fasted: no
- How many animals: 5 males and 5 females
- parameters in table 2 were examined

- samples are be analysed in a randomised sequence
- samples may be deep frozen (-18°C) for later analysis

CLINICAL CHEMISTRY: yes
- Time schedule for collection of blood: towards the end of week two of treatment (shortly before the start of the mating period)
- Animals fasted: no
- How many animals: 5 males and 5 females
- parameters in table 3 were examined

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: shortly before sacrifice in males and towards the end of week two of treatment (shortly before the start of the mating period) in females
- Battery of functions tested: see tables 4 and 5

Litter observations:

Intravital investigations: clinical observations
- litters are inspected at least once per day
- cages in which dams or litters exhibit unusual behavior or appearance are tagged for inspection by the veterinarian or his designate

Intravital investigations: survival and pup weight
- number of offspring and the occurrence of any dead pups or abnormalities are recorded for all groups
- individual weight of the pups is recorded to the nearest 0.1 g on days 0 and 4 p.p.

Postmortem examinations (parental animals):

see tables 6 for "gross pathology", table 7 for "organ weights", and table 8 for "histopathology"

Postmortem examinations (offspring):

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

Statistics:

- statistical comparison of groups is performed for each sex at the level of α < 0.05
- body weights, food consumption, clinico-chemical, hematological and activity data are analysed using analysis of variance
- if the group means differ significantly with this method the means of the treatment groups are 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 are applied in case of non-homogeneous data
- qualitative reproduction data are analysed using the two tailed FISHER test with Bonferroni correction or Chi-square test
- histopathology data are analysed using the Chi-square and the FISHER test, both two tailed
- in all instances, the dam or litter are used as the basic unit

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

but only in female rats, coincident with increased food consumption and only in high and medium dose group on several days, considered to be of no toxicological relevance

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

effects were observed in female rats only, coincident with increased food consumption and only in high and medium dose group on several days; these findings are considered to be of no toxicological relevance

Food efficiency:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

See attached background material "Tables.pdf" for further information.

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): The summary of the weekly F0 premating clinical observations is provided in Table 3, the summary of clinical observations during pregnancy and lactation is given in Tables 4 and 5.
The only clinical sign observed was red eye discharge in animal no. 1107 (control) beginning on day 22 and persisting until final sacrifice.
No other clinical signs were observed during the study, consequently there was no indication of any clinical observation related to the test item.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): The summary of the weekly F0 premating body weights and body weight gains is provided in Tables 6 and 7. The summary of the maternal gestation and postpartum body weights and body weight gains is given in Tables 8, 9, 10 and 11.
No effects of test item exposure were observed on body weight or body weight gain of the male animals at any time point of the study.
In females, statistically significantly increased body weight was observed for the high and medium dose (groups 3 and 4) in the premating period starting with day 7 which was also reflected by increased body weight gains between day 0 and 7 in all substance exposed females (groups 2-4).
During pregnancy, statistically significantly increased body weight was observed in the high dose group 4 on days 0, 7, and 14 p.c., and in the medium dose group 3 on day 14 only. During lactation, this finding was noticed on day 0 and 4 p.p. in medium dose animals (group 3) only. There was, however, no effect on body weight gain during gestation or lactation in any of the groups exposed to the test item.
Taking into account that the observed effects on body weight represent a (temporary) increase and they coincide with increased food consumption in these groups, none of them can be considered adverse.
Consequently, no adverse effects of test item exposure could be observed on body weight during the study.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): The summary of the weekly F0 premating food consumption data is provided in Table 12. The summary of the maternal gestation and postpartum food consumption data is given in Tables 13 and 14.
No effects of test item exposure were observed on food consumption of the male animals at any time point of the study, with the exception of a decreased food consumption from day 14–21 in the low dose group 1, which is considered incidental.
In females, statistically significantly increased food consumption was observed for the high and medium dose (groups 3 and 4) in the premating period from day 7 to 14 as well during the whole premating period.
During pregnancy, statistically significantly increased food consumption was observed in the high and medium dose group (groups 3 and 4) between days 0 and 7 p.c. and during days 7 to 14 p.c. as well as for the whole period of pregnancy (days 0-20 p.c.).
No statistically significant effects on food consumption were observed during lactation.
The observed (temporary) increase in food consumption in medium and high dose animals (groups 3 and 4) are in good concordance with the effects observed on body weight. These effects were not considered adverse.
Consequently, no adverse effects of exposure to the test item could be observed on food consumption during the study.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS): The mean substance intake calculations are summarised in Table 2.
Based on mean body weights and food consumption data this resulted in the following actual substance intake: 85, 256, and 859 mg/kg bw/day for males; 106, 317, and 1051 mg/kg bw/day for females in the premating period; 94, 292, and 994 mg/kg bw/day for females during gestation; and 153, 438, and 1521 mg/kg bw/day for females during lactation, respectively.

REPRODUCTIVE FUNCTION: The summary of the F0 mating data is provided in Table 19.
For the four groups, mating yielded 10, 10, 8, and 10 sperm positive females in groups 1-4, respectively. This resulted in 10, 9, 9, and 10 pregnancies; 10, 9, 9, and 10 females with liveborn offspring in F0 dams, and 10, 9, 9, and 10 females with live litters on day 4 p.p., respectively (Table 20).
Animal no. 3202 gave birth, although no mating date was determined, while animal 2201 was not pregnant although it was found sperm positive.
Precoital time was statistically significantly increased in high dose pairs (group 4) but since all matings were observed within the first 4 days of mating (representing a normal estrus cycle) this finding is considered incidental and without toxicological relevance.
Consequently, the results show that test item exposure did not produce any adverse effects 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).

ORGAN WEIGHTS (PARENTAL ANIMALS): A summary of the F0 organ weight data is provided in Table 25.
No statistically significant differences from the control group were observed in this study.

GROSS PATHOLOGY (PARENTAL ANIMALS): A summary of the F0 necropsy observations is provided in Table 24.
Findings included sporadical enlargement of the adrenal glands, discoloration and nodules of the epididymis, cysts and reduction in kidney size and focal discoloration of the thymus.
None of the macroscopic findings were considered to be induced by the test item.

HISTOPATHOLOGY (PARENTAL ANIMALS): There were no treatment-related microscopic findings. All of the histopathological findings encountered were considered to have arisen spontaneously or post mortem.
Occasional stage IX-XIV tubules in both controls and treated animals contained a single retained spermatid. No treatment-related abnormalities were noted during examination of the testes.

Dose descriptor:

NOAEL

Effect level:

>= 859 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: clinical signs, mortality, body weight, food consumption, food efficiency, haematology, clinical chemistry, gross pathology, organ weights, histopathology

Remarks on result:

other: no adverse effects observed

Dose descriptor:

NOAEL

Effect level:

>= 1 051 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: females in the premating period: clinical signs, mortality, body weight, food consumption, food efficiency, haematology, clinical chemistry, gross pathology, organ weights, histopathology

Remarks on result:

other: no adverse effects observed

Dose descriptor:

NOAEL

Effect level:

>= 994 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: females during gestation: clinical signs, mortality, body weight, food consumption, food efficiency, haematology, clinical chemistry, gross pathology, organ weights, histopathology

Remarks on result:

other: no adverse effects observed

Dose descriptor:

NOAEL

Effect level:

>= 1 521 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: females during lactation: clinical signs, mortality, body weight, food consumption, food efficiency, haematology, clinical chemistry, gross pathology, organ weights, histopathology

Remarks on result:

other: no adverse effects observed

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

See attached background material "Tables.pdf" for further information.
VIABILITY (OFFSPRING): a summary of the delivery and litter data is given in Tables 20-23
BODY WEIGHT (OFFSPRING); CLINICAL SIGNS (OFFSPRING): No effect of exposure to the test item could be observed on litter size and pup survival as well as on pup body weight. Pup clinical observations included sporadical subcutaneous hemorrhage, bad condition, and pale pups. None of these findings were considered to be related to the test item, and no abnormal pups were observed.

SEXUAL MATURATION (OFFSPRING): not examined

ORGAN WEIGHTS (OFFSPRING): not examined

GROSS PATHOLOGY (OFFSPRING): not examined

HISTOPATHOLOGY (OFFSPRING): not examined

Key result

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

Because no deviations from the test protocol are reported and the study has been performed according to the principles of Good Laboratory Practice the study is regarded as valid.
Based on the study results the NOAEL value (parental, reproductive and developmental) was determined as the high (limit) target dose level of 12000 mg/kg food. This corresponds to the following actual substance intake: 859 mg/kg bw/day for males; 1051 mg/kg bw/day for females in the premating period; 994 mg/kg bw/day for females during gestation; 1521 mg/kg bw/day for females during lactation.

Executive summary:

The aim of this study was to evaluate possible adverse effects of the test item (milled carbonised PAN based fibre) 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 according to the OECD Guideline 422 "Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test" (March 1996) and in compliance with the Principles of Good Laboratory Practice (German Chemicals Law, § 19a, Appendix 1, 02 July 2008), with the exception of the determination of the test item in food which was performed as a non-GLP analysis, as well as with the German Animal Protection Law (Tierschutzgesetz of 18 May 2006). The dose levels were based on a palatability study in which the animals accepted food containing 12 g test item/kg food (estimated target dose of 1000 mg/kg/day) without any problems. The dose of 1000 mg/kg/day is also recommended as limit dose in the OECD Guideline 422. Consequently, this dose was chosen as the high dose in the present study. The other doses were determined using a stagger of approx. 3. The concentration in food was documented by the quality assurance unit. The analyses showed that the test item was homogenous in the preparations.

Based on mean body weights and food consumption data this resulted in the following actual substance intake: 85, 256, and 859 mg/kg bw/day for males; 106, 317, and 1051 mg/kg bw/day for females in the premating period; 94, 292, and 994 mg/kg bw/day for females during gestation; and 153, 438, and 1521 mg/kg bw/day for females during lactation. Wistar rats [Crl:Wistar(Han)], purchased from Charles River Deutschland, Sulzfeld, Germany, 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 groups exposed to the test item. The animals were housed individually in polycarbonate cages type III. Cages and absorbing softwood bedding material (Lignocel BK 8-15) was changed at least twice a week. Tap water from the Hannover city water supplier was offered fresh at least once weekly. In the control group, a closed formula in pellet form identified as V1324 was taken as the diet for this study. For the treatment groups, the test item was added to the same diet 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. The animals were inspected at least once daily and the individual body weight and food consumption of all animals were recorded weekly until sacrifice of the animals.

After successful mating, body weight and food consumption of the females were determined on days 0, 7, 14, and 20 p.c., as well as on days 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, the following parameters were investigated: hematocrit, hemoglobin, mean erythrocyte volume, mean erythrocyte hemoglobin mass, mean erythrocyte hemoglobin concentration, erythrocyte count, total and differential leucocyte count, platelet count, prothrombin time, sodium, potassium, glucose, total cholesterol, urea, creatinine, total protein and albumin, alanine aminotransferase, aspartate aminotransferase, and bile acid. Due to technical problems of the prothrombin time (PT) determination observed in samples collected in life, an additional blood sample was collected from the vena cava caudalis during terminal sacrifice for the determination of the PT.

Spontaneous locomotor activity over 60 minutes using the „Actimot“ computerised light-beam system 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. The data were analysed in 15 minutes intervals. In addition, the total values for distance, time in rest, time in movement, rearing time, and number of rearings were determined.

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. In addition to the determination of forelimb grip strength, the FOB included 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.

The animals were mated after two weeks of treatment for two consecutive weeks or until successful mating. Overnight mating was performed with 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. The time to successful insemination (precoital time) was determined.

Litters were inspected at least once daily. The number of offspring and the occurrence of any dead pups or abnormalities were 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.

The treatment of all animals was continued until their sacrifice. 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 CO2 overdose and subsequent exsanguination.

Necropsy was 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.

Weights of the following organs were determined: liver, kidneys, adrenals, thymus, spleen, brain, heart, testes, and epididymides.

In 5 males and 5 females of the high dose group 4 and the control group 1 a complete histopathological investigation according to OECD Guideline 422 was performed. In testes, staging of the seminal epithelium was performed. The following results were obtained (see also Summary Table 26):

None of the sporadically observed clinical findings were regarded as related to the test item. No adverse effects after exposure to the test item were observed on body weight, body weight gain or food consumption of the animals at any time point of the study. Overall, hematological and 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.

No toxicologically relevant effects of the exposure to the test item were observed on locomotor activity, neither for any of the investigated time intervals nor for the whole test period. No effects of exposure to the test item were observed on any of the investigated endpoints of the Functional Observational Battery.

For the investigation of mating data, each group consisted of 10 pairs for mating. For the four groups, mating yielded 10, 10, 8, and 10 sperm positive females in groups 1-4, respectively. This resulted in 10, 9, 9, and 10 pregnancies, 10, 9, 9, and 10 females with liveborn offspring in F0 dams, and 10, 9, 9, and 10 females with live litters on day 4 p.p., respectively.

No effect of exposure to the test item 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 exposure to the test item was observed on litter size and pup survival as well as on pup body weight. None of the sporadically observed clinical findings in pups were considered to be related to the test item and no abnormal pups were observed.

None of the sporadically observed necropsy findings were considered to be related to the test item and statistically significant differences in organ weights were not found. Substance-related findings were not observed in the histologically examined organs of males and females of the control and high dose group. Occasional stage IX-XIV tubules in both controls and treated animals contained a single retained spermatid. No treatment-related abnormalities were noted during examination of the testes.

Based on the results described above, the NOAEL for this study (parental, reproductive and developmental) was determined as the high (limit) target dose level of 12.000 mg/kg food. This corresponds to the following actual substance intake: 859 mg/kg body weight/day for males; 1051 mg/kg body weight/day for females in the premating period; 994 mg/kg body weight/day for females during gestation; and 1521 mg/kg body weight/day for females during lactation, respectively.

Reference 2

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because (i) the substance is of low toxicological activity (no evidence of toxicity seen in any of the tests available), (ii) it can be proven from toxicokinetic data that no systemic absorption occurs via relevant routes of exposure (e.g. plasma/blood concentrations below detection limit using a sensitive method and absence of the substance and of metabolites of the substance in urine, bile or exhaled air) and (iii) there is no or no significant human exposure

Reproductive effects observed:

not specified

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

Based on the results from an OECD 422 guideline study, the NOAEL (parental, reproductive and developmental) was determined as the high (limit) target dose level of 12.000 mg/kg food (equals 1000 mg/kg bw/day (nominal). This corresponds to the following actual substance intake: 859 mg/kg body weight/day for males; 1051 mg/kg body weight/day for females in the premating period; 994 mg/kg body weight/day for females during gestation; and 1521 mg/kg body weight/day for females during lactation, respectively.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-08-10 to 2013-01-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: 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:

yes

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Germany
- Age at study initiation: 8 weeks at delivery
- Housing: individually in polycarbonate (Makrolon) cages type III in the conventional area of the animal house, except during mating or dams with their litters
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 2 °C
- Humidity: 55 +/- 15 %
- Air changes: 15 per hour
- Photoperiod: 12 hrs dark/ 12 hrs light

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS

DIET PREPARATION
- Rate of preparation of diet (frequency): food mixtures are prepared and delivered once by ssniff Spezialdiaeten GmbH, Soest, Germany
- Mixing appropriate amounts with (type of food): V1324 (closed formula) from ssniff Spezialdiaeten GmbH, Soest, Germany. The concentration is documented by ssniff Spezialdiaeten GmbH under the supervision of the contract research organisation's quality assurance unit.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- food mixtures are prepared and delivered once by ssniff Spezialdiaeten GmbH, Soest, Germany
- concentration is documented by ssniff under supervision of the contract research organisation's quality assurance unit
- from each concentration 3 samples are taken from 3 different sites of the 1st container after delivery for checking the homogeneity
- these samples are investigated by repeated determination
- based on the chemical properties of the test item an investigation of its stability is not regarded necessary
- homogeneity of the test item in food is analysed by base-digestion (4M NaOH) followed by turbidity measurement in aqueous media
- the content of test item is determined photometrically at a wavelength of 860 nm using a calibration curve
- the determination is performed as a non-GLP analysis
- the method is validated to allow an evaluation of the precision and accuracy of the results
- the relative standard error of the carbon fibre homogeneity determination is confirmed to be below ± 20% within the operative concentrations
- consequently, acceptance criteria for homogeneity are set at ± 20 % of the target value

Details on mating procedure:

- animals are first mated after two weeks of treatment for two consecutive weeks or until successful mating
- overnight mating is performed with 1 male : 1 female of the same dose group
- vaginal smears are taken the next morning
- mating is considered successful if sperm and/or a vaginal plug is found

Duration of treatment / exposure:

- animals are 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 is continued until day 4 post partum (p.p.) and subsequent sacrifice
- males are sacrificed after successful mating, but not before day 28 of treatment; females are sacrificed as soon as possible after day 4 p.p.
- exposure of all males and females not successfully mated is continued until their sacrifice

Frequency of treatment:

see "duration of treatment/exposure"

Duration of test:

see "duration of treatment/exposure"

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

= 1200 mg/kg food

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

= 3600 mg/kg food

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

= 12000 mg/kg food

No. of animals per sex per dose:

10 males and 10 females per dose and in the control group

Control animals:

yes, plain diet

Details on study design:

- dose selection rationale: Dose levels are based on a palatability study (CRO's study number: 12N12507) in which the animals accepted a target dose of 1000 mg/kg bw/day (12 g/kg food) without any problems. This dose is also recommended as limit dose in the OECD Guideline 422. Consequently, this dose was chosen as the high dose in the present study. The other doses were determined using a stagger of approx. 3.
- rationale for animal assignment (if not random): not applicable, randomised assignment
- rationale for selecting satellite groups: no satellite groups
- post-exposure recovery period in satellite groups: not applicable

Maternal examinations:

CAGE SIDE OBSERVATIONS: yes
- Time schedule: clinical observations at least once daily

DETAILED CLINICAL OBSERVATIONS: yes
- Time schedule: once a week all animals are inspected outside their home cages and carefully examined for clinical symptoms

BODY WEIGHT: yes
- Time schedule for examinations: weekly to the nearest 0.1 g until sacrifice of the animals
- After successful mating, body weight of the females is determined on days 0, 7, 14, and 20 p.c., as well as on days 0 and 4 p.p.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: yes
- Actual test item intake of the animals will be calculated based on food consumption: yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): no

POST-MORTEM EXAMINATIONS: yes
- Sacrifice on gestation day: Males are sacrificed after successful mating but not before day 28 of treatment. Females are sacrificed as soon as possible after day 4 p.p.. Females not successfully mated are sacrificed not earlier than on day 25 p.c. or three weeks after last mating. All animals are sacrificed by CO2 overdose and subsequent exsanguination. The treatment of all animals is continued until their sacrifice.

- Organs examined: The following organs and tissues are collected from all animals and fixed in 10% neutral buffered formalin:
brain, pituitary, tongue, eyes, lacrimal glands, nasal and paranasal cavities, larynx, pharynx, trachea, thyroid, parathyroids, lungs, thymus, heart, aorta, lung associated lymph nodes, salivary glands, mandibular lymph nodes, liver, pancreas, spleen, kidneys, adrenals, oesophagus, forestomach, glandular stomach, duodenum, jejunum, ileum, caecum, colon, rectum, mesenterium and lymph nodes, urinary bladder, testes (fixation in modified Davidson‘s fluid), epididymides (fixation in modified Davidson‘s fluid), prostate incl. coagulating glands, seminal vesicles, ovaries with oviduct (fixation in modified Davidson‘s fluid), uterus, vagina, mammary glands, skeletal muscle, femur including joint, vertebrae with spinal cord, skin, peripheral nerve, sternum with bone marrow

Ovaries and uterine content:

Ovaries and uterine content are examined after termination.
The examinations include:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No data
- Number of late resorptions: No data

Fetal examinations:

- External examinations: yes, all per litter
Litters are inspected at least once daily. Number of offspring and the occurrence of any dead pups or abnormalities are recorded for all groups. Individual pup sex is determined and individual weight of the pups is recorded to the nearest 0.1 g on days 0 and 4 p.p.. All pups are humanely sacrificed as soon as possible after day 4 p.p. and carefully examined for gross abnormalities.

Statistics:

- statistical comparison of groups is performed for each sex at the level of α < 0.05
- body weights, food consumption, clinico-chemical, hematological and activity data are analysed using analysis of variance
- if the group means differ significantly with this method the means of the treatment groups are 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 are applied in case of non-homogeneous data
- qualitative reproduction data are analysed using the two tailed FISHER test with Bonferroni correction or Chi-square test
- histopathology data are analysed using the Chi-square and the FISHER test, both two tailed
- in all instances, the dam or litter are used as the basic unit

Details on maternal toxic effects:

- Maternal toxic effects: no effects observed
- Details on maternal toxic effects: see attached background material "Tables.pdf" for further information

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): The summary of the weekly F0 premating clinical observations is provided in Table 3, the summary of clinical observations during pregnancy and lactation is given in Tables 4 and 5.
The only clinical sign observed was red eye discharge in animal no. 1107 (control) beginning on day 22 and persisting until final sacrifice.
No other clinical signs were observed during the study, consequently there was no indication of any clinical observation related to the test item.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): The summary of the weekly F0 premating body weights and body weight gains is provided in Tables 6 and 7. The summary of the maternal gestation and postpartum body weights and body weight gains is given in Tables 8, 9, 10 and 11.
No effects of test item exposure were observed on body weight or body weight gain of the male animals at any time point of the study.
In females, statistically significantly increased body weight was observed for the high and medium dose (groups 3 and 4) in the premating period starting with day 7 which was also reflected by increased body weight gains between day 0 and 7 in all substance exposed females (groups 2-4).
During pregnancy, statistically significantly increased body weight was observed in the high dose group 4 on days 0, 7, and 14 p.c., and in the medium dose group 3 on day 14 only. During lactation, this finding was noticed on day 0 and 4 p.p. in medium dose animals (group 3) only. There was, however, no effect on body weight gain during gestation or lactation in any of the groups exposed to the test item.
Taking into account that the observed effects on body weight represent a (temporary) increase and they coincide with increased food consumption in these groups, none of them can be considered adverse.
Consequently, no adverse effects of test item exposure could be observed on body weight during the study.
The summary of the weekly F0 premating food consumption data is provided in Table 12. The summary of the maternal gestation and postpartum food consumption data is given in Tables 13 and 14.
No effects of test item exposure were observed on food consumption of the male animals at any time point of the study, with the exception of a decreased food consumption from day 14–21 in the low dose group 1, which is considered incidental.
In females, statistically significantly increased food consumption was observed for the high and medium dose (groups 3 and 4) in the premating period from day 7 to 14 as well during the whole premating period.
During pregnancy, statistically significantly increased food consumption was observed in the high and medium dose group (groups 3 and 4) between days 0 and 7 p.c. and during days 7 to 14 p.c. as well as for the whole period of pregnancy (days 0-20 p.c.).
No statistically significant effects on food consumption were observed during lactation.
The observed (temporary) increase in food consumption in medium and high dose animals (groups 3 and 4) are in good concordance with the effects observed on body weight. These effects were not considered adverse.
Consequently, no adverse effects of exposure to the test item could be observed on food consumption during the study.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS): TThe mean substance intake calculations are summarised in Table 2.
Based on mean body weights and food consumption data this resulted in the following actual substance intake: 85, 256, and 859 mg/kg bw/day for males; 106, 317, and 1051 mg/kg bw/day for females in the premating period; 94, 292, and 994 mg/kg bw/day for females during gestation; and 153, 438, and 1521 mg/kg bw/day for females during lactation, respectively.

REPRODUCTIVE FUNCTION: The summary of the F0 mating data is provided in Table 19.
For the four groups, mating yielded 10, 10, 8, and 10 sperm positive females in groups 1-4, respectively. This resulted in 10, 9, 9, and 10 pregnancies; 10, 9, 9, and 10 females with liveborn offspring in F0 dams, and 10, 9, 9, and 10 females with live litters on day 4 p.p., respectively (Table 20).
Animal no. 3202 gave birth, although no mating date was determined, while animal 2201 was not pregnant although it was found sperm positive.
Precoital time was statistically significantly increased in high dose pairs (group 4) but since all matings were observed within the first 4 days of mating (representing a normal estrus cycle) this finding is considered incidental and without toxicological relevance.
Consequently, the results show that test item exposure did not produce any adverse effects 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).

ORGAN WEIGHTS (PARENTAL ANIMALS): A summary of the F0 organ weight data is provided in Table 25.
No statistically significant differences from the control group were observed in this study.

GROSS PATHOLOGY (PARENTAL ANIMALS): A summary of the F0 necropsy observations is provided in Table 24.
Findings included sporadical enlargement of the adrenal glands, discoloration and nodules of the epididymis, cysts and reduction in kidney size and focal discoloration of the thymus.
None of the macroscopic findings were considered to be induced by the test item.

HISTOPATHOLOGY (PARENTAL ANIMALS): There were no treatment-related microscopic findings. All of the histopathological findings encountered were considered to have arisen spontaneously or post mortem.
Occasional stage IX-XIV tubules in both controls and treated animals contained a single retained spermatid. No treatment-related abnormalities were noted during examination of the testes.

Dose descriptor:

NOAEL

Effect level:

>= 1 051 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Remarks on result:

other: no adverse effects observed

Dose descriptor:

NOAEL

Effect level:

>= 994 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Remarks on result:

other: no adverse effects observed

Dose descriptor:

NOAEL

Effect level:

>= 1 521 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Remarks on result:

other: no adverse effects observed

Abnormalities:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects: no effects observed; see attached background material "Tables.pdf" for further information
VIABILITY (OFFSPRING): a summary of the delivery and litter data is given in Tables 20-23
BODY WEIGHT (OFFSPRING); CLINICAL SIGNS (OFFSPRING): No effect of exposure to the test item could be observed on litter size and pup survival as well as on pup body weight. Pup clinical observations included sporadical subcutaneous hemorrhage, bad condition, and pale pups. None of these findings were considered to be related to the test item, and no abnormal pups were observed.

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

Because no deviations from the test protocol are reported and the study has been performed according to the principles of Good Laboratory Practice the study is regarded as valid.
Based on the study results the NOAEL value (parental, reproductive and developmental) was determined as the high (limit) target dose level of 12000 mg/kg food. This corresponds to the following actual substance intake: 859 mg/kg bw/day for males; 1051 mg/kg bw/day for females in the premating period; 994 mg/kg bw/day for females during gestation; 1521 mg/kg bw/day for females during lactation.

Executive summary:

The aim of this study was to evaluate possible adverse effects of the test item (milled carbonised PAN based fibre) 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 according to the OECD Guideline 422 "Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test" (March 1996) and in compliance with the Principles of Good Laboratory Practice (German Chemicals Law, § 19a, Appendix 1, 02 July 2008), with the exception of the determination of the test item in food which was performed as a non-GLP analysis, as well as with the German Animal Protection Law (Tierschutzgesetz of 18 May 2006). The dose levels were based on a palatability study in which the animals accepted food containing 12 g test item/kg food (estimated target dose of 1000 mg/kg/day) without any problems. The dose of 1000 mg/kg/day is also recommended as limit dose in the OECD Guideline 422. Consequently, this dose was chosen as the high dose in the present study. The other doses were determined using a stagger of approx. 3. The concentration in food was documented by the quality assurance unit. The analyses showed that the test item was homogenous in the preparations.

Based on mean body weights and food consumption data this resulted in the following actual substance intake: 85, 256, and 859 mg/kg bw/day for males; 106, 317, and 1051 mg/kg bw/day for females in the premating period; 94, 292, and 994 mg/kg bw/day for females during gestation; and 153, 438, and 1521 mg/kg bw/day for females during lactation. Wistar rats [Crl:Wistar(Han)], purchased from Charles River Deutschland, Sulzfeld, Germany, 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 groups exposed to the test item. The animals were housed individually in polycarbonate cages type III. Cages and absorbing softwood bedding material (Lignocel BK 8-15) was changed at least twice a week. Tap water from the Hannover city water supplier was offered fresh at least once weekly. In the control group, a closed formula in pellet form identified as V1324 was taken as the diet for this study. For the treatment groups, the test item was added to the same diet 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. The animals were inspected at least once daily and the individual body weight and food consumption of all animals were recorded weekly until sacrifice of the animals.

After successful mating, body weight and food consumption of the females were determined on days 0, 7, 14, and 20 p.c., as well as on days 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, the following parameters were investigated: hematocrit, hemoglobin, mean erythrocyte volume, mean erythrocyte hemoglobin mass, mean erythrocyte hemoglobin concentration, erythrocyte count, total and differential leucocyte count, platelet count, prothrombin time, sodium, potassium, glucose, total cholesterol, urea, creatinine, total protein and albumin, alanine aminotransferase, aspartate aminotransferase, and bile acid. Due to technical problems of the prothrombin time (PT) determination observed in samples collected in life, an additional blood sample was collected from the vena cava caudalis during terminal sacrifice for the determination of the PT.

Spontaneous locomotor activity over 60 minutes using the „Actimot“ computerised light-beam system 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. The data were analysed in 15 minutes intervals. In addition, the total values for distance, time in rest, time in movement, rearing time, and number of rearings were determined.

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. In addition to the determination of forelimb grip strength, the FOB included 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.

The animals were mated after two weeks of treatment for two consecutive weeks or until successful mating. Overnight mating was performed with 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. The time to successful insemination (precoital time) was determined.

Litters were inspected at least once daily. The number of offspring and the occurrence of any dead pups or abnormalities were 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.

The treatment of all animals was continued until their sacrifice. 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 CO2overdose and subsequent exsanguination.

Necropsy was performed in all F0males 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.

Weights of the following organs were determined: liver, kidneys, adrenals, thymus, spleen, brain, heart, testes, and epididymides.

In 5 males and 5 females of the high dose group 4 and the control group 1 a complete histopathological investigation according to OECD Guideline 422 was performed. In testes, staging of the seminal epithelium was performed. The following results were obtained (see also Summary Table 26):

None of the sporadically observed clinical findings were regarded as related to the test item. No adverse effects after exposure to the test item were observed on body weight, body weight gain or food consumption of the animals at any time point of the study. Overall, hematological and 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.

No toxicologically relevant effects of the exposure to the test item were observed on locomotor activity, neither for any of the investigated time intervals nor for the whole test period. No effects of exposure to the test item were observed on any of the investigated endpoints of the Functional Observational Battery.

For the investigation of mating data, each group consisted of 10 pairs for mating. For the four groups, mating yielded 10, 10, 8, and 10 sperm positive females in groups 1-4, respectively. This resulted in 10, 9, 9, and 10 pregnancies, 10, 9, 9, and 10 females with liveborn offspring in F0dams, and 10, 9, 9, and 10 females with live litters on day 4 p.p., respectively.

No effect of exposure to the test item 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 exposure to the test item was observed on litter size and pup survival as well as on pup body weight. None of the sporadically observed clinical findings in pups were considered to be related to the test item and no abnormal pups were observed.

None of the sporadically observed necropsy findings were considered to be related to the test item and statistically significant differences in organ weights were not found. Substance-related findings were not observed in the histologically examined organs of males and females of the control and high dose group. Occasional stage IX-XIV tubules in both controls and treated animals contained a single retained spermatid. No treatment-related abnormalities were noted during examination of the testes.

Based on the results described above, the NOAEL for this study (parental, reproductive and developmental) was determined as the high (limit) target dose level of 12000 mg/kg food. This corresponds to the following actual substance intake: 859 mg/kg body weight/day for males; 1051 mg/kg body weight/day for females in the premating period; 994 mg/kg body weight/day for females during gestation; and 1521 mg/kg body weight/day for females during lactation, respectively.

Reference 2

Endpoint:

developmental toxicity

Remarks:

rat

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because the substance is of low toxicological activity (no evidence of toxicity seen in any of the tests available), it can be proven from toxicokinetic data that no systemic absorption occurs via relevant routes of exposure (e.g. plasma/blood concentrations below detection limit using a sensitive method and absence of the substance and of metabolites of the substance in urine, bile or exhaled air) and there is no or no significant human exposure

Species:

rabbit

Abnormalities:

not specified

Developmental effects observed:

not specified

Reference 3

Endpoint:

developmental toxicity

Remarks:

rabbit

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because the substance is of low toxicological activity (no evidence of toxicity seen in any of the tests available), it can be proven from toxicokinetic data that no systemic absorption occurs via relevant routes of exposure (e.g. plasma/blood concentrations below detection limit using a sensitive method and absence of the substance and of metabolites of the substance in urine, bile or exhaled air) and there is no or no significant human exposure

Species:

rabbit

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Toxicity to reproduction: other studies

Additional information

To determine and evaluate the potential toxicity to reproduction/development of non-graphitic carbon fibres within the scope of this registration a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD Guideline 422 has been performed. As test substance the milled carbonised PAN based fibre has been chosen. It falls within the typical range of the substance definition including the impurities (in total < 0.3%) which in principle consist of metal and mixed metal oxides (mainly Al, Ca, Fe, Na, and Si oxides). These substances are of negligible concern regarding human health and the environment.

The different manufacturing processes and precursor materials for non-graphitic carbon fibres within the scope of this registration lead to slightly different compositions. Accordingly, the choice of the test material is based on the substance with the highest nitrogen content. The obtained test results are therefore regarded to also cover all other qualities of the substance non-graphitic carbon fibre within the scope of this registration.

The available study is regarded as valid because no deviations from the test protocol are reported and the study has been performed according to the principles of Good Laboratory Practice.

Based on the study results the NOAEL value (parental, reproductive and developmental) was determined as the high (limit) target dose level of 12.000 mg/kg food. This corresponds to the following actual substance intake: 859 mg/kg bw/day for males; 1051 mg/kg bw/day for females in the premating period; 994 mg/kg bw/day for females during gestation; 1521 mg/kg bw/day for females during lactation.

No signs of developmental toxicity were observed as there were no effects on litter size and pup survival as well as on pup body weight. None of the sporadically observed clinical findings in pups were considered to be related to the test item and no abnormal pups were observed.

Justification for classification or non-classification

Based on the results of a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422) the test substance (milled carbonised PAN based fibre) is regarded to be neither toxic to reproduction nor toxic to development or fertility and therefore should not be classified as toxic to reproduction according to the criteria of the Regulation (EC) No 1272/2008 (CLP Regulation).