Single Wall Carbon Nanotubes (SWCNT)

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Justification for type of information:

The Acute Oral Toxicity study on test item, was not performed as the test item was found to be impossible to formulate satisfactorily in a suitable vehicle for oral dosing.

It is the opinion of Envigo Research Limited that the bio-availability of the test item is negligible, due to its inert nature, and it is reasonable to assume that the test item presents no significant acute toxicity risk under the conditions of the test.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

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:

other: CD / Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

For this study CD rats, bred by Charles River Laboratories Germany GmbH, were used. The healthy nulliparous adult animals were randomised and assigned to the treatment groups and cages. The body weight range did not exceed 20% of the mean weight for each sex at the time of selection. The animals were held for 7 days for adaptation. Health checks were performed on the day of delivery and at first administration.
Test species / Strain / Stock: Rat / CD / Crl:CD(SD)
Breeder: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
Body weight (at 1st administration, TD15): Males: 416.4 g - 482.5 g; Females: 205.2 g - 255.2 g
Age (at 1st administration): Males: 75 days; Females: 75 days
Selection of species: The rat is a commonly used rodent species for such studies.
Number and sex of animals: Pre-exposure period (TD 1 to TD 14): 60 female animals, a sufficient number of animals in order to grant at least 40 females with a normal oestrus cycle for the main study.
Main study (start on TD 15): 80 animals (40 males and 40 females); a sufficient number in order to grant at least 8 pregnant females per group for evaluation of the F0 generation.
Adaptation period: 7 days
Animal identification: For identification, each rat received a continuous number according to a differentiated number scheme. Points were set on paws and/or tail by tattoo. Additionally, the animal cages were labelled with the tattooed serial number, sex, study code number, type of study, route of administration, dose level, date of conception and dates of administration.
With the exception of the mating period, the males and females (F0-Generation) were kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm. The room temperature was 22 °C ±3 °C (maximum range) and the relative humidity was 55% ±15% (maximum range).
Deviations from the maximum range caused, for example, during cleaning procedures are dealt with in SOPs. No values exceeding the maximum range were noted during the course of the study.
Except during the mating period the animals were placed in the animal room as follows:
Male animals: On one side of the room with each dose group separated by an empty row.
Female animals: On the other side of the room with each dose group separated by an empty row.
Granulated textured wood released for animal bedding (Granulat A2, J. Brandenburg, 49424 Goldenstedt/Arkeburg, Germany) was used as bedding material in the cages. The cages were changed and cleaned once a week. Periodic analysis of the bedding material for contaminants based on EPA/USA is conducted at least once a year by LUFA-ITL. Certificates of analysis of the bedding material are included in the raw data.
The rooms were alternately lit (about 150 lux at approximately 1.5 m room height) and darkened in a 12 hours dark/12 hours light cycle. The ventilation rate of the animal room was between fifteen to twenty air changes per hour.

Route of administration:

oral: feed

Details on route of administration:

As exposure via gavage was technically not feasible (material clocked up the gavage tubes), administration via diet had to be performed.

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

Preparation of test item-diet mixtures and administration
Route of administration: Oral, via the diet
Frequency of administration: Daily via the diet
Duration of administration: Plain diet was provided during Pre-exposure period (14 days); thereafter, males and females received diet including test material at doses pre-defined (low dose, mid dose and high dose) during pre-mating period (days 15 to 28) and during mating (days 30 to 33). Males continued to receive test material via diet until necropsy at day 44, whereas females received test material during gestation and lactation period (until test days 66 and 69, respectively.
Selection of route of administration: According to international guidelines
The test item-diet mixtures were freshly prepared once a week. The appropriate amount of test item was weighed into a glass container. Some of the test item and diet was mixed with an impact mill to produce a premix. This process was repeated until the whole quantity of test compound was distributed in the diet. Then the premix was added to the diet, mixed with a mixer (Röhnradmischer; Typ ELTE 650; J. Engelsmann AG, Ludwigshafen, Germany) for 15 minutes and then transferred to a closable bucket. Each bucket was labelled according to group and dose. Samples were taken and analysed at CURRENTA GmbH & Co, Germany as described below.
It is technical not feasible to quantitatively distinguish SWCNT from carbon impurities and from the organic carbon in the diet itself. Therefore, iron as major metal impurity of the test item was used as a marker substance for the test item, by quantification of the iron content in the pure test item as well as in the diet formulations and in blank diet, allowing to determine test item concentration and homogeneity of the test item in the diet.
To maintain a constant dose level in relation to the animal body weight the concentration of the test item in the diet was adjusted based on the mean group food consumption per sex. The concentration was adjusted weekly using the food consumption values from the previous week.
The actual test item-intake for each test week is given in this report as the test item intake in mg per kg body weight and day.
Actual test item intake (mg test item/kg b.w./day) = Relative food consumption * test item concentration per day (g food/kg b.w./day in mg/kg food/ 1000
For calculation of the relative food consumption per day from the total food consumption per week see below. The test item-concentration in the diet during the test weeks is given in the analytical report.
The control animals received the standard diet only.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Exposure diet analysis - determination of iron concentration
For the determination of the iron content as a marker substance for the test item in the exposure diets, approximately 10 g samples of exposure diet divided in 2 aliquots (the aliquots were taken independently from each other) were taken at the following times and stored at minus 20 °C ±10% until shipment to the analysis laboratory.
Concentration and homogeneity as well as the stability of the test item-diet mixtures were determined.
Determination of concentration and homogeneity in the test item diet mixtures was confirmed as follows, immediately after preparation of the weekly new charge of the test item-diet mixture:
TW3 At the start of treatment (TD15) Control: 1 from the feed sack for each sex  2 samples (4 aliquots)
TW3 At the start of treatment (TD15) Dose groups (males and females separated): 1 from the top, 1 from the middle and 1 from the bottom of the bucket for each dose group and sex  18 samples (36 aliquots)
TW5 At the start of pairing (TD29) Control: 1 from the feed sack for each sex  2 samples (4 aliquots)
TW5 At the start of pairing (TD29) Dose groups (males and females separated): 1 from the top, 1 from the middle and 1 from the bottom of the bucket for each dose group  18 samples (36 aliquots
TW7 At necropsy of males (TD43) Control: 1 from the feed sack for males  1 sample (2 aliquots)
TW7 At necropsy of males (TD43) Dose groups: (only males) 1 from the top, 1 from the middle and 1 from the bottom of the bucket for each dose group  9 samples (18 aliquots)
TW10 At necropsy of females (TD64) Control: 1 from the feed sack for males  1 sample (2 aliquots)
TW10 At necropsy of females (TD64) Dose groups: (only females) 1 from the top, 1 from the middle and 1 from the bottom of the bucket for each dose group  9 samples (18 aliquots)
Concentration and stability was also confirmed 7days after start of use of diet mixtures from left-over diet, to confirm stability of the dose formulations as follows:
TW3 + 7 days Control: 1 from the feed sack of each sex 2 samples (4 aliquots); Dose groups: 1 from the bucket of each dose and for each sex 6 samples (12 aliquots)
TW5 + 7 days Control: 1 from the feed sack of each sex 2 samples (4 aliquots); Dose groups: 1 from the bucket of each dose and for each sex 6 samples (12 aliquots)
TW7 + 7 days Control: 1 from the feed sack of the males 1 sample (2 aliquots); Dose groups: 1 from the bucket of each dose 3 samples (6 aliquots)
TW10 + 7 days Control: 1 from the feed sack of the females 1 sample (2 aliquots); Dose groups: 1 from the bucket of each dose 3 samples (6 aliquots)
The samples were labelled with the study number, species, sex, type of sample, concentration, test week, test day, location (top/middle/bottom), and date. Following advance notice the exposure diet samples were shipped on dry ice via courier to the analytical laboratory for determination of iron concentration by AAS.

Duration of treatment / exposure:

Males were exposed to the test substance during pre-mating phase (14 days), mating phase (2 - 5 days) and post-mating phase (11 - 14 days); Females were exposed to the test substance during pre-mating phase (14 days), mating phase (2 - 5 days) and gestation and lactation phase (36 days).

Frequency of treatment:

Daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

vehicle control group

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

low dose group

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

mid dose group

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

high dose group

No. of animals per sex per dose:

Each dose group (including controls) consisted of 10 males and 10 females

Control animals:

yes, plain diet

Details on study design:

Principle: The test item was administered in graduated doses to 3 groups of males and females prior to, during and after mating to generate limited information concerning the effects of the test item on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition.
At the time of sacrifice or death, the animals were examined macroscopically. The adult animals were examined externally and internally. Endocrine disruptor effects of the test item were examined by using appropriate laboratory methods (e.g. determination of the T4 level by ELISA). The reproductive organs (a detailed examination of the male gonads is mandatory) and all organs of the adult animals showing macroscopic lesions were preserved.
A histopathological examination was performed for selected organs from the animals of group 1 and group 4 and for all organs of the adult animals showing macroscopic lesions. The pups were examined externally.
Justification for dose selection: The dose levels were selected in agreement with the Sponsor based on the results of the 14-day dose range finding study (LPT Study No. 36823).
In this dose-range finding study, the test item Tuball™ was administered orally via diet to the rats at dose levels of 100, 300 or 1000 mg/kg b.w./day for 14 days.
Dark discoloured faeces were noted for all male and female animals that were dosed with 300 or 1000 mg/kg from test day 2 onwards until the end of the study on test day 15. The discolouration is most probably due to the characteristics of the test item (black powder). No further observations were noted.
No test item-related differences in body weight and food consumption were noted for all male and female animals during the whole study.
Autopsy revealed dark content in the gastrointestinal tract of all male and female animals that were dosed with 300 or 1000 mg/kg, which is most probably due to the characteristics of the test item (black powder). No further observations were noted during the macroscopic inspection during autopsy.
Mating procedure: Sexually mature male and female rats of the same dose group were paired for mating. Mating was monogamous: 1 male and 1 female animal were placed in one cage during the dark period. The female was placed with the same male until pregnancy had occurred or 2 weeks had elapsed. Each morning the females were examined for the presence of sperm or a vaginal plug. The day of conception (day 0 of gestation or GD0) was considered to be the day on which sperm was found.
In case pairing would have been unsuccessful, re-mating of females with proven males of the same group could have been considered after approximately 2 weeks of unsuccessful mating. However, all males successfully inseminated their female partners.
Housing and feeding: A certified commercial diet (ssniff® R/Z V1324, ssniff Spezialdiäten GmbH, 59494 Soest, Germany) served as food. This food was offered ad libitum. Food residue was removed and weighed.
Samples of the food are analysed for contaminants based on EPA/USA by LUFA-ITL at least twice a year. Certificates of analysis of the composition and for contaminants are provided by the manufacturer and are included in the raw data. No contaminants above the limitations were noted.
Tap water was offered ad libitum.
Samples of the drinking water are taken periodically by the Wasserwerk Wankendorf and periodic analyses are performed by LUFA-ITL according to the 'Deutsche Trinkwasserverordnung, Bundesgesetzblatt 2001' [German Regulations on drinking water, public notice of the law, 2001].
In addition, drinking water samples taken at LPT are analysed by LUFA-ITL once a year for means of bacteriological investigations according to the "Deutsche Trinkwasserverordnung 2001, Anlage 1" [German Regulations on Drinking Water 2001, Addendum 1]. No contaminants above the limitations were noted.
Housing: With the exception of the mating period, the males and females (F0-Generation) were kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm. The room temperature was 22 °C ±3 °C (maximum range) and the relative humidity was 55% ±15% (maximum range). Deviations from the maximum range caused, for example, during cleaning procedures are dealt with in SOPs. No values exceeding the maximum range were noted during the course of the study.
Except during the mating period the animals were placed in the animal room as follows: Male animals on one side of the room with each dose group separated by an empty row and female animals on the other side of the room with each dose group separated by an empty row.
Granulated textured wood released for animal bedding (Granulat A2, J. Brandenburg, 49424 Goldenstedt/Arkeburg, Germany) was used as bedding material in the cages. The cages were changed and cleaned once a week.
Periodic analysis of the bedding material for contaminants based on EPA/USA is conducted at least once a year by LUFA-ITL. Certificates of analysis of the bedding material are included in the raw data.
The rooms were alternately lit (about 150 lux at approximately 1.5 m room height) and darkened in a 12 hours dark/12 hours light cycle. The ventilation rate of the animal room was between fifteen to twenty air changes per hour.

Positive control:

None

Observations and examinations performed and frequency:

Daily observations: Throughout the test period, each animal (parental animals and pups) was observed for clinical signs at least once daily. Behavioural changes, signs of difficult or prolonged parturition, and all signs of toxicity were recorded. Mortality was recorded twice daily. Animals which died prematurely were necropsied as soon as possible after exitus.
Individual animals were observed before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment or illness. Any signs of illness or reaction to treatment were recorded for each individual animal.
Cage-side observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded. In addition, animals were checked regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays animals were checked regularly from 7:00 a.m. to 11:00 a.m. with a final check performed at approximately 3:30 p.m.
Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals.
Detailed clinical observations: Additionally, once before the first exposure (to allow for within-subject comparisons) and once a week thereafter, detailed clinical observations were made in all animals of the parental generation. These detailed clinical observations were performed at least 2 hours after dosing and were made outside the home cage in a standard arena and at the same time, each time preferably by observers unaware of the treatment. Signs observed included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypy (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.
Dated and signed records of appearance, change, and disappearance of clinical signs were maintained on clinical history sheets for individual animals.

Sacrifice and pathology:

The animals were euthanized by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis, weighed, dissected and inspected macroscopically (gross necropsy) at the following times:
Males on test day 44
Pups on PND 13
Dams on lactation days 14 - 16
Examination of the pups See endpoint study record for reproductive endpoint
Dissection of adult animals: At the time of sacrifice or premature death during the study, the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system. During necropsy the number of implantation sites was recorded in the female animals.
Apparently non-pregnant uteri were placed in a 10% aqueous solution of ammonium sulfide for about 10 minutes to stain possible implantation sites in the endometrium according to SALEWSKI.
All superficial tissues were examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection all subcutaneous tissues were examined. The condition to the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and the caecum were incised and examined. The lungs were removed and all pleural surfaces were examined under suitable illumination.
The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.
Organs weighed: The weight of the following organs of all adult male and female animals was determined. Paired organs were weighed individually and identified as left or right.
Adrenal gland (2), Brain, Epididymis (2), Heart, Kidney (2), Liver, Ovary (2), Spleen, Testicle (2), Thyroid (1), Thymus, As a whole: prostate, seminal vesicles with coagulating glands, Uterus including cervix
The weights of the organs were determined before fixation. Only the weight of the thyroid glands was determined after fixation.
Organs fixed for preservation: The following organ(s) or parts thereof of all adult male and female animals were fixed in modified Davidson's solution or 7% buffered formalin:
Fixative: modified Davidson'solution for Epididymis (2)and Testicle (2);
Fixative: 7 % buffered formalin for Gross lesions observed, Ovary and oviduct (2), Prostate, Seminal vesicles with coagulating glands, Thyroid (including parathyroids), Uterus (including cervix), Vagina
Any other organs displaying macroscopic changes were also preserved.
Selection of animals and organs for histopathology: 5 male and female animals from each group were randomly selected for histopathology examination:
The following organs or parts thereof from the above selected animals and from every deceased or prematurely sacrificed animal were fixed for histopathology examination.
Fixed organs from the 5 randomly selected male and female animals per group:
Fixative: Davidson'solution for Eye with optic nerve (2)
Fixative: modified Davidson'solution for Epididymis (2) and Testicle (2)
Fixative: 7 % buffered formalin for Adrenal gland (2), Bone, Bone marrow (os femoris), Brain (cerebrum, cerebellum, brain stem (pons)), Gross lesions observed, Heart (3 levels: right and left ventricle, septum), Intestine, small (duodenum, jejunum, ileum, incl. Peyer's patches, Swiss roll method), Intestine, large (colon, rectum), Kidney and ureter (2), Liver, Lungs (with mainstem bronchi and bronchioles), Lymph node (1, cervical), Lymph node (1, mesenteric), Mammary gland, Muscle (skeletal), Nerve (sciatic), Oesophagus, Ovary and oviduct, Pituitary, Prostate and seminal vesicles with coagulating glands, Spinal cord (3 sections), Spleen, Stomach, Thyroid (including parathyroids), Thymus, Tissue masses or tumors, Tongue (including base), Trachea (including larynx), Urinary bladder, Uterus (including cervix), Vagina
Histopathology
Animals to be examined and preparation of slides: Full histopathology was performed on the preserved organs of the selected parental animals of groups 1 and, and the thyroids of the selected pups.
The organs listed above were examined histologically after preparation of paraffin sections and haematoxylin-eosin staining. Parathyroids cannot always be identified macroscopically. They were examined microscopically if in the plane of section and in all cases where they are noted were grossly enlarged. In addition, frozen sections of the heart, liver and one kidney were prepared, stained with Oil Red O and examined histologically.
Detailed histopathologic examination was performed on one testicle and one epididymis (with special emphasis on the qualitative stages of spermatogenesis and histopathology or interstitial testicular structure) of the selected males of groups 1 and 4 following H-E and PAS staining.
The blood smears prepared from all animals during the haematological examination were available for possible examination of pathological changes but examined and evaluated only depending on necropsy findings and upon agreement with the Sponsor. So far, no examination was performed.
Histopathological evaluation: The histotechnique was performed by LPT. The slides (labelled with study number, species, animal number, block number) were dispatched to Global Pathology Support (GPS) on July 05, 2019. The transport of slides for the histopathology work performed by GPS to the Test Site was arranged by LPT, whereas the return transport to the Test Facility was arranged by GPS. Histopathology was performed by GPS, Dr. R.J.M.M. Thoolen, Benoordenhoutseweg 23, 2596 BA The Hague, The Netherlands, under the Test Site Reference No. 537 according to all relevant GPS SOPs.

Other examinations:

Neurological screening: Screening of sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli) (based on Gad ), as well as the assessment of grip strength (Meyer ) and motor activity assessment were conducted as described on the following pages in five males and five females randomly selected from each study group.
The screening was conducted on the following test days:
5 male F0 animals per group (randomly selected) on test day 42.
5 female F0 animals per group (randomly selected) on test day 65 (between lactation days 11 to 13).

Statistics:

The following data were captured or calculated by the departmental computerized system (Provantis® integrated preclinical software, version 10.2.1, Instem LSS Ltd): Parental clinical signs, body weight, body weight gain, food consumption, haematological and biochemical parameters.
Raw data not fully compatible with the computerized system (e.g. data from the neurological screening or pup data) were maintained on paper according to the appropriate SOPs. Data maintained on paper (e.g. data from the neurological screening or pup data) was entered in Provantis in a retrospective manner using the laboratory records according to the appropriate SOPs.
Parametrical data: The statistical evaluation of the parametrical values was done by Provantis (Provantis® integrated preclinical software, version 10.2.1, Instem LSS Ltd) using the following settings: Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILK’s test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).
Non-parametrical data: The statistical evaluation of non-parametrical values was done by comparison of the group values using the FISHER or the Chi2 test with the following software: Using Provantis for statistical evaluation of histopathology findings and using StatXact 4.0.1 for statistical evaluation of the fertility index, the gestation index, the birth and live birth index, the viability indices and the post-implantation loss.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No changes in behaviour, the external appearance and the consistency of the faeces were noted for the male and female animals of the control group and the treatment groups (100, 300 or 1000 mg Tuball/kg bw/day). Dark discoloured faeces were noted continuously from test day 18 (4 days after the start of dosing) until the end of the study for all male and female animals of the high dose group. This was due to the high concentration of the administered test item (black powder) and was not considered to be of toxicological relevance. No external observations, no changes in body posture, movement and coordination and in behaviour were noted for the male and female animals of the treatment groups (100, 300 or 1000 mg Tuball/kg bw/day) and the control group.

Mortality:

no mortality observed

Description (incidence):

No premature death was noted in the control group and in the treatment groups (100, 300 or 1000 mg Tuball/kg b.w./day).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test item-related changes in body weight and body weight gain were noted for the male rats between the control group and the treatment groups (100, 300 or 1000 mg Tuball/kg b.w./day) and no test item-related differences in body weight and body weight gain were noted between the female rats of the control group and the female rats of the treatment groups (100, 300 or 1000 mg Tuball/kg bw/day) during the pre-mating, the gestation and the lactation period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

During the pre-mating period (test days 15 to 28) no test item-related difference in food consumption was noted between the male rats of the control group and the rats of the treatment groups (100, 300 or 1000 mg Tuball/kg bw/day). No test item-related differences in food consumption were noted between the female rats of the control group and the female rats of the treatment groups (100, 300 or 1000 mg Tuball/kg bw/day) during the pre-mating, the gestation and the lactation period.

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

no effects observed

Description (incidence and severity):

No test item-related changes in the consumption of drinking water was noted for the male and female rats treated with 100, 300 or 1000 mg Tuball/kg bw/day by visual appraisal.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related differences for the examined haematological parameters were noted between the control group and the treatment groups (100, 300 or 1000 mg Tubal/kg bw/day).
For the male animals slight but statistically significantly increased values were noted at the low dose level for the haemoglobin concentration (5.8% above the control value) and at the high dose level for the number of red blood cells (7.5% above the control value). However, as the increases were only small, and nearly all individual values were within the LPT background range (only the number of red blood cells of the high dose animal no. 63 was slightly above the background range), these observations were considered to be spontaneous.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related differences for the examined biochemical parameters were noted between the control group and the treatment groups (100, 300 or 1000 mg Tuball/kg bw/day).
A slight but statistically significantly decreased albumin concentration was noted at all dose levels (between 6.2% and 6.7% below the control value) and a statistically significantly increased aP activity at the low and the high dose level (56.8% or 53.3% above the control value). However, as all individual values were within the LPT background data, these observations were considered to be spontaneous.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No test item-related observations of abnormal behaviour, no adverse effects on motoric skills, changes in the external appearance and the consistency of the faces were noted for the male and female animals of all treatment groups (100, 300 or 1000 mg Tuball/kg bw/day). No test item-related differences were noted in body temperature or the hind-leg splay in comparison to the control group. No test item-related influence on the fore- and hindlimb grip strength or on the spontaneous motility was noted for the male and female animals of all treatment groups (100, 300 or 1000 mg Tuball/kg bw/day).

Immunological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related differences were noted between the control group and the treatment groups (100, 300 or 1000 mg Tuball/kg bw/day) for the T4 levels of the parental males.
A slight, but statistically significantly reduced T4 level was noted at the high dose level (15.8% below the control). This was mainly due to animal no. 66 with a markedly reduced T4 level (30.2 nmol/L) that was below the LPT background range. As the T4 levels of the remaining 9 high dosed males were all in the LPT background range, the reduced T4 level that was noted for the females of the high dose group was considered to be spontaneous.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No test item-related differences were noted for body weight at autopsy between the control group and the treatment groups (100, 300 or 1000 mg Tuball/kg bw/day). No test item-related differences for the examined absolute and relative organ weights were noted between the control group and the treatment groups (100, 300 or 1000 mg Tuball/kg b.w./day).

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No macroscopic (post-mortem) test item-related observations were noted for the male and female animals of of the treatment groups (100, 300 or 1000 mg Tuball/kg bw/day).
The following observations that were noted for the male and female animals were considered to be spontaneous:
Control: Dilated uterus horns that were filled with clear liquid were noted for female animal no. 17.
Group 2: A small left testes of soft consistency was noted for male animal no. 28.
Group 4: Several black coloured foci (approx. 2 mm in diameter) were noted at the fundus region of the stomach of male animal no. 61.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No test item-related changes were observed for the examined male and female animals of the high dose group (1000 mg Tuball/kg bw/day) during the microscopic examination. No test item-related microscopic changes were observed for the reproductive organs of males and females of the high dose group (1000 mg Tuball/kg bw/day) that were examined microscopically.
The histopathological examination performed on one testicle and one epididymis of the selected males of groups 1 and 4 (with special emphasis on the qualitative stages of spermatogenesis (proliferative, meiotic and spermiogenic phases) and histopathology of the interstitial testicular structure), did not reveal any test item-related effects. The mammary glands of the observed female animals showed prominent mammary development.

Details on results:

The actual mean test item intake during the whole study was in the range of the nominal values for all treatment groups (100, 300 or 1000 mg Tuball/kg bw/day).
No significantly lower actual values in comparison to the nominal values were noted, but significantly higher actual values as the nominal values were noted for some test weeks (i.e. actual test item intake in test week 8 for the females of the intermediate and the high dose group). However, as no signs of toxicity were noted in the whole study, the temporary higher actual dose levels were not relevant, moreover, as the mean values for the whole study period were in the range of the nominal values as mentioned above.

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

The calculation of the concentration of the test item Tuball in the test item diet mixtures was performed by determination of the iron content as lead substance for Tuball™. The received values were corrected for the iron content in pure diet samples.

In total 72 samples of test item diet mixtures (24 per group; groups 2 to 4) were analyzed. This corresponds to 144 aliquots. Each aliquot was separately taken from the test item-diet mixture.

Some samples revealed a high variation between both aliquots (taken independently from each other). This may result from a demixing of the samples during the transport to the analytical laboratory. Another possibility were inhomogeneity hot spots in the samples, as it was difficult to generate a fine distribution of the coarse Tuball material with the diet.

However, the result of each sample is given as the mean value from its two aliquots. For most samples the quotient of the actual to the nominal concentration was within the admissible limits of 80% to 120%.

In some samples (16 of 72) the actual values were more than 20% above the nominal concentrations (actual / nominal quotient between 1.21 and 1.79). However, as no toxic findings were observed a slight overdosing in a few samples is not of toxicological relevance.

Values below the admissible limit of 80% were noted for 2 samples of group 2 from test week 10 (bottom of the bucket with 0.52 and left over from the bucket after 7 test days with 0.46). However, the samples from the top of the bucket and the middle of the bucket were well in the admissible range. Furthermore, test week 10 was the last test week of the study. During this test week the female animals were successively removed and did not receive a possible underdosing for the whole test week. Finally, the 10 week samples from test groups 3 and 4 were within the admissible range and no signs of toxicity were noted in these test groups. Hence, the low actual / nominal quotient that was noted for 2 samples of group 2 in test week 10 was not of toxicological relevance.

Conclusions:

In this study according to OECD 422 no effects by the Single-walled Carbon Nanotubes (SWCNT) Tuball wer obsered at any of the dose groups investigated (0, 100, 300 and 1000 mg/kg bw/d). Thus, the NOAEL for repeated dose toxicity was set to >1000 mg/kg bw Tuball™/d.

Executive summary:

The aim of the study was to obtain information on possible effects of the test item on general toxicity, reproduction and/or development according to OECD guideline 422. The test item Tuball™ was administered orally to rats via the diet at dose levels of 100, 300 or 1000 mg Tuball™/kg bw/day.

General toxicity to parental male and female animals

None of the animals died prematurely.

No changes were noted in behaviour, the external appearance and the consistency of the faeces. Black discoloured faeces that were noted for all males and females at the high dose level were due to the high concentration of the administered test item (black powder) and not of toxicological relevance.

No influence was noted for the male and female animals on body weight, food consumption, on the results of the neurological screening, the haematological and biochemical parameters and the T4 level of the male animals.

No test item-related changes were noted during the macroscopic and the microscopic examinations and for the relative and absolute organ weights.

The mean actual intake of the test item via the diet over the whole study period was 102, 305 and 1026 mg Tuball™/kg bw/day for the male and 117, 341 and 1195 mg Tuball™/kg bw/day for the female animals. Hence, the actual values were in the range of the nominal values with 100, 300 or 1000 mg Tuball™/kg bw/day.

Reproductive toxicity to parental females

No influence was noted on the fertility index, the gestation index, the duration of the pre-coital time interval and the gestation period.

No test item-related effect was noted on the prenatal development of the pups (birth and live birth index, percentage of post implantation loss).

Furthermore, no test item-related effect was noted on the postnatal development of the pups with regarding to the viability indices (pre- and post-cull), the pup body weight, the ano-genital distance, the nipple retention, the T4 level and the histopathological examination of the pup thyroids. No variations or malformations were noted during the macroscopic external examination at necropsy.

The following no-observed-adverse-effect levels (NOAEL) were established:

General toxicity: NOAEL for systemic toxicity is above 1000 mg Tuball™/kg bw/day via the diet

Reproductive toxicity:

a) for adverse effects on the reproductive parameters of the parental females a NOAEL above 1000 mg Tuball™/kg bw/day via the diet

b) adverse effects on pre- and postnatal development

- for adverse effects on prenatal development (conceptus to birth) a NOAEL above 1000 mg Tuball™/kg bw/day via the diet was found.

- for adverse effects on postnatal development (pup) a NOAEL above 1000 mg Tuball™/kg bw/day via the diet was defined.