Boron, (benzenemethanamine)trifluoro-, (T-4)-, reaction products with Bu glycidyl ether

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-01-16 to 2019-03-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 29, 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Boron, (benzenemethanamine)trifluoro-, (T-4)-, reaction products with Bu glycidyl ether

EC Number:

270-821-9

EC Name:

Boron, (benzenemethanamine)trifluoro-, (T-4)-, reaction products with Bu glycidyl ether

Cas Number:

68478-46-6

Molecular formula:

C14H23BF3NO2

IUPAC Name:

1-phenylmethanamine; 7-[(benzylamino)methyl]-10-(butoxymethyl)-5,8,11,15-tetraoxaicosan-13-ol; benzyl(3-butoxy-2-hydroxypropyl)amine; benzyl[3-butoxy-2-(3-butoxy-2-hydroxypropoxy)propyl]amine; dibenzyl(3-butoxy-2-hydroxypropyl)amine

Test material form:

liquid: viscous

Details on test material:

Boron, (benzenemethanamine)trifluoro-, (T-4)-, reaction products with Bu glycidyl ether, Batch: NC18201915

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

The rat is the common species used for the OECD 422 study.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Breeder Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany

Strain: Sprague-Dawley CD®/ Crl:CD (SD)

Body weight (at 1st administration, TD15)
Males: 420.2 g - 482.7 g
Females: 211.6 g - 260.4 g

Age (at 1st administration)
Males: 77 days
Females: 77 days

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 estrus 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

ENVIRONMENTAL CONDITIONS
Animals were kept singly in MAKROLON cages with exception of the mating period
Temperature: 22°C ± 3°C (maximum range)
Relative humidity: 55% ± 10% (maximum range)
Dark/Light period 12 hours each per day
Ventilation rate between fifteen to twenty air changes per hour
Diet: ssniff® R/Z V1324
Food and Water ad libitum

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Tap water

Details on exposure:

The test item was diluted in the vehicle (tap water) to provide dose concentrations of 3, 10 or 30/40 mg/mL (administration volume was 10 ml/kgbw/d). The test item was administered orally at a constant volume once daily. The test item formulations were freshly prepared every day and were adjusted to the animal's actual body weight daily. The control animals received the vehicle at the same administration volume daily in the same way.

Details on mating procedure:

Sexually mature male and female rats were randomly 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.


Sex Period Test days
Males + Females Pre-mating 15 to 29 (pairing was in the evening of test day 29)
Mating 30 to 35 #1 (first mating day, confirmed by positive sperm detection,
was in the morning of test day 30)
Males Post-mating until test day 43 (one day before necropsy on test day 44)
Females Gestation and lactation period until test days 64 to 70 #2 (corresponding to lactation days 13#3 to 15#3).
The last dosing was always one day before necropsy (necropsy was between test days 65 to 71).
#1: Test days on which a positive sperm detection was noted (mating day or gestation day 0), see Table B 'Overview of Female Test Days - Individual Data; column: Mating (Test Day)'.
#2: The removal days of the female animals in relation to the start date of the study are given in Table B 'Overview of Female Test Days - Individual Data'.
#3:The removal days of the female animals in relation to lactation day 1 are given in Table B 'Overview of Female Test Days - Removal Days Related to Lactation Day 1'.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For the analysis of the test item-vehicle formulations, samples of 10 mL were taken at the following times and stored at ≤-20°C ± 10% until analysis: At start of the treatment period, at any dose change and towards the end of the treatment period. Samples have been collected immediatly after preparation, after 8 & 24 h of storage at RT. The samples were labelled with the study number, species, type of sample, concentration, test day, sampling time and date.
An analytical method was validated at LPT for the detection of Boron,… using HPLC-UV detection.
The following parameters were determined during the validation:
- Linearity
- Accuracy
- Precision
- Sensitivity
- Specificity
- Stability

Duration of treatment / exposure:

Males were exposed for 43 days and females until test days 64 - 70 (corresponding to lactation days 13 to 15). The last dosing was one day before necropsy (necropsy was between test days 65 to 71).

Frequency of treatment:

once daily

Details on study schedule:

During the 14-day pre-exposure period (TD 1 to TD 14), the oestrus cycle of the female animals was monitored to gain at least 40 animals with a normal oestrus cycle. Animals that failed to exhibit typical 4 to 5 day cycles were excluded from the randomisation process on test day 14.


Groups of 10 male and 10 female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable).
- On Day 15, all animals were paired on a 1 male:1 female basis within each dose group until sperm or vagial plug
- Following evidence of mating, the males were returned to their original cages and females were transferred to individual cages.
- Pregnant females were allowed to give birth and maintain their offspring
- at Day 4 post partum the litter size, litter weight, pup sex, amount of live births, runts, gross abnormalities and the angogenital distance
- Following completion of the female gestation and lactation phases, the male dose groups were killed and examined macroscopically on Day 44.
- At Day 13 post partum, all females and offspring were killed and examined macroscopically.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control group

Doses / concentrations 2

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

low dose group

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

medium dose group

Doses / concentrations 4

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

high dose group; dose increased on test day 28 to 400

Doses / concentrations 5

Dose / conc.:

400 mg/kg bw/day (nominal)

Remarks:

Highest dose increased on test day 28

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
The dose levels were chosen based on the results of a preliminary dose range finding study. The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test material, and the results of the study are believed to be of value in predicting the likely toxicity of the test material to man and to screen for potential adverse effects on reproduction.

Positive control:

not required

Examinations

Parental animals: Observations and examinations:

CAGE SIDE 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.
These observations 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 between approx. 2.0 hours and 4.0 hours after dosing and before any blood sampling for laboratory examinations: Males on test day 42; females between lactation days 10 to 12.

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.
An overview of the observational screen components is given in the table on the following page.

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.




BODY WEIGHT:
The adult animals were weighed on each day of dosing for dose adjustment and at sacrifice; 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 were determined on the following days:
Test days 15, 22, 29. Gestation days 0, 7, 14, 20 and on lacatation days 1, 4, 8 and 13.


FOOD CONSUMPTION AND WATER INTAKE (if feeding study):
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 using the following formula:
Relative food consumption Total food given (g) - Total food left (g)
(g/kg b.w./day) =
Number of animal days# x Body weight (kg)

Drinking water consumption was monitored daily by visual appraisal throughout the study.

Oestrous cyclicity (parental animals):

During the 14-day pre-exposure period (TD 1 to TD 14), the estrus cycle of the female animals was monitored to yield study groups of at least 10 animals each with a normal estrus cycle. Animals that failed to exhibit typical 4 to 5 day cycles were excluded from the randomization process on test day 14 that was performed to allocate the female animals to the test groups of the main study.
Most of the female animals that were used for the main study revealed 2 or 3 typical 4 to 5 day cycles during the 14-day pre-exposure period.
After the allocation of the animals to the study groups and the start of treatment, the estrus cycle was further monitored during the pre-mating period and the mating period until one day before a positive mating sign was noted.
Finally, a vaginal smear was taken in the morning of the day of scheduled necropsy.

Sperm parameters (parental animals):

Organ weights and histopathology see below.

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.
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.
-Male nipples counting
Nipples were counted in all male pups on PND 13 (shortly before scheduled sacrifice).

Postmortem examinations (parental animals):

SACRIFICE
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

LABORATORY EXAMINATIONS
Blood samples were taken from the retrobulbar venous plexus under isoflurane anaesthesia from animals fasted overnight at the following times:
At study termination (before necropsy) 5 male and 5 female F0 animals randomly selected from each group.
HAEMATOLOGY: All relevant parameters were deterimed.
CLILICAL BIOCHEMISTRY: All relevant parameters were determined.
THYROID HORMONE DETERMINATION:
Blood samples were taken under isoflurane anaesthesia from animals fasted overnight always at the same time of day (in the morning between 7.30 a.m. and 9.30 a.m. for the adult animals) as scheduled below.
Blood withdrawal was performed by randomization of the parental male and female animals. The male animals of all test groups were completely randomized in a one- step process, the female animals in different staggers according to their litter day.
Determination of the pups used for blood withdrawal:
On lactation day 4 (PND4) and lactation day 13 (PND13) the litter sequence of pup blood withdrawal was determined by randomization of the dams. The collection of the pups for blood withdrawal from the individual litters occurred in an ascending order (the male and female pups per dam with the lowest number were used, if possible).


GROSS NECROPSY
Examination of the pups
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.
On 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 dam nos. 20 and 55 were taken. The same pups were used for T4 ELISA sampling.

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.

ORGAN WEIGHTS
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, Spleen, Brain, Testicle, Epididymis, Thyroid, Heart, Thymus, Kidney, As a whole: prostate, seminal vesicles, Liver with coagulating glands, Ovary, Uterus including cervix
The weights of the organs were determined before fixation. Only the weight of the thyroid glands was determined after fixation.


HISTOPATHOLOGY
Samples of the following tissues were preserved from five males and five females from each dose group, in buffered 10% formalin. The tissues shown in bold were also removed from the remaining animals:
Adrenals
Aorta (thoracic)
Bone & bone marrow (femur including stifle joint)
Bone & bone marrow (sternum)
Brain (including cerebrum, cerebellum and pons)
Caecum
Coagulating gland *
Colon
Duodenum
Epididymides *
Eyes
Gross lesions
Heart
Ileum
Jejunum
Kidneys
Liver
Lungs (with bronchi)
Lymph nodes (cervical and mesenteric)
Mammary gland
Muscle (skeletal)
Ovaries *
Pancreas
Pituitary *
Prostate *
Oesophagus
Rectum
Salivary glands (submaxillary)
Sciatic nerve
Seminal vesicles *
Skin (hind limb)
Spinal cord (cervical)
Spleen
Stomach
Thyroidlparathyroid
Trachea
Testes *
Thymus
Urinary bladder
UterusICewix
Vagina
Full histopathology was performed on the preserved organs of the selected parental animals of groups 1 and 4, and the thyroids of the selected pups.
Due to test item-related findings in the target organs of the high dosed animals, HE-stained paraffin sections of the stomach and kidneys of the selected main study animals of the low and the intermediate dose level group (groups 2 and 3, n = 20) were examined histopathologically.
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.

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.
On 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 dam nos. 20 and 55 were taken. Additionall, due to the death of the pups of dams no. 76 and 75 on blood could be taken. at day 13.
The same pups were used for T4 ELISA sampling.

Statistics:

Parametrical data: Provantis (Provantis® integrated preclinical software, version 10.2.1, Instem LSS Ltd)
settings: Homogeneity of variances and normality of distribution with BARTLETT’s and SHAPIRO-WILK’s test
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: FISHER or the Chi2 test with
- Provantis: Statistical evaluation of histopathology findings (see histopathology report; see Appendix 5 'Histopathology Phase Report')
- StatXact 4.0.1:Statistical evaluation of the fertility index, the gestation index, the birth and live birth index, the viability indices and the post-implantation loss (group indices)

Reproductive indices:

per group: Femal Fertility Index (%)
Gestation Index (%)

Offspring viability indices:

for each litter and group:
Birth index (%)
Live Birth Index (%)
Viability Indeex (%) per- and post-cull
Post-implantation loss

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Male animals: No changes in behaviour, the external appearance or the appearance of the faeces were noted of the control group and the treatment groups (30, 100 or 300/400 mg/kg b.w./day).

Femal animals: No changes in behaviour, the external appearance or the appearance of the faeces were noted for the control group and the female animals of the low and the intermediate dose group (30 or 100 mg /kg b.w./day). At the high dose level (300/400 mg /kg b.w./day) one (female no. 76) of 9 females that littered live pups revealed piloerection during nearly the whole lactation period (on lactation days 1 to 3 (test days 56 - 58) and from lactation days 5 to 14 (test days 60 - 69)). This observation is considered to be test item-related and can be correlated with the premature death of all pups from dam no. 76 during the early phase of the lactation period.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Male rats: 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 (30, 100 or 300/400 mg Boron,.../kg b.w./day).
At the high dose level a slightly and statistically not significantly reduced body weight was noted on test day 43 (4.7% below the value of the control group). However, this slight reduction was considered to be in the range of normal variability and not to be test item-related. In accordance with the slightly reduced body weight on test day 43 that was noted at the high dose level, the percentage of body weight gain was also slightly decreased in the high dose group in comparison to the control group and the low and the intermediate dose group. As mentioned in the section above, this observation was not considered to be test item-related.

Female: 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 low and the intermediate dose group (30 or 100 mg Boron,.../kg b.w./day) during the pre-mating, the gestation and the lactation period.
At the high dose level (300/400 mg Boron,.../kg b.w./day) a slightly reduced body weight was noted at the end of the gestation period,and during the lactation period (maternal toxicity). In detail, the body weight of the high dosed animals was 6.2% below the control group on gestation day 20 (test days 50 - 55) (statistically not significant). During the lactation period the body weight of the high dosed animals was 3.5% (lactation day 1), 9.9% (lactation day 4), (7.0% lactation day 8) and 6.7% (lactation day 13) below the control value, statistically significant on lactation day 4 (p ≤ 0.01). In accordance with the slightly reduced body weights that were noted at the high dose level at the end of the gestation period and during the lactation period, body weight gain for these periods was slightly reduced at the high dose level in comparison to the control group.
The reductions in body weight and body weight gain that were noted at the high dose level (300/400 mg Boron,.../kg b.w./day) were considered to be test item related (maternal toxicity).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Male: During the pre-mating period (test days 15 to 29) no test item-related difference in food consumption was noted between the male rats of the control group and the male rats of the treatment groups (30, 100 or 300/400 mg/kg b.w./day).

Female: 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 (30, 100 or 300/400 mg/kg b.w./day) during the pre-mating, the gestation and the lactation period.
However, a slightly reduced food consumption was noted at the high dose level during the first week of gestation (8.3% below the control; p ≤ 0.01) and during the lactation period (13.6% and 11.5%, respectivly first and the second lactation week; statistically not significant). However, dams no. 75 and 76 lost all pups of their litters early in the lactation phase and therefore did no longer have to produce milk to feed their offspring. It is reasonable to assume that this causes a reduction in food consumption compared to other dams that still have suckling pups. Furthermore, for dam no. 76 piloerection was noted on almost all days of the lactation period, indicating animal stress and / or discomfort that might also cause reduced food consumption. Accordingly, after exclusion of dams no. 75 and 76 the mean value for food consumption of the high dose group is no longer different from the other treatment groups and the control (1.8% below and 0.4% above the control value during the first and the second lactation week). Thus, while the reduced food consumption observed in dams no. 75 and 76 might be a secondary effect of the reduced viability of their offspring, it is no adverse effect of the test item in general.

Food efficiency:

not examined

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 30, 100 or 300/400 mg/kg b.w./day by visual appraisal.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No test item-related differences for the examined haematological parameters were noted between the control group and the treatment groups (30, 100 or 300/400 mg/kg b.w./day).
Slightly but statistically significantly decreased values were noted for the male rats in the red blood cell concentration at the intermediate and the high dose level. Simelar oberservations have been noticed for the MCH and MCHC concentrations of the female rats (statistically significantly). However, these slight changes in males and females were considered to be spontaneous.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No test item-related differences for the examined biochemical parameters were noted between the control group and the treatment groups (30, 100 or 300/400 mg Boron,.../kg b.w./day).
Slightly but statistically significantly decreased values were noted for the protein (total), albumin and chloride concentrations, as well as the aP activity in the male rats. However, these changes were not considered to be test item-related.

No test item-related changes were noted between the control group and the treatment groups (30, 100 or 300/400 mg/kg b.w./day) for the T4 levels of the parental males.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No test item-related observations of abnormal behaviour, no adverse effects on motoric skills, or changes in the appearance of the faeces were noted for the male and female animals of all treatment groups (30, 100 or 300/400 mg/kg b.w./day), approx. 2 hours to 4 hours after treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Test item-related changes were noted for the kidneys and the stomach of the male and the female animals of the high dose group (300/400 mg/kg b.w./day).

Kidneys: In 3/5 group 4 males (300/400 mg /kg b.w./day) basophilic tubules were observed in the renal cortex (unilateral and bilateral) versus 1/5 in the control group.
In females, there were basophilic tubules in the renal cortex in 4/6 group 4 (300/400 mg /kg b.w./day) animals versus 0/5 in the control group. In addition, in females only, there was bilateral tubule degeneration observed of proximal and distal tubules in 3/6 group 4 females versus 1/5 in the control group.

Stomach: The submucosa of the glandular stomach had statistically significant neutrophil infiltrate in 4/5 male and 5/6 females of the high dose group, whereas no infiltration of neutrophilic granulocytes was noted in the stomach of the male and female animals of the control group.

Due to the histopathological effects in the high dose group, additional examinations of group 2 and 3 animals of the kidneys and the stomach were performed.
Test item-related changes in the form of basophilic tubules in the kidneys were noted for the male animals of the intermediate dose group (100 mg /kg b.w./day).
No test item-related changes were noted for the kidneys of the female animals of the intermediate dose group and the male and female animals of the low dose group.
Furthermore, no test item-related changes were noted for the stomach of the male and female animals of the low and the intermediate dose groups.

Results of a detailed histopathological analysis in a peer review by Dr. Weber from Anapath:
The report from Dr. Weber (histopathologist at Anapath) was attached to the experimental study report. Dr. Weber re-assessed the histopathological effects in the stomach and the kidney of the low to high dosed animals. Findings in the stomach in animals at 100 and 300/400 mg/g b.w./day, consisting of an increased inflammatory infiltrate (mainly granulocytes) in the sub-/mucosa of the glandular stomach, as well as one cases of forestomach inflammation at 300/400 mg/kg b.w./day are deemed to be test item-related, and are indicative for a local irritative potential, secondary to corrosive properties of the test item.

Findings in the kidneys consisted of tubular basophilia associated in a few cases with inflammatory cell infiltrated and minor infarction (cortical scarring), as well as in one case by hyaline tubular casts. In most cases, the findings were unilaterally present only that is not indicating systemic toxicity. Tubular basophilia was noted throughout all groups and was noted at minor severity only.
Tubular basophilia was, in almost all cases unilaterally, but in most cases of a minimal degree.
Therefore, the tubular basophilia is deemed to be indicative for the early onset of chronic progressive nephropathy (CPN), which is a spontaneous background lesion in rodents.

The local NOAEL is deemed to be 30 mg/kg b.w./day due to the gastric lesions. Renal lesions were not considered to be related to treatment with the test item. Therefore, based on pathology evaluation, a systemic NOAEL is established at 300/400 mg/kg b.w./day.


Reproductive organs: No test item-related microscopic changes were observed for the reproductive organs of males and females of the high dose group (300/400 mg /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 did not reveal any test item-related effects.
The mammary glands of the observed female animals showed prominent mammary development in 5/5 control group animals, whereas in group 4 females only 3/6 animals had marked mammary development, with 2 animals showing minimal mammary development in this group (nos. 76 and 77).
In addition, also the oestrus stages that were noted during the histopathological examination of the vagina revealed differences between the control group and the high dose group (5/5 metoestrus in control versus 4/6 metoestrus in group 4 with dioestrus in no. 76 and oestrus in no. 77).
However, the differences that were noted in mammary development and for the oestrus stages for animal nos. 76 and 77 in comparison to the other examined females of the high dose group were due to the non-pregnancy status of no. 77

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No test item-related differences were noted for the mean length and the mean number of oestrus 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 (30, 100 or 300/400 mg/kg b.w./day). I

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):

Female fertility:
No test item-related influence on the fertility index of the female rats was noted for any of the treatment groups (30, 100 or 300/400 mg /kg b.w./day).
In the control group and in the low and the intermediate dose group 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%.
At the high dose level 9 of 10 females that were paired with their male partner became pregnant, leading to a female fertility index of 90%.
The occurrence of one non-pregnant female (no. 77) at the high dose level was considered as spontaneous, due to a positive sperm detectionwhich was received on mating day 2 for female no. 77.

Gestation:
No test item-related influence on the gestation index, pre-coital time or gestation length was noted for the female rats of the treatment groups (30, 100 or 300/400 mg /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.

Birth indices and pots-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 (30, 100 or 300/400 mg /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 (30, 100 or 300/400 mg /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) is nearly equal between the control group (1.8 per dam) and the high dose group (1.6 per dam).
A slightly increased birth index and correlating a slightly decreased post-implantation loss in comparison to the control group were noted at the intermediate dose level (statistically significant at the group level), indicating a reduced number of resorptions and / or stillbirths at the intermediate dose group in comparison to the control group. As such an observation is not of toxicological relevance the observed statistically significant changes that were noted at the intermediate dose level for the birth index and the post-implantation loss were considered to be spontaneous.

Details on results (P0)

None of the animals died prematurely.
In the male rats no changes were noted in behaviour, the external appearance and the appearance of the faeces. Piloerection was noted for one dam of the high dose group (300/400 mg /kg b.w./day) during nearly the whole lactation period (between test days 56 to 69), whereas no changes in behaviour, the external appearance and the appearance of the faeces were noted for the male animals.
A slightly reduced body weight was noted for the female animals of the high dose group (300/400 mg /kg b.w./day) at the end of the gestation period and during the whole lactation period. No test item-related influence on body weight was noted for the male animals.
No test item-related influence was noted for the male and female animals 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 examination and for the relative and absolute organ weights.
At the high dose level (300/400 mg /kg b.w./day) the microscopic examination revealed test item-related changes in the kidneys in the form of basophilic tubules (both sexes) and tubule degeneration (females only) as well as in the stomach in the form of infiltration of neutrophilic granulocytes in the submucosa of the glandular part (both sexes).
The additional examination of the kidneys and the stomach of male and female animals of the low and intermediate dose level (30 or 100 mg Boron,.../kg b.w./day) revealed test item-related kidney changes in the form of basophilic tubules in males treated with 100 mg Boron,.../kg b.w./day.
No test ite-related changes were observed for oestrus cyle, fertility index, gestation index, pre-coital time and the gestation length.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

3

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive performance

Effect level:

> 300 - < 400 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

No difference between the control group and the low and the intermediate dose group (30 or 100 mg /kg b.w./day) was noted for the viability indices between lactation days 0/1 and 4 (pre-cull) and lactation days 5 and 13 (post-cull).
At the high dose level (300/400 mg /kg b.w./day) a statistically significant reduced viability index was noted during the pre-cull period and not significant during the post-cull period. This was mainly due to 2 of 9 high dosed dams with a total loss of all their pups (nos. 75 and 76). In detail, all pups of dam no. 76 died during the pre-cull period, whereas the pups from dam no. 75 died during the pre-cull period and at the beginning of the post-cull period. The reduced pup viability can be considered as a secondary effect due to the maternal toxicity (dam no. 76 revealed piloerection during the whole lactation period and dam no. 75 a reduced body weight gain at the beginning of the lactation period).
For the remaining 7 high dosed dams the number of prematurely deceased pups (1 and 2 prematurely deceased pups for no. 74 and no. 80) were in the range of the number of prematurely deceased pups that were noted for the dams of the control group and the low and the intermediate dose group..
During the later phase of the lactation period (>day 6), only one prematurely deceased pup was still noted at the high dose level (no. 80-14 on lactation day 10). Hence, during the later period of the lactation period (lactation week 2) an adverse effect of the test item on pup survival is no longer recognisable.

Body weight and weight changes:

effects observed, non-treatment-related

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 low and the intermediate dose group (30 or 100 mg /kg b.w./day) on lactation days 1, 4 and 13.
Slight but statistically not significant reductions in pup body weight were noted for the male and female pups of the high dose group (300/400 mg /kg b.w./day) on lactation days 1, 4 and 13, which are also below the background values of the test facility. Nevertheless, the reduced values on lactation days 1 and 4 (6.5% and 7.4% below the control) were due to the low pup body weights of dam nos. 75 (all pups deceased until lactation day 2) and 76 (all pups deceased until lactation day 6). Excluding dam nos. 75 and 76, no difference between the high dose group and the control group was noted for the remaining 7 high dosed dams with live pups on lactation days 1 and 4.
However, the remaining 7 high dosed dams showed a slightly reduced pup body weight on lactation day 13 (8.4% below the control value).

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No test item-related differences betweem the control group and the treatment groups (30, 100 or 300/400 mg /kg b.w./day) were noted for the T4 levels of the male and female pups at day 13.

Urinalysis findings:

not examined

Sexual maturation:

not examined

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 (30, 100 or 300/400 mg /kg b.w./day).

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No test item-related difference in the number of nipples was noted between the male pups of the control group and in the male pups of the treatment groups (30, 100 or 300/400 mg /kg b.w./day).

Organ weight findings including organ / body weight ratios:

not examined

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 30, 100 or 300/400 mg /kg b.w./day after terminal sacrifice on lactation day 13 or for the pups that died (stillbirth or found dead) during the lactation period.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

male to female ratio:
No test item-related influence on the male to female ratio was noted for all treatment groups (30, 100 or 300/400 mg/kg b.w./day).
However, a male to female ratio that markedly differed from the expected value (around 1.0) was noted at the intermediate dose group (0.75). However, as no dose-response relationship was noted, the reduced male to female ratio that was noted at the intermediate dose group was not considered to be test item-related but spontaneous.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

No adverse effect was noted on the prenatal development of the pups (birth and live birth index, percentage of post implantation loss).
At 300/400 mg /kg b.w./day an adverse effect was noted on the postnatal development of the pups in form of a reduced viability (due to dams no. 75 and 76 with total litter loss) during the first lactation week and a slightly reduced pup body weight as a consequence of the maternal effects (e.g. reduced body weight). However, the reduced pup viability can be considered as a secondary effect of the maternal toxicity (dam no. 76 revealed piloerection during the whole lactation period and damn no. 75 a reduced body weight gain at the beginning of the lactation period)
No influence was noted on the ano-genital distance and the number of nipples per male pup.

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 300 - < 400 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: prenatal development

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 300 - < 400 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

The oral administration of test item to rats by gavage at a maximum dose level of 300/400 mg/kg/day resulted in a reduced body weight (statistically significant only on lactadion day 4) . Histopathological changes have been observed for kidney and the stomach in the intermediate and high dose. The inflammaton of the stomach was induced by the corrosive propeties of the test substance and these effects are local effects. The effects in the kidneys are considered to be an early onset of chronic progressive nephropathy (CPN), which is a spontaneous background lesion in rodents. Piloerection was noted for one dam of the high dose group (300/400 mg /kg b.w./day) during nearly the whole lactation period (between test days 56 to 69), whereas no changes in behaviour, the external appearance and the appearance of the faeces were noted for the male animals.
A slightly reduced body weight was noted for the female animals of the high dose group (300/400 mg /kg b.w./day) at the end of the gestation period and during the whole lactation period.
The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was therefore considered to be 100 mg/kg/d.

At 300/400 mg /kg b.w./day an adverse effect was noted on the postnatal development of the pups in form of a reduced viability during the first lactation week and a slightly reduced pup body weight as a consequence of the maternal effects (reduced body weight during lactation).
The NOAEL for reproductive toxicity was therefore, also considered to be >300/400 mg/kg/d (prenatal and postnatal development).

Executive summary:

 

Introduction.The study was designed to investigate the systemic toxicity and potential adverse effects on reproduction (including offspring development) of the test material and complies with the recommendations of the OECD Guidelines for Testing of Chemicals No. 422 "Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test" (adopted July 29 2016).

 

Methods.The test material was administered by gavage to three groups each of ten male and ten female Sprague-Dawley Crl.:CD (SD) strain rats, for up day 15 until one day before sacrifice, at dose levels of 30, 100 and 300/400 mg/kg/day. A control group of ten males and ten females was dosed with vehicle alone (tap water).Clinical signs, behavioural assessments, bodyweight development, food and water consumption were monitored during the study.Pairing of animals within each dose group was undertaken on a one male to one female basis on Day 15 of the study, to produce litters.During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of developmental landmarks.Functional observations were performed on five selected parental males from each dose group after the completion of the mating phase, and for five selected parental females from each dose group during postpartum.Males were terminated on Day 44, followed by the termination of all surviving females on Day 14 -16 (relative to littering) and offspring on Day 13 postpartum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed. On these days blood samples have been take as well as organs from the adult animals.

 

 

 

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 Boron, (benzenemethanamine)trifluoro-, (T-4)-, reaction products with BU glycidyl ether was administered orally to rats at dose levels of 30, 100 or 300 mg Test item/kg b.w./day.

As no signs of toxicity were observed, the high dose level was increased to 400 mg Test item/kg b.w./day, starting on test day 28.

General toxicity

Parental male and female animals

None of the animals died prematurely.

Piloerection was noted for one dam of the high dose group (300/400 mg Test item/kg b.w./day) during nearly the whole lactation period (between test days 56 to 69). Whereas no changes in behaviour, the external appearance and the appearance of the faeces were noted for the male animals.

A slightly reduced body weight was noted for the female animals of the high dose group (300/400 mg Test item/kg b.w./day) at the end of the gestation period (not significant) and during the whole lactation period (significant at LD 4). No test item-related influence on body weight was noted for the male animals.

No test item-related influence was noted for the male and female animals on the results of the neurological screening, the haematological and biochemical parameters and the T4 level of the male animals. However, changes in the biochemical values of albumin, total protein and chloride were observed. These were considered secondary to maldigestion due to stomach inflammation. Similar was thought for the decrease of red blood cells, this was also considered secondary to stomach inflammation.

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

Histopathology revealed local changes in the stomach at dose levels of 100 and 300/400 mg/g b.w./day in form of an increased inflammatory infiltrate (mainly granulocytes) in the sub-/mucosa of the glandular stomach, as well as one case of forestomach inflammation at 300/400 mg/kg b.w./day which are deemed to be test item-related, and are indicative for a local irritative potential, secondary to corrosive properties of the test item. Additionally, rodent strain-related changes in the kidneys in form of tubular basophilia (both sexes) and tubule regeneration (females only) were noted in all groups.

Reproductive toxicity

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.

Pups

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

At 300/400 mg Test item/kg b.w./day an adverse effect was noted on the postnatal development of the pups in form of a reduced viability (due to dams no. 75 and 76 with total litter loss) during the first lactation week. Secondly, a slightly reduced pup body weight as a consequence of the maternal effects (e.g. reduced body weight) was noticed.

However, the reduced pup viability can be considered as a secondary effect of the maternal toxicity (dam no. 76 revealed piloerection during the whole lactation period and dam no. 75 a reduced body weight gain at the beginning of the lactation period).

No influence was noted on the ano-genital distance and the number of nipples per male pup.

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

 

General toxicity

NOAEL dams for systemic toxicity: 100 mg test material/kg b.w./day, p.o.

Based on: body weight, body weight gain and piloerection in females.

 

NOAEL males for systemic toxicity: above 300/400 mg test material/kg b.w./day, p.o.

 

NOAEL local: The local NOAEL is deemed to be 30 mg test material/kg b.w./day, p.o. due to the gastric lesions.

 

Reproductive toxicity

a) adverse effects on the reproductive parameters of the parental females
NOAEL
above 300/400 mg Boron, (benzenemethanamine)trifluoro-, (T-4)-, reaction products with BU glycidyl ether/kg b.w./day, p.o.

b) adverse effects on pre- and postnatal development
- b1) adverse effects on prenatal development (conceptus to birth)
NOAEL
above 300/400 mg Boron, (benzenemethanamine)trifluoro-, (T-4)-, reaction products with BU glycidyl ether/kg b.w./day, p.o.

- b2) adverse effects on postnatal development (pup)
NOAEL
above 300/400 mg Boron, (benzenemethanamine)trifluoro-, (T-4)-, reaction products with BU glycidyl ether/kg b.w./day, p.o.

 

 

 

 

 

 

 Findings

General toxicity - Parental animals
Mortality	Males and females No animal of the control group and the treatment groups (30, 100 or 300/400 mg Test item/kg b.w./day) died prematurely.
Clinical signs	Males No changes were noted in behaviour, the external appearance and the appearance of the faeces. Females At 300/400 mg Test item/kg b.w./day piloerection  was noted for 1 of 9 dams during nearly the whole lactation period (between test days 56 and 69) (sign of maternal toxicity).
Neurological screening
Observational screening	Males and females No test item-related effects were noted.
Functional screening
Grip strength	Males and females No test item-related influence was noted.
Spontaneous motility	Males and females No test item-related differences were noted.

 

Body weight and body weight gain	Males No test item-related differences were noted. Females At 300/400 mg Test item/kg b.w./day a slightly reduced body weight was noted on gestation day 20 (test days 50 - 55) (6.2% below the control) and during the lactation period (3.5%, 9.9%, 7.0% and 6.7% below the control on lactation days 1, 4, 8 and 13) (sign of maternal toxicity). A statistically significant difference was only noted on lactation day 4 with p ≤ 0.01. Accordingly, body weight gain at 300/400 mg Test item/kg b.w./day was slightly reduced in comparison to the control group during the gestation period (52.2% in comparison to 62.4% in the control group) and during the lactation period (11.8% in comparison to 15.8% in the control group) (signs of maternal toxicity).
Food consumption		Males and females No test-item related changes were noted.
Drinking water consumption		Males and females No test-item related changes were noted.
Haematology (at necropsy)		Males and females No test-item related changes were noted.
Clinical biochemistry (at necropsy)		Males and females No test-item related changes were noted.
Examination at termination
Macroscopic post mortem findings		Males and females No test item-related observations were noted.
Organ weights		Males and females No test item-related differences were noted.
T4 determination (at necropsy)		Males and females No test item-related differences were noted.
Histopathological examinations (control and high dose group) and additional examination of kidneys and stomach (groups 2 and 3) after retrospective histopatho-logical review		Males and females Histopathology revealed local changes in the stomach at dose levels of 100 and 300/400 mg/g b.w./day which are deemed to be test item-related and rodent strain-related changes in the kidneys in all groups. Findings in the stomach in animals at 100 and 300/400 mg/g b.w./day, consisting of an increased inflammatory infiltrate (mainly granulocytes) in the sub-/mucosa of the glandular stomach, as well as one case of forestomach inflammation at 300/400 mg/kg b.w./day are deemed to be test item-related, and are indicative for a local irritative potential, secondary to corrosive properties of the test item. Findings in the kidneys consisted of tubular basophilia associated in a few cases with inflammatory cell infiltrates and minor infarction (cortical scarring), as well as in one case by hyaline tubular casts. In most cases, the findings were unilaterally present only that is not indicating systemic toxicity. Tubular basophilia was noted throughout all groups and was noted at minor severity only. Tubular basophilia was in almost all cases unilateral, but in most cases of a minimal degree. Therefore, the tubular basophilia is deemed to be indicative for the early onset of chronic progressive nephropathy (CPN), which is a spontaneous background lesion in rodents.

 

Target organ lesions after review

Target organ lesions after review
Group	Sex	Kidneys				Stomach
		Tubular basophilia	Hyaline cast	Infiltrate	Infarction	Infiltrate	Forestomach inflammation
1	m	3 / 1.0	0	0	0	0	0
	f	1 / 1.0	0	0	0	0	0
2	m	5 / 1.0	0	1 / 1.0	1 / 1.0	1 / 1.0	0
	f	1 / 1.0	0	0	0	2 / 1.0	0
3	m	5 / 1.0	0	1 / 1.0	0	1 / 2.0	0
	f	1 / 1.0	0	0	0	1 / 1.0	0
4	m	3 / 1.0	1 / 1.0	2 / 1.0	1 / 1.0	4 / 1.8	1 / 2.0
	f	4 / 1.5	0	3 / 1.0	1 / 1.0	5 / 1.8	0

Numbers in tables indicate for each finding: Incidence / Mean Severity

m:      male

f:       female

 

Reproductive toxicity Reproductive Parameters of the Parental Females
Oestrus cycle monitoring	No test item-related influence was noted.
Fertility index	No test item-related influence was noted on the fertility index of the female animals.
Gestation index	No test item-related influence was noted.
Pre-coital time	No test item-related influence was noted.
Gestation length	No test item-related influence was noted.

Pups - Pre- and Postnatal Development
- Prenatal development (from conceptus to birth)
Reproductive parameters		No test item-related influence was noted on the birth index, the live birth index and the post-implantation loss.
- Postnatal development (pup)
Mortality (Viability index)		Pre-cull period (lactation days 0/1 - 4) At 300/400 mg Test item/kg b.w./day a reduced viability index (pre-cull) was noted (84.8% in comparison to 99.3% in the control group; statistically significant at p ≤ 0.01). Post-cull period (lactation days 4 - 13) At 300/400 mg Test item/kg b.w./day a reduced viability index (post-cull) was noted (84.4% in comparison to 93.0% in the control group; statistically not significant). The reduced viability indices at the high dose level were due to 2 high dosed females that lost all their pups during the first week of lactation (until and including lactation day 6), considered to be a consequence of the maternal effects. During the second lactation week, the number of prematurely deceased pups at the high dose level was in the range of the control group.
Pup body weight		Slight reductions on pup body weight were noted at the high dose level (300/400 mg Test item/kg b.w./day) on lactation days 1, 4 and 13 (6.5%, 7.4% and 8.4% below the control value; statistically not significant).
Ano-genital distance		No test item-related influence was noted.
Count of male nipples		No test item-related difference was noted.
T4 determination (lactation day 13)		No test item-related differences were noted.
Histopathological examination of the thyroid glands of the pups		No test item-related changes were noted.
External examination		The external macroscopic examination of the prematurely deceased pups and after scheduled sacrifice on lactation day 13 revealed no gross abnormalities.
Analysis of test item formulation	The measured concentrations of Boron, (benzenemethanamine)trifluoro-, (T-4)-, reaction products with BU glycidyl ether in the test item-vehicle mixtures were between 101.9% and 109.1% of the nominal concentration, indicating correctly prepared test item-vehicle formulations that were stable for at least 24 hours at room temperature.