Single Wall Carbon Nanotubes (SWCNT)

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

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 to obtain information on possible effects of the test item on general toxicity, reproduction and/or development according to OECD guideline 422.

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.

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 for reproductive parameters:

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.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018 - 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

Vehicle:

unchanged (no vehicle)

Details on 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.

Details on 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.

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 via feed

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control group, plain diet

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

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

Parental animals: Observations and examinations:

Dated and signed records of all activities relating to the day by day running and maintenance of the study within the animal unit as well as to the group observations and examinations outlined in the Study Plan were recorded in the appropriate documentation. In addition, observations relating to individual animals made throughout the study were recorded.
The following observations were made during the course of the study:
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.
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).
Observational screening
Righting reflex: The animal was grasped by its tail and flipped in the air approximately 60 cm above the cart surface so that it turned head over heels. The normal animal should land squarely on its feet; that means zero (0) points were scored. If it landed on its side, 1 point was scored; if it landed on its back, 2 points were scored. This test was repeated five times and the total scores were recorded.
Body temperature: An electronic probe thermometer with a blunt probe was used to take the rectal temperature, being allowed to equilibrate for 30 seconds before the reading was recorded.
Salivation: Discharge of clear fluid from the mouth is most frequently seen as beads of moisture on lips in rats. The normal state is to see none, in which case a zero (0) was recorded in the blank space of the scoring sheet. If present, a plus sign (+) was recorded in the blank.
Startle response: With the animal on the cart, the metal cage was struck with the blunt probe. The normal animal should exhibit a marked but short-lasting response, whereby a zero (0) was recorded in the blank space of the scoring sheet. If there was no response, a plus sign (+) was recorded.
Respiration: While at rest on the cart, the animal's respiration cycle was observed and evaluated in terms of a scale from 1 (reduced) to 5 (increased), with 3 being normal.
Mouth breathing: Rats are normally obligatory nose-breathers. Each animal was observed whether or not it was breathing through its mouth. If the rat was breathing through its nose, a zero (0) was recorded; mouth breathing was documented by a plus sign (+).
Urination: When an animal was removed from its cage, the pan beneath the animal's cage was examined while returning the animal to its cage. The signs of urination were evaluated on a scale of 0 (lacking) to 5 (polyurea) with 3 being normal.
Convulsions: If clonic or tonic convulsions were observed, their intensity was graded on a scale of 1 (minor) to 5 (marked) and the type was recorded. In the normal animal no convulsions should be observed, in which case a score of zero (0) was recorded.
Pilo-erection: The fur of the animal's back was observed whether it was raised or elevated. In the normal animal no pilo-erection should be observed and a score of zero (0) was recorded. If pilo-erection was present, a plus sign (+) was recorded.
Diarrhoea: In examining the pan beneath an animal's cage, it was noted if there were any signs of loose or liquid stools. The normal state is for there to be none (0); in case of diarrhoea the intensity was recorded on a scale of 1 (slight) to 5 (much increased).
Pupil size: It was determined if the pupils were constricted or dilated and the observations were evaluated in terms of a scale from 1 (constricted) to 5 (dilated), with 3 being normal.
Pupil response: The beam of light from the pen light was played across the eyes of the animal and the changes in pupil size were noted. In the normal animal, the pupil is constricted when the beam is on it and then dilates back to normal when the light is removed, whereby a score of zero (0) was recorded. If there was no pupil response, a minus sign (-) was recorded in the blank space.
Lacrimation: The animal was observed for the secretion and discharge of tears. The tears of rats contain a reddish pigment. No discharge is normal, whereby a score of zero (0) was recorded in the blank space of the scoring sheet. If a discharge was present, a plus sign (+) was recorded.
Impaired gait: The occurrence of abnormal gait was evaluated. The most frequent impairments are waddling (W), hunched gait (H), or ataxia (A, the inability of all the muscles to act in unison). The extent of any impairment was recorded on a scale of 1 (slight) to 5 (marked). A normal gait was documented by a score of zero (0).
Stereotypy: Each animal was evaluated for stereotypic behaviour (isolated motor acts or partial sequences of more complex behavioural patterns occurring out of context and with an abnormally high frequency). These were graded on a scale of 1 (slight) to 5 (marked). Normal behaviour was documented by a score of zero (0).
Toe pinch: The blunt probe was used to bring pressure to bear on one of the digits of the hind limb. This should evoke a response from the normal animal. The response or lack thereof was graded on a scale from 1 (absent) to 5 (exaggerated) with 3 being the normal response.
Tail pinch: The toe pinch procedure was utilized with the animal's tail instead of its hind limb and was graded on the same scale from 1 (absent) to 5 (exaggerated), with 3 being the normal response.
Wire maneuver: The animal was placed on the metal rod suspended parallel to the cart approximately 60 cm above the cart's surface. The animal's ability to move along the rod was evaluated. If impaired, a score from 1 (slightly impaired) to 5 (unable to stay on wire) was recorded. Normal movement was documented by a score of zero (0).
Hind leg splay: The hind paws were marked with ink using an ink pad. The rat was then held 30 cm above a sheet of blotting paper on the cart. The animal was dropped and the distance between the prints of the two hind paws was measured (in cm).
Positional passivity: The animal was observed after being placed in an awkward position, such as on the edge of the top of the wire-bottomed cage on the cart surface. If the animal immediately moved into a normal position, a score of zero (0) was recorded. If not, a score was recorded on a scale of 1 (slightly impaired) to 5 (cataleptic).
Tremors: Periods of continued fine movements, usually starting in the limbs and perhaps limited to them. The normal case is to have none, in which case a score of zero (0) was recorded. If tremors were present, they were graded on a scale of 1 (slight and infrequent) to 5 (continuous and marked).
Positive geotropism: The animal was placed on the inclined (approximately 30°) top surface of the wire cage with its head facing downward. It should turn 180° and face "uphill", in which case a score of zero (0) was recorded in the blank space of the scoring sheet. If this did not occur, a negative sign was recorded in the blank.
Limb rotation: One of the animal's hind limbs was taken and moved through its normal plane of rotation. In the normal state, it should rotate readily but there should be some resistance. The variations from normal were from no resistance (1) to markedly increased resistance or rigidity (5), with 3 being normal.
Auditory function: Each animal was placed in a container and observed for Preyer's reflex (twitching of the pinna) in response to a high frequency sound stimulus. The stimulus was repeated, if necessary, up to 3 times. A normal response was recorded with a plus sign (+); if there was no response a zero (0) was recorded.
Functional tests
Grip strength: Prior to testing, the gauge (Chatillon, Modell DPP - 1.0 kg) was calibrated with a set of known weights and the apparatus adjusted for the size of the animal (about 1 cm clearance on both sides of the animal). After the strain gauge was zeroed and set in the record mode, the animal was placed into the trough with the forepaws inside the triangular grasping ring. Using one hand, the animal was grasped about 2.5 cm of the way up toward the base of the tail and steadily pulled (approx. 2.5 cm/sec) away from the ring until the grip was broken. It was continued to pull the animal along the trough until the hind limbs grasp the T-bar. The trial was completed when grip of the hind limbs was broken. Three successive readings were taken for each animal with an intertrial interval long enough to record the data and zero both meters for the next trial.
Locomotor activity: The motility was measured using the TSE InfraMot system . The infrared sensor was placed on the cage and any movements were measured for a duration of 12 min by sensing the body heat image, i.e. the infrared radiation, and its spatial displacement over time. Any movements within the cage, even brief movement events of only a few milliseconds duration, were detected and included in the activity data.
Mortality
Further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. This allowed post-mortem examination to be carried out during the working period of that day. On Saturdays and Sundays, a similar procedure was followed with a final check at approximately 3.30 p.m. For the prematurely deceased pups a post-mortem examination was performed as described in Section 4.1.1 'Examination of the pups'.
Body weight: The adult animals were weighed weekly; the individual body weights were recorded.
The report included weekly values for the male animals (starting on test day 15) and the body weight on the day of sacrifice. For the female animals the body weights on the following days were given in the report on test days 15,22, and 29 during pre-mating period, gestation days 0, 7, 14, and 20 during gestation period and on lactation days 1, 4, 8 and 13 during lactation period.
Food and drinking water consumption: Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each rat in each group upon completion of a treatment week (pre-mating and gestation) or treatment period (lactation). From these data the relative food consumption (in g/kg b.w./day) was determined. Drinking water consumption was monitored daily by visual appraisal throughout the study.

Oestrous cyclicity (parental animals):

Vaginal smears were examined on the day of necropsy to determine the stage of the oestrus cycle and allow correlation with the histopathology of the female reproductive organs.

Sperm parameters (parental animals):

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.

Litter observations:

As soon as possible after delivery, each litter was examined to establish the number and sex of pups, stillbirths, live births, runts (pups were considered as runts if their weight was less than 70% of the mean litter weight) and the presence of gross abnormalities. Any abnormal behaviour of the offspring would have been recorded. However, no abnormal behaviour was noted for the pups. Litter adjustment on PND 4: After counting on PND 4, the litters were adjusted to 10 pups per litter by eliminating (culling) surplus pups using a randomization scheme generated by Provantis®.

Postmortem examinations (parental animals):

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

Postmortem examinations (offspring):

Dead pups and pups sacrificed at day 13 post-partum were carefully examined externally for gross abnormalities. The external reproductive genitals were examined for signs of altered development.
At lactation day 13, the thyroid of 1 male and 1 female pup from each litter was fixed in 7% formalin, if possible. However, in case of dam nos. 20 and 55 no male pup was available on lactation day 13. Therefore, the thyroids from 2 female pups from dams nos. 20 and 55 were taken. The same pups were used for T4 ELISA sampling.

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.

Reproductive indices:

The following parameters and indices were evaluated:
Reproductive parameters: Pre-coital time and gestation length
- number of pregnant females
- duration of pre-coital time
- gestation length
(The duration of gestation was calculated from gestation day 0 (day of positive sperm detection) until (but not including) lactation day 1 (lactation day 1: morning after littering when no signs of littering were noted anymore)).
Implantation sites
- number per dam
- distribution in the uterine horns
- absolute number per group
- mean per group
Number of pups absolute
- at birth (alive and dead)
- after 4 days of life (pre- and post cull) and 13 days of life
Number of pups per dam
- at birth (alive and dead)
- after 4 days of life (pre- and post cull) and 13 days of life
Number of male and female pups
- at birth
- after 4 days of life (pre- and post cull) and 13 days of life
Number of stillbirths
- absolute
- per dam
Number of pups with malformations
- absolute
- per dam
Reproductive indices
The following indices were calculated for each group:
Female Fertility Index [%] = Number of pregnant rats x 100 / Number of rats paired with a male
The female fertility index reflects the total number of dams that had achieved pregnancy, including dams which delivered at term, aborted or had fully resorbed litters.
Gestation Index [%] = Number of dams with live pups x 100 / Number of pregnant rats
For each litter and group the following indices were determined:
Birth Index [%] = Total number of pups born (alive + dead) x 100 / Number of implantation scars
Live Birth Index [%] = Number of pups alive on day 0/1 of lactation x 100 / Total number of pups (alive + dead)
Viability Index [%] pre-cull = Number of pups alive on day 4 (pre cull) x 100 / Number of pups alive on day 0/1
Viability Index [%] post cull = Number of pups alive on day 13 x 100 / Number of pups alive on day 4 (post cull)
Post-implantation loss [%] = Implantations - number of pups born alive x 100 / Implantations

Offspring viability indices:

As soon as possible after delivery, each litter was examined to establish the number and sex of pups, stillbirths, live births, runts (pups were considered as runts if their weight was less than 70% of the mean litter weight) and the presence of gross abnormalities. Any abnormal behaviour of the offspring would have been recorded. However, no abnormal behaviour was noted for the pups.
The following examinations/observations were done for the offspring.
Counting: Live pups were counted, sexed and weighed on post-natal days 1, 4 and 13.
Ano-genital distance: On post-natal day 4 before litter adjustment the ano-genital distance (AGD) of all pups was determined using a scale.
Litter adjustment on PND 4: After counting on PND 4, the litters were adjusted to 10 pups per litter by eliminating (culling) surplus pups using a randomization scheme generated by Provantis®.
Blood sampling for thyroid hormone (T4) determination: On PND 4 and on PND 13 blood samples for T4 hormone level determination were taken from 2 selected pups per litter, if possible from one male and one female pup. On PND 4 the culled surplus pups were used for blood collection. The selection procedure of the pups is described in section 3.24 'Thyroid hormone (T4) determination'.
Male nipples counting: Nipples were counted in all male pups on PND 13 (shortly before scheduled sacrifice).

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 b.w./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 b.w./day) and the control group.

Dermal irritation (if dermal study):

not examined

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 b.w./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 b.w./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 b.w./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 b.w./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 b.w./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 b.w./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 b.w./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 b.w./day).

Immunological findings:

effects observed, non-treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

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 b.w./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 b.w./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.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Monitoring of estrus cycle - Exposure period
After the allocation of the animals to the test groups and the start of treatment on test day 15, the estrus cycles were further monitored during the pre-mating and mating period until one day before a positive mating sign was noted. No test item-related differences were noted for the mean length and the mean number of estrus cycles per dam during the pre-mating and mating period between the female animals of the control group and the female animals of the treatment groups (100, 300 or 1000 mg Tuball/kg b.w./day).
The mean number of estrus cycles (from test day 15 = start of treatment until test day 29 = pairing) for group 1 (control) were 1.9, for group 2 (low dose) 1.9, for group 3 (mid dose) 2.0 and for group 4 (high dose) 1.6 and the mean cycle length were 4.62, 5.31, 5.24 and 5.00 days, respectively.
Abnormal estrus cycles in the form of estrus cycles with an elongated diestrus period (more than 5 consecutive test days in the diestrus stage) were noted for 3 animals of the control group and the intermediate dose group, one animal of the low dose group and 5 animals of the high dose group.
As the incidences of females with elongated diestrus cycles between the control group and the high dose group were similar, the occurences of estrus cycles with elongated diestrus periods can be considered as spontaneous.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

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.

Reproductive performance:

no effects observed

Description (incidence and severity):

No test item-related influence on the fertility index of the female rats was noted for any of the treatment groups (100, 300 or 1000 mg Tuball/kg b.w./day).
In the control group and in the treatment groups all female animals (10 of 10) that were paired with their male partner on test day 29 became pregnant, leading to a female fertility index of 100% for all groups.

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 b.w./day) (see text table 7-3 on the following page; last column 'Mean intake').
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.
Gestation index: No test item-related influence on the gestation index was noted for the female rats of the treatment groups (100, 300 or 1000 mg Tuball/kg b.w./day. All pregnant animals of the control group and the treatment groups delivered live pups, leading to a gestation index of 100% for all groups (see text table 7-10 below).
Pre-coital time: No test item-related differences were noted in the length of the pre-coital time between the control group and the treatment groups (100, 300 or 1000 mg Tuball/kg b.w./day).
Gestation length: No test item-related differences were noted for the length of the gestation period between the rats of the control group and the treatment groups (100, 300 or 1000 mg Tuball/kg b.w./day).
Birth indices and post-implantation loss: No test item-related differences were noted for the mean number of implantation sites per dam, the mean number of pups born (alive and dead) per dam and the mean number of live born pups per dam between the control group and the treatment groups (100, 300 or 1000 mg Tuball/kg b.w./day). Also the reproductive indices as the birth index, the live birth index and the percentage of post implantation loss revealed no test item-related differences between the control group and the treatment groups (100, 300 or 1000 mg Tuball/kg b.w./day). The mean number of resorptions and stillbirths per dam (calculated from the difference between the number of implantation sites and the number of live born pups) of the high dose level (0.6 per dam) is even slightly below the control group (0.9 per dam).

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

Viability index: Pre-cull period - No test item-related differences between the control group and the treatment groups (100, 300 or 1000 mg Tuball/kg b.w./day) were noted for the viability index between lactation days 0/1 and 4 (pre-cull); the viability indices were 98.0, 100.0, 99.3 and 97.2 for groups 1 - 4, respectively. The total number of ligfe born pups were 150, 142, 146 and 143, accordingly.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

No test item-related differences between the control group and the treatment groups (100, 300 or 1000 mg Tuball/kg b.w./day) were noted for the viability index during the post-cull period. However, increased values of prematurely deceased animals during the post-cull period were noted in the control group and in all treatment groups, leading to viability indices that were below the LPT background range for the control group and the treatment groups. Whereas the decreased viability indices of the low and the intermediate dose groups were at the level of the control group (and have to be considered as spontaneous), a slightly further reduction in comparison to the control group was noted for the viability index of the high dose group (p ≤ 0.05 at the group level). However, with regard to the assessment of the reduced viability indices that were noted in the control group and the low and the intermediate dose group as spontaneous, the slightly further decrease that was noted in the high dose group was also considered to be spontaneous. Due to the slightly increased number of prematurely deceased pups during the post-cull period a slightly reduced mean number of pups per dam was noted at the high dose level on lactation day 13 (statistically not significant).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test item-related difference was noted between the mean body weight of the pups from the dams of the control group and the mean body weight of the pups from the treatment groups (100, 300 or 1000 mg Tuball/kg b.w./day) on lactation days 1, 4 and 13. One runt each was noted in the control group, the intermediate and the high dose group. The observation of one runt in a treatment group is considered to be spontaneous, especially as one runt was noted in the control group. No test item-related differences in litter weight were noted between the control group and the treatment groups (100, 300 or 1000 mg Tuball/kg b.w./day).

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

No test item-related difference in the absolute and the relative ano-genital distance of the male and the female pups was noted between the control group and the treatment groups (100, 300 or 1000 mg Tuball/kg b.w./day).

Nipple retention in male pups:

effects observed, non-treatment-related

Description (incidence and severity):

Male pups with nipple retention on lactation day 13 were noted for 4 dams of the control group, 2 of the low dose group, 5 of the intermediate dose group and 4 of the high dose group.
However, whereas only 1 male pup with nipples was noted for each of the 4 affected dams of the control group, the 5 or 4 affected dams of the intermediate and the high dose group mostly showed 2 or 3 male pups with nipple retention per affected dam. An increase was also noted for the number of nipples per male pup. Whereas in the control group and the low dose group only 1 or 2 nipples were noted for an affected pup, 3 or 2 male pups with 3 or 4 nipples were noted at the intermediate and the high dose level. The resulting group mean values were at the upper range or above the upper range of the LPT background data.
The increased nipple retention that was noted at the intermediate and the high dose level was considered to be most likely spontaneous and not test item-related.
Assessment of increased nipple retention:
An increased number of pups with nipple retention were noted at the intermediate and the high dose level. The toxicological relevance of this finding is unclear, but limited by the following facts:
- The finding is completely isolated, no influence at a related endpoint, anogenital distance, was found to form a pattern with the increased number of retained nipples.
- The number of litters displaying retained nipples as the statistical unit is not influenced.
- Though there is a dose factor of more than 3 between the medium and high dose group (300 mg/kg and 1000 mg/kg), no dose response relationship at all for nipple retention was found.
- For all calculated mean values the standard deviation is higher than the mean value, indicating large variety.
- The investigation of this endpoint within this study type was only introduced in 2015, so that the data base of historical control data is limited as well as the interpretation of the described pattern of effects.
Based on all these considerations, the observation is most likely spontaneous and the likelihood of this effect as being test item-related is very low, though it cannot be excluded with certainty.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No gross abnormalities (e.g. malformations or variations) were noted during the macroscopic external examination of the control pups and the pups from the dams treated with 100, 300 or 1000 mg Tuball/kg b.w./day after terminal sacrifice on lactation day 13 or for the pups that died during the lactation period.

Other effects:

no effects observed

Description (incidence and severity):

No test item-related influence on the male to female ratio was noted for all treatment groups (100, 300 or 1000 mg Tuball/kg b.w./day).

Developmental immunotoxicity:

no effects observed

Description (incidence and severity):

No test item-related changes were noted for the thyroid glands of the pups of the treatment groups (100, 300 or 1000 mg Tuball/kg b.w./day).
Also no changes were noted for the thyroids of the pups of the control group.
No test item-related differences betweem the control group and the treatment groups (100, 300 or 1000 mg Tuball/kg b.w./day) were noted for the T4 levels of the male and female pups.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

No test-item related effects were observed on reproductive arameter in parental animals or in pups. Thus, the NOAEL in this study was found being >1000 mg/kg b.w./d for reproductive toxicity.

Executive summary:

The test item Tuball™ (Batch no. 01RW02.N1.381) was investigated for subacute toxicity and reproductive effects in an OECD 422 study in rats (CD / Crl:CD(SD).

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. A slight overdosing in a few samples was not of toxicological relevance as no toxic findings were noted in the study.

80 animals (40 males and 40 females) in order to grant at least 8 females per group for evaluation of F0 generation were used and distributed to 4 groups (group 1 control (vehicle), group 2 (low dose): 100 mg/kg b.w./day, group 3 (mid dose): 300 mg/kg b.w./day, and group 4 (high dose): 1000 mg/kg b.w./day) that were exposed orally via feed/diet on a daily basis.

Findings

General toxicity - Parental animals (males and females): No animal of the control group and the treatment groups (100, 300 or 1000 mg Tuball/kg b.w./day) died prematurely.

Clinical signs (males and females): No changes were noted in behaviour, the external appearance and the consistency of the faeces for the male and female animals. At 1000 mg Tuball/kg b.w./day black discoloured faeces were noted for all male and female animals, starting 4 days after the start of dosing and lasting until the end of the study. This was due to the high concentration of the administered test item (black powder) and was not considered to be of toxicological relevance.

Neurological screening (males and females): No test-item related effects were observed. Also the functional screening on grip strength and spontaneous motility revealed no effects.

Body weight and body weight gain for males and females was not affected in all dose groups and so was food and drinking water consumption. The mean actual intake of the test item via the diet for the whole study period was in the range of the nominal values:

group 2 (nominal 100 mg/kg b.w./d: actual 102 mg/kg b.w./d. for males and 117 mg/kg b.w./d females.

group 3 (nominal 300 mg/kg b.w./day): actual 305 mg/kg b.w./d. for males and 341 mg/kg b.w./d females

group 4 (nominal 1000 mg/kg b.w./day): actual 1026 mg/kg b.w./d. for males and 1195 mg/kg b.w./d females

No test-item related effects were seen in all dose groups for Haematology (TD 29), Clinical biochemistry (TD 29), and following examination at termination no test item-related observations were noted macroscopic post mortem, on organ weights or on T4 determination (at necropsy). Also histopathological examinations (control and high dose group) of males and females revealed no test item-related effects.

Regarding reproductive toxicity no influence was seen on oestrus cycle monitoring, fertility index, gestation index, pre-coital time or on gestation length.

Pups (pre- and postnatal development) investigated showed no test item-related influence on the birth index, the live birth index and the percentage of post-implantation loss. Mortality (Viability index) in pre- and post-cull period was not influenced, nor was pup body weight, ano-genital distance or count of male nipples (nipple retention). T4 determination (lactation day 13) showed no effects and neither did histopathological examination of the thyroid glands of the pups show and effects. External examination of pups also did not indicate malformations or variations during the macroscopic external examination of the pups at terminal sacrifice or the external examination of the pups that died prematurely.

Conclusion

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 b.w./day.

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 and 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 b.w./day for the male and 117, 341 and 1195 mg Tuball/kg b.w./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 b.w./day.

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 above 1000 mg Tuball/kg b.w./day via the diet

Reproductive toxicity:

a) adverse effects on the reproductive parameters of the parental females NOAEL above 1000 mg Tuball/kg b.w./day via the diet

b) adverse effects on pre- and postnatal development

- b1) adverse effects on prenatal development (conceptus to birth) NOAEL above 1000 mg Tuball/kg b.w./day via the diet

- b2) adverse effects on postnatal development (pup) NOAEL above 1000 mg Tuball/kg b.w./day via the diet

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Experimental exposure time per week (hours/week):

144

Species:

rat

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

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 to obtain information on possible effects of the test item on general toxicity, reproduction and/or development according to OECD guideline 422.

None of the animals died prematurely.

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.

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.

Justification for classification or non-classification

Based on absence of any adverse effect in a reproductive toxicity screen study according to OECD 422, the test substance is not required to be classified for reproductive toxicity according to CLP (Regulation EC No. 1272/2008).