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

Endpoint summary

Description of key information

The toxicity of the test substance was first analyzed in a 14 day Dose Range Finding Study based on OECD 422 guideline by oral administration to rats. The selected dose groups were 0, 15, 50, 150 mg/kg bw/d, however, because of the low toxicity of the substance the dose levels were increased to 300 mg/kg bw/d (intermediate dose) and 450 mg/kg bw/d (high dose).

 The clinical symptoms observed in the study were post dosing salivation from 50 mg/kg bw/d group onwards and piloerection and haemorrhagic canthi from 300 mg/kg bw/d onwards in males. In the higher dose groups also breathing sounds and piloerection could be observed (LPT, 2019). 

In the subsequently performed OCED 422 study with female Sprague-Dawley Crl.:CD (SD) strain rats, for up day 15 until one day before sacrifice, the dose levels were 0, 30, 300/400 mg/kg bw/d (Provivo 2022).

The administration resulted in a reduced body weight in females. 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). Histopathological changes have been observed for kidney and the stomach in the intermediat and high dose. The kidney effects are indicative for the early onset of chronic progressive nephropathy (CPN), which is a spontaneous background lesion in rodents and therefore the kidney effects are not tretament related. The inflammatory effects in the somach are caused by the corrosive properties of the test compound and are considered to be local effects.
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. The local NOEAL was considered to be 30 mg/kg bw/d.

 

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

repeated dose toxicity: oral, other

Remarks:

OECD 422 Dose Range Finding Study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

18-11-06 to 18-12-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

3 animals per dose group used, limited scope of examination

Justification for type of information:

This DRF toxicity study is used as dose range finding study for the OECD 422 main study (LPT, 2019).

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

adopted July 29, 2016

Deviations:

yes

Remarks:

Dose Range Finding Study, 3 animals per dose group

GLP compliance:

no

Remarks:

The study was performed according to SOPs which are in line with the 'Good Laboratory Practice' regulations.

Limit test:

no

Species:

rat

Strain:

other: CD/Crt:CD(SD)

Details on species / strain selection:

Species was selected because of its proven suitability in toxicology and reproduction studies and its compliance with regulatory requirements for testing in a rodent animal species.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Breeder: Charles River Laboratories Research Models and Services Germany GmbH
Number and sex of animals: 36 (18 male and 18 female) animals, 3 animals per sex and group
Additionally, 6 (3 male and 3 female) spare animals were available for possible replacement during the adaptation period

Body weight (at 1st dosing)
Males
Groups 1 to 4: 320.0 g - 350.6 g
Groups 5 and 6: 329.8 g - 383.8 g
Females
Groups 1 to 4: 207.8 g - 229.9 g
Groups 5 and 6: 224.5 g - 263.1 g

Age (at 1st dosing)
Males
Groups 1 to 4: 53 days
Groups 5 and 6: 62 days
Females
Groups 1 to 4: 53 days
Groups 5 and 6: 62 days

Identification of animals Each rat received a continuous number from 1 to 36. Points were set on paws and/or tail by tattoo; additionally, the animal cages were labelled with study number, animal number, sex and treatment group.

Adaptation period 5 or 8 days

ENVIRONMENTAL CONDITIONS
Animals were kept singly in MAKROLON cages (type III plus)
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

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

tap water

Details on oral exposure:

ADMINISTRATION:
Dose levels
Group 1: Control (vehicle)
Group 2: 15 mg/kg b.w./day
Group 3: 50 mg/kg b.w./day
Group 4: 150 mg/kg b.w./day
Group 5: 300 mg/kg b.w./day (add)
Group 6: 450 mg/kg b.w./day (add)

Route of administration Oral, by gavage

Frequency of administration: Once daily for 14 days

Administration volume 10 mL/kg b.w./day

Duration of study - 5 or 8 adaptation days - 14 dosing days

DOSAGE PREPARATION:
The test item formulations were freshly prepared on every administration day.
The test item was diluted in the vehicle (groups 2 to 4) or suspended in the vehicle (groups 5 and 6) to the appropriate concentrations.
The amount of the test item was adjusted to the animal's current body weight on each administration day.

Analytical verification of doses or concentrations:

no

Remarks:

Tests by appropriate analytical methods will be carried out for the main study

Details on analytical verification of doses or concentrations:

The test item formulations were freshly prepared on every administration day.
The test item was diluted in the vehicle (groups 2 to 4) or suspended in the vehicle (groups 5 and 6) to the appropriate concentrations.
The amount of the test item was adjusted to the animal's current body weight on each administration day.

Duration of treatment / exposure:

14 days

Frequency of treatment:

Once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control (vehicle)

Dose / conc.:

15 mg/kg bw/day (nominal)

Remarks:

low dose

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

intermediate dose

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

high dose

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

additional dose

Dose / conc.:

450 mg/kg bw/day (nominal)

Remarks:

additional dose

No. of animals per sex per dose:

36 (18 male and 18 female) animals,
3 animals per sex and group
Additionally, 6 (3 male and 3 female) spare animals were available for possible replacement during the adaptation period.

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were selected in agreement with the Sponsor based on available data. In an acute toxicity study the LD50 was determined to be 750 mg Boron/kg b.w.
The dose level groups 5 and 6 treated with 300 or 450 mg Boron/kg b.w./day were added at a later time point as no toxicity was observed in groups 2 to 4.

Positive control:

no

Observations and examinations performed and frequency:

OBSERVATIONS
Dated and signed records of all activities related 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 appropriate documentation. In addition, observations related to individual animals
were made throughout the study and recorded.
- Clinical signs
Each animal was observed before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment or illness.
In addition, the animals were checked regularly throughout the working day from 7.00 a.m. to 3.45 p.m. by cageside observations. On Saturdays and Sundays, the
animals were checked regularly from 7.00 a.m. to 11.00 noon with a final check performed at approximately 3.30 p.m.
Cageside 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. Dated and signed records of appearance, change and disappearance of clinical
signs of each animal were maintained on clinical history sheets.
- 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 would have 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. The post mortem examination was performed as described in section 'PATHOLOGY'.
- Body weight
The body weight of each rat was recorded at the time of group allocation and daily thereafter during the treatment period for a possible dose adjustment. However, no
dose adjustment was carried out.
-Food and drinking water consumption
The quantity of food left by individual animals was recorded on a weekly basis throughout the experimental period. The food intake per animal (g/animal/week)
was calculated using the total amount of food given to and left by each animal in each group on completion of a treatment week. The drinking water consumption was monitored daily by visual appraisal
throughout the study.

Sacrifice and pathology:

On test day 15 (approximately 24 hours after the last administration) all animals were euthanised by carbon dioxide (CO2) inhalation, exsanguinated by cutting the
aorta abdominalis, weighed, dissected and inspected macroscopically under the direction of a pathologist.
All superficial tissues were examined visually and by palpation. The cranial roof was 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 conditions of the thoracic viscera were 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 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, adrenal glands, uterus, intra-abdominal lymph nodes, and accessory reproductive organs were recorded.
In the prematurely deceased animal, the examinations were performed after the animal had been found dead in the morning.
The weights of the following organs of all animals were determined before fixation:
Adrenal gland, Brain, Epididymis, Heart, Kidney, Liver, Ovary, Spleen, Testis, Thymus, Uterus including cervix, Prostate and seminal vesicles with coagulating glands

Paired organs were weighed individually and identified as left or right. Special attention was paid to the organs of the reproductive system.
Organs of the prematurely deceased animal were recorded but not included into the mean value comparison.
The following organs or parts of organs of all animals were fixed in 7% buffered formalin except for the testes and epididymides, which were preserved in modified
Davidson's solution.
Ovary, epididymis, Testis, Uterus incl. cervix, Vagina, accesory sex organs (prostate, seminal vesicles with coagulation glands) and all organs showing macroscopic lessions

Statistics:

Toxicology and Pathology data were captured, as far as possible, using the departmental computerised systems (Provantis® Integrated preclinical software, Instem LSS Ltd., version 10.2.1). Raw data not fully compatible with the
computerized systems were maintained on paper according to appropriate SOPs.
The following comparison was carried out:
The test item groups 2 to 6 were compared to the control group 1 using the following statistical method:
Test : Multiple t-test based on DUNNETT, C. W. New tables for multiple comparisons Biometrics, 482-491 (September 1964)
Parameter : Body weight / Food consumption / Relative and absolute organ weights (p ≤ 0.05 and p ≤ 0.01)
Homogeneity of variances and normality of distribution were tested using BARTLETT's test and the 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 DUNNETT's test (see above).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Males
At 50 mg Boron/kg b.w./day, a short lasting slight salivation after dosing was noted in 1 of 3 males on test day 13.
From a dose level of 150 mg Boron/kg b.w./day onwards, a short lasting slight or moderate - on one test day extreme - salivation was noted on several test
days in 2 of 3 males each.
Treatment with 300 or 450 mg Boron/kg b.w./day caused salivation and piloerection on several or nearly all test days. Furthermore, a
haemorrhagic canthus (both eyes) were noted on several test days in 1 of 3 males treated with 300 mg Boron/kg b.w./day.

Females
At 15, 50 or 150 mg Boron/kg b.w./day (groups 2, 3 and 4), the following symptoms were noted in individual animals on only one test day: A short lasting
slight salivation after dosing was noted in 1 of 3 females at 15 mg Boron/kg b.w./day and in 2 of 3 females at 150 mg Boron/kg b.w./day. Furthermore,
breathing sounds were noted in 1 of 3 females at 50 mg Boron/kg b.w./day.
At 300 mg Boron/kg b.w./day, a short lasting slight or moderate - on one test day extreme - salivation was noted on a few to several test days in 3 of 3 females.
The two surviving high dose females (450 mg Boron/kg b.w./day) revealed salivation, piloerection and breathing sounds on one to several test days.

Mortality:

mortality observed, treatment-related

Description (incidence):

One of 3 female animals (no. 34) from the high tested dose level (450 mg Boron/kg b.w./day) was found dead in the morning of test day 10. Slightly reduced
motility (consistently during the day), piloerection and a haemorrhagic nose/snout were noted before death (test days 8 and 9). One day before death the animal was
cold to touch. A marked body weight loss was noted from test day 7 onwards. Necropsy revealed changes in the gastro-intestinal tract in the form of a thickened
and reddened mucosa.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain: At 300 or 450 mg Boron/kg b.w./day, a distinct and dose related slight reduction was noted for the body weight gain of all male and female rats.
Body weight: No changes in body weight for dose group 2, 3, and 4. No comparison to the control group was done for the test groups 5 and 6 (300 and 450 mg Boron/kg b.w./day) as these dose groups started at a later time point with higher body weights than those of the control and the test groups 2, 3 and 4.
A pronounced reduction was noted for the body weight at autopsy of the prematurely deceased female no. 34 from the high tested dose level (group 6: 450 mg Boron/kg b.w./day)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Statistically significant reductions in food intake (at p ≤ 0.05 or p ≤ 0.01 compared to the control group) were noted for the male and female rats treated with 300 or
450 mg Boron/kg b.w./day during the first test week. The relative food intake in these groups was decreased by 30.6% or 23.9% for the males
and by 16.2% or 29.2% for the females in test week 1. During the second test week, the food consumption of the male and female rats was in the range of the control group.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant increase (at p ≤ 0.05 compared to the control) was noted for the mean absolute thymus weights of the female rats treated with 15 mg Boron/kg b.w./day. This finding is considered to be not test itemrelated but spontaneous.

The relative and/or absolute mean liver weights appeared to be slightly increased in the male rats treated with 300 or 450 mg Boron/kg b.w./day,
being statistically significant at p ≤ 0.05 for the relative liver weights at 300 mg Boron/kg b.w./day. Corresponding increases were noted for the relative liver and
absolute weights of the two surviving female rats treated with 450 mg Boron/kg b.w./day. However, increased values compared to the control data
were only noted in individual male or female animals and macroscopic inspection at necropsy revealed no hepatic lesions. Hence, these changes are considered to be
not test item-related.
Reductions were noted for the absolute and relative thymus weights of both sexes treated with 300 or 450 mg Boron/kg b.w./day (statistically significant at p ≤ 0.05
or p ≤ 0.01 compared to the control in the male rats). The absolute and relative values of the individual animals were below those of the individual control animals.
This effect is considered to be test-item related.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The following changes are considered to be within the spontaneous variability: Dark-red discoloured lungs were observed for the female rat no. 16 (50 mg Boron/kg b.w./day).
External macroscopic inspection of the male rat no. 27 (300 mg Boron/kg b.w./day) confirmed the already mentioned clinical
observation in the form of a haemorrhagic canthus at both eyes. Macroscopic inspection of the prematurely deceased female no. 34 (450 mg Boron/kg b.w./day)
on test day 10 revealed external changes in the form of a haemorrhagic snout and a haemorrhagic canthus at both eyes. Internal inspection revealed changes in the gastro-intestinal
tract (inflated, thickened or reddened mucosa), a reddened brain and a reddened mesenteric lymph node.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

There was only one dead female animal in the high dose group on test day 10. The clinical symptoms observed in the study were post dosing salivation from 50 mg/kg bw/d group onwards and piloerection and haemorrhagic canthi from 300 mg/kg bw/d onwards in males. In females the post dose salivation startet in the 15 mg/kg bw/d group. In the higher dose groups also breathing sounds and piloerection could be observed.
A dose-related reduction in body weight gain was noted for the male and female rats treated with 300 or 450 mg Boron/kg b.w./day. In these dose groups also statistically significant reductions (at
p ≤ 0.05 or p ≤ 0.01) in the relative food consumption by 30.6% or 23.9% in the male rats and by 16.2% or 29.2% in the female rats during the first treatment week (reversible in 2nd test week). At end of the study no macroscopic finding was noted, however, the deceased female rat of the group 450 mg Boron/kg b.w./day revealed changes in the gastro-intestinal tract in the form of a thickened and reddened mucosa.
The organ weight anlysis revealed reductions for the absolute and relative thymus weights of both sexes treated with 300 or 450 mg Baron/kg b.w./day.
After consideration of these data, the following dose levels are suggested for the oral toxicity study with Boron, (benzenemethanamine)trifluoro-, (T-4)-, reaction products with Bu glycidyl ether (called for short Boron) in CD® rats based on OECD guideline 422: 0 mg Boron/kg b.w./day (control), 30 mg Boron/kg b.w./day (group 1), 100 mg Boron/kg b.w./day (group 2), 300 mg Boron/kg b.w./day (group 3).

Key result

Dose descriptor:

LOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Conclusions:

After consideration of all data, the following dose levels are suggested for the main oral toxicity study with the test item in CD® rats based on OECD guideline 422: 0 mg /kg b.w./day (control), 30 mg kg b.w./day (group 1), 100 mg kg b.w./day (group 2), 300 mg /kg b.w./day (group 3).

Executive summary:

 

The toxicity of the test substance was analyzed in a 14 day Dose Range Finding Study based on OECD 422 guideline by oral administration to rats. The selected dose groups were 0, 15, 50, 150 mg/kg bw/d, however, because of the low toxicity of the substance the dose levels were increased to 300 mg/kg bw/d (intermediate dose) and 450 mg/kg bw/d (high dose).

There was only one dead female animal of the high dose on test day 10. The clinical symptoms observed in the study were post dosing salivation from 50 mg/kg bw/d group onwards and piloerection and haemorrhagic canthi from 300 mg/kg bw/d onwards in males. In females the post dose salivation startet in  the 15 mg/kg bw/d group. In the higher dose groups also breathing sounds and piloerection could be observed.

A dose-related reduction in body weight gain was noted for the male and female rats treated with 300 or 450 mg/kg b.w./day. In these dose groups also statistically significant reductions (at

p ≤ 0.05 or p ≤ 0.01) in the relative food consumption by 30.6% or 23.9% in the male rats and by 16.2% or 29.2% in the female rats during the first treatment week (reversible in 2nd test week). At end of the study no macroscopic finding was noted, however, the deceased female rat of the group 450 mg/kg b.w./day revealed changes in the gastro-intestinal tract in the form of a thickened and reddened mucosa.

The organ weight anlysis revealed reductions for the absolute and relative thymus weights of both sexes treated with 300 or 450 mg/kg b.w./day.

After consideration of these data, the following dose levels are suggested for the oral toxicity study with the test item in CD® rats based on OECD guideline 422: 0 mg/kg b.w./day (control), 30 mg/kg b.w./day (group 1), 100 mg/kg b.w./day (group 2), 300 mg/kg b.w./day (group 3).

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

Combinded repeated dose/developmental toxicity study according to OECD 422

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

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

Species:

rat

Strain:

other: Crt: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

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

tap water

Details on oral 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.

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 the test item 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

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control group (vehicle)

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

low dose group

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

medium dose group

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

high dose group, dose increased on test day 28 to 400 mg/kg bw/d

Dose / conc.:

400 mg/kg bw/day (nominal)

Remarks:

Highest dose increased to 400 mg/kg bw d 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

Observations and examinations performed and frequency:

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.

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.

Sacrifice and pathology:

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.

Other examinations:

NEUROLOGICAL SCREENING
Screening of sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli) (based on Gad ), as well as the assessment of grip strength (Meyer ) and motor activity assessment were conducted as described on the following pages in five males and five females randomly selected from each study group.
The screening was conducted 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.

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)

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.

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/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. I

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/kg b.w./day) during the pre-mating, the gestation and the lactation period.
At the high dose level (300/400 mg/kg b.w./day) a slightly reduced body weight was noted at the end of the gestation period,andduring 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/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 /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

Description (incidence and severity):

No test item-related differences for the examined absolute and relative organ weights were noted between the control group and the treatment groups (30, 100 or 300/400 mg/kg b.w./day).
Slightly to moderately (in some cases statistically significantly) increased relative and absolute organ weights of the adrenal glands and the liver were noted for the male and / or female animals of the high dose group. However, as the differences were only slight or moderately and no changes were noted for the adrenal glands and the liver during the histo-pathological examination, the increased absolute and relative organ weights of the adrenal glands and the liver were considered to be spontaneous and not test item-related.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic post mortem findings
Males: No observations were noted for the male animals of the control group and of the treatment groups (30, 100 or 300/400 mg/kg b.w./day).

Females : No test item-related observations were noted for the female animals of the treatment groups. (30, 100 or 300/400 mg /kg b.w./day).
The following observations that were noted for the female animals were considered to be spontaneous:
Control: - Two haemorrhagic foci were noted in the stomach of animal no. 13.
Group 3: - Multiple red to brown coloured foci were noted in the lungs of animal nos. 56 and 58.
Group 4: - A reddened duodenum was noted for animal no. 72.
- An uterus cyst was noted for animal no. 75.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, 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.

Additional examinations of group 2 and 3 animals (kidneys and stomach)
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

Details on results:

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. 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 (maternal toxicity). 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 /kg b.w./day) revealed test item-related kidney changes in the form of basophilic tubules in males treated with 100 mg/kg b.w./day.
In a peer review analysis of Dr. Weber, he concluded that the effects in the stomach were caused by the corrosive properties of the test compound and can be considered as local effects. The kidney effects are indicative for the early onset of chronic progressive nephropathy (CPN), which is a spontaneous background lesion in rodents.
No test ite-related changes were observed for oestrus cyle, fertility index, gestation index, pre-coital time and the gestation length.

Key result

Dose descriptor:

NOAEL

Remarks:

local effects

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

Critical effects observed:

no

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) . 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 (maternal toxicity). 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.

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,maternal toxicity). 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.

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of the repeated dose toxicity studies, the test substance has not to be classified  according to CLP regulation 1272/2008.