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Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

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Endpoint:
repeated dose toxicity: oral, other
Remarks:
Fourteen Day Repeated Dose Oral (Gavage) Range-Finding Toxicity Study in the Rat
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
25th August 2016 to 07th October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Justification for type of information:
The study was designed and conducted to cause the minimum suffering or distress to the
animals consistent with the scientific objectives and in accordance with the Envigo Research
Limited, Shardlow, UK policy on animal welfare and the requirements of the United
Kingdom’s Animals (Scientific Procedure) Act 1986 Amendment Regulations 2012. The
conduct of the study may be reviewed, as part of the Envigo Research Limited, Shardlow,
UK Ethical Review Process.
Qualifier:
no guideline required
Version / remarks:
The study was performed according to the Study Plan and was designed to provide
information for further repeated dose toxicity studies.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Identification : (μ(5-amino-1,3,3-trimethylcycloihexylamine-
N,N’)hexafluorodiboron
The test item is also known as Aradur HZ 5933.
Physical State/Appearance : Test Facility description: Clear colorless liquid
Sponsor description: Liquid
Chemical Name : (μ(5-amino-1,3,3-trimethylcycloihexylamine-
N,N’)hexafluorodiboron
Purity : not supplied
Batch Number : AEF0009100
Date Received : 11 July 2016
Storage Conditions : Room temperature in the dark; used/formulated in light
Expiry Date : 29 August 2017
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were
obtained from Envigo RMS (UK) Limited, Oxon, UK. On receipt the animals were
examined for signs of ill-health or injury. The animals used in the main part of the study
were acclimatized for twenty-three days during which time their health status was assessed.
A total of twenty-four animals (twelve males and twelve females) were accepted into the
main study. At the start of treatment (main study), the males weighed 357 to 397g, the
females weighed 212 to 240g, and were approximately fourteen weeks old.

The animals were housed in groups of three by sex (main study) in solid floor polypropylene
cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire,
UK). The animals were allowed free access to food and water. A pelleted diet (Rodent
2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK) was used.
Certificates of analysis of the batches of diet used are given in Annex 1. Mains drinking
water was supplied from polycarbonate bottles attached to the cage. Environmental
enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels
(Datesand Ltd., Cheshire, UK). The diet, drinking water, bedding and environmental
enrichment was considered not to contain any contaminant at a level that might have affected
the purpose or integrity of the study.
The animals were housed in a single air-conditioned room within the Envigo Research
Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at
least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to
give twelve hours continuous light and twelve hours darkness. Environmental conditions
were continuously monitored by a computerized system, and print-outs of hourly
temperatures and humidities are included in the study records. The Study Plan target ranges
for temperature and relative humidity were 22 ± 3 °C and 50 ± 20%. Short term deviations
from these targets were considered not to have affected the purpose or integrity of the study;
see deviations from Study Plan.
The animals used in the main part of the study were allocated to dose groups using a
randomization procedure based on stratified body weights and the group mean body weights
were then determined to ensure similarity between the dose groups. The animals were
uniquely identified within the study, by an ear punch system routinely used in these
laboratories.
Route of administration:
oral: gavage
Details on route of administration:
In order to increase the toxicity information, preliminary sighting work was undertaken,
during which the test item was administered by gavage to one male and one female Wistar
Han™:RccHan™:WIST strain rats at a dose level of 1000 mg/kg bw/day, using a dose
volume of 5 mL/kg (vehicle: Distilled water).
Vehicle:
water
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
The test item was administered once daily, for up to fourteen consecutive days, by gavage
using a stainless steel cannula attached to a disposable plastic syringe. Control animals were
treated in an identical manner with 5 mL/kg of Distilled water.
The volume of test and control item administered to each animal was based on the most
recent scheduled body weight and was adjusted on Days 4, 8 and 11.
Frequency of treatment:
The test item was administered once daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
3
Control animals:
yes
Details on study design:
The animals used in the main part of the study were allocated to dose groups using a
randomization procedure based on stratified body weights and the group mean body weights
were then determined to ensure similarity between the dose groups. The animals were
uniquely identified within the study, by an ear punch system routinely used in these
laboratories.
Observations and examinations performed and frequency:
All animals were examined for overt signs of toxicity, ill health or behavioral change
immediately before dosing, up to thirty minutes after dosing and one hour after dosing.
Additional observations were made four hours following dosing (not at weekends or on
public holidays). All observations were recorded.

Body Weight
Individual body weights were recorded on Days 1, 4, 8, 11 and 15.
Food Consumption
Food consumption was recorded for each cage group for Days 1 to 4, 4 to 8, 8 to 11 and 11 to
15. Food conversion efficiency was calculated retrospectively.

Water Consumption
Water intake was measured and recorded daily for each cage group; see deviations from
Study Plan.
Sacrifice and pathology:
On completion of the dosing period, all surviving animals were killed by intravenous
overdose of a suitable barbiturate agent followed by exsanguination and subjected to an
internal and external macroscopic examination. Animals that died during the study were also
necropsied. Any tissues showing macroscopic abnormalities were preserved in buffered 10%
formalin. These will be discarded following issue of the final report.
Other examinations:
The following computerized systems were used on the study:
Provantis
Delta BMS
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 250 mg/kg bw/day, one male showed clinical signs of hunched posture at one and four
hours after dosing on Day 5 relative to the start of treatment. This animal was found dead on
the following day (before dosing). The remaining animals from this dose group did not show
any clinical signs, but were terminated on the same day as this dose level was deemed to be
excessive for repeat dosing.
At 150 mg/kg bw/day, isolated instances of increased post-dose salivation were observed in
one male (Day 8) and one female (Day 6). Such observations are common in this type of
study and are generally considered to be due to an irritant nature of the test item and/or
formulations.
There were no clinical signs for animals of either sex treated with 50 mg/kg bw/day
throughout the dose administration period.
Mortality:
mortality observed, treatment-related
Description (incidence):
At 250 mg/kg bw/day, one male (Male 21) was found dead on Day 6 (before dosing) relative
to the start of treatment. Prior to death, clinical signs for this animal included hunched
posture at one and four hours after dosing on Day 5 whilst macroscopic observations at
necropsy were confined to dark liver, sloughing of the stomach, thinning of the non-glandular
region of the stomach and gaseous distension of the stomach and small intestine with the
latter also filled with yellow fluid. A body weight loss of approximately 20% relative to Day
1 was also evident for this male and this death was considered to be treatment-related. There
were no clinical signs for the remaining animals from this dose group, but marked body
weight losses (more prominent in males) or stasis were evident over Days 1 to 6. The effect
on body weight development was associated with a marked reduction in dietary intake for the
males over Days 1 to 4 and a marked increase in water consumption for either sex. Taking
into consideration the overall results from this dose level, it was considered to be too high for
continued dosing and the remaining animals from this dose group were also killed on the
same day; these animals were not dosed on Day 6. At necropsy, macroscopic observation for
individual animals included dark or reddened patches in the stomach with one male and one
female also showing thickened or pale (with dark patches) glandular region in the stomach,
respectively.
There were no further unscheduled deaths during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Male 1 from the control group showed a body weight loss of 45 g over the first week of
dosing followed by a body weight gain of 42 g over the second week. As body weight gains
for this animal were considered to be atypical, data for the test item-treated males are
compared with the remaining control animals.
At 250 mg/kg bw/day, marked body weight losses were evident in males over Days 1 to 6 of
dosing. Individual females from this dose group also exhibited slight body weight losses or
stasis over the corresponding period. Following the premature death of one male, which had
shown a body weight loss of approximately 20% relative to Day 1, the remaining animals
from this dose group were also killed on Day 6.
Group mean body weight gains for study Days 1-4 in males treated with the test item at dose
levels of 150 or 50 mg/kg bw/day were slightly lower than controls in a dose-related manner.
Subsequent improvement was noted, however, body weight gains in these animals were again
lower than controls towards the end of the treatment period. Overall body weight gains in
these males were lower than controls in a dose-related manner. When compared with
controls, females receiving 150 mg/kg bw/day also showed lower group mean body weight
gains or actual body weight losses up to Day 11 which resulted in an overall group mean
body weight loss of 4.3g. At 50 mg/kg bw/day, group mean body weight gains in females
were generally comparable with controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At the start of dosing (Days 1 to 4), males given 250 mg/kg bw/day showed marked reduction
in dietary intake in relation to controls whilst the corresponding value in females was similar
to controls. A marked reduction in food conversion efficiency for either sex was deemed to
be reflective of intergroup differences in body weight gain and/or food intake.
At 150 mg/kg bw/day, food consumption in males was generally slightly lower than controls
throughout the treatment period. A slightly lower dietary intake was also noted for the
corresponding females but only over the first week of dosing. Food consumption for animals
of either sex treated with 50 mg/kg bw/day was generally comparable with controls. Any
differences in food conversion efficiency were deemed to reflect intergroup differences in
body weight gain and/or food intake.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
At 250 mg/kg bw/day, water consumption for animals of either sex was higher than controls
with the effect appearing to be more prominent in males.
When compared with controls, animals of either sex receiving 150 mg/kg bw/day also
showed sporadic instances of slightly higher water consumption which resulted in an overall
increase for these animals. Fluctuations in water consumption were evident at 50 mg/kg
bw/day, but no increase in overall water intake was observed for these animals.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At 250 mg/kg bw/day, the male found dead on Day 6 of dosing, was noted to have dark liver,
sloughing of the stomach, thinning of the non-glandular region of the stomach and gaseous
distension of the stomach and small intestine with the latter also filled with yellow fluid. The
remaining animals from this dose group were also killed on the same day with necropsy
findings confined to the stomach; these included dark or reddened patches in the stomach
with one male and one female also showing thickened or pale (with dark patches) glandular
region in the stomach, respectively.
At 150 mg/kg bw/day, 2/3 males exhibited pale and thickened glandular region in the
stomach. No macroscopic abnormalities were observed in females treated with 150 mg/kg
bw/day or in animal of either sex given 50 mg/kg bw/day.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Details on results:
Administration of (μ(5-amino-1,3,3-trimethylcycloihexylamine-N,N')hexafluorodiboron to
Wistar Han™:RccHan™:WIST strain rats at a dose level of 250 mg/kg bw/day resulted in
the premature death of one male on Day 6 (before dose) relative to the start of dosing. The
remaining animals in this dose group, in particular the males, showed appreciable body
weight losses or stasis which was associated with a marked reduction in food intake for males
and an increase in water consumption for either sex. Taking into consideration the overall
results, this dose level was considered to be too high for continued use and the surviving
males and females were terminated on the same day. At necropsy, macroscopic findings in
these animals were confined to the stomach and included dark or reddened patches in the
stomach with one male and one female also showing thickened or pale (with dark patches)
glandular regions in the stomach, respectively.
At 150 mg/kg bw/day, animals of either sex generally showed reduced body weight gains or
actual body weight losses which resulted in lower overall body weight gain in males and
actual body weight loss in females over the fifteen day treatment period. This was associated
with marginally lower dietary intake in both sexes (evident in females over the first week of
dosing only) and an increase in overall water consumption. At necropsy, 2/3 males were
observed with pale/thickened glandular region in the stomach.
At 50 mg/kg bw/day, males showed slightly lower overall body weight gain but this was not
associated with an effect on food or water intake. The corresponding values in females from
this dose group were generally comparable with controls. None of these animals were
observed with any macroscopic findings at necropsy.
Key result
Dose descriptor:
dose level: high Dose
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
gross pathology
mortality
Key result
Dose descriptor:
dose level: Low Dose
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
gross pathology
mortality
Key result
Dose descriptor:
dose level: Intermediate
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
gross pathology
mortality
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
150 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes
Conclusions:
Administration of (μ(5-amino-1,3,3-trimethylcycloihexylamine-N,N')hexafluorodiboron to
Wistar Han™:RccHan™:WIST strain rats at a dose level of 250 mg/kg bw/day resulted in
the premature death of one male on Day 6 (before dose) relative to the start of dosing and this
dose level was deemed to be excessive for repeat dosing.
Treatment at 150 mg/kg bw/day was associated with reduced overall body weight gain in
males and an overall body weight loss in females. The dietary intake for animals of either
sex was marginally lower than controls (limited to the first week of dosing in females) with
these animals also showing an overall increase in water consumption. Macroscopic findings
at necropsy were confined to two males exhibiting pale/thickened glandular region in the
stomach.
Taking into account the overall results from this study and in consultation with the Sponsor, a
high dose level of 100 mg/kg bw/day was considered to be suitable for use in a subsequent
OECD 422 study (Envigo Study Number: VN75RW). Furthermore, dose levels of 25 and 50
mg/kg bw/day were considered to be appropriate as the low and intermediate doses,
respectively.
Executive summary:

Introduction

The study was designed to provide information for further repeated dose toxicity studies.

Methods

Following preliminary sighting work, the test item was administered by gavage to three

groups, each of three male and three female Wistar Han™:RccHan™:WIST strain rats, for up

to fourteen consecutive days, at dose levels of 50, 150 or 250 mg/kg bw/day. A control group

of three males and three females was dosed with vehicle alone (Distilled water). One male

from the 250 mg/kg bw/day dose group was found dead on Day 6 (before dosing) relative to

the start of dosing. The remaining animals from this dose group showed adverse effects on

body weight and/or dietary intake and this dose level was deemed to be excessive for repeat

dosing; the surviving animals were not dosed on Day 6 and terminated on the same day.

Clinical signs, body weight change, dietary intake and water consumption were monitored

during the study. All animals were subjected to gross necropsy examination.

Results

Mortality

One male treated with 250 mg/kg bw/day was found dead on Day 6 (before dosing) relative

to the start of dosing. Clinical signs for this animal had included hunched posture at one and

four hours after dosing on Day 5. Macroscopic examination at necropsy showed dark liver,

sloughing of the stomach, thinning of the non-glandular region of the stomach and gaseous

distension of the stomach and small intestine with the latter filled with yellow fluid. A body

weight loss of approximately 20% relative to Day 1 was also evident and this death was

considered to be related to treatment with the test item. As this dose level was deemed to be

too high for repeat dosing, the remaining animals from this group were not dosed on Day 6

and terminated on the same day.

There were no unscheduled deaths at 150 or 50 mg/kg bw/day throughout the dose

administration period.

Clinical Observations

At 250 mg/kg bw/day, clinical signs were confined to hunched posture observed in one male

at one and four hours after dosing on Day 5; this animal was found dead before dosing on the

following day. None of the remaining animals from this dose group showed any clinical

signs prior to termination on Day 6.

At 150 or 50 mg/kg bw/day, there were no clinical signs considered to be related to the

toxicity of the test item.

Body Weight

Males treated with the test item at a dose level of 250 mg/kg bw/day showed marked body

weight losses over Days 1 to 6 with the corresponding females also exhibiting body weight

losses or stasis over this period; these animals were only dosed up to Day 5 and terminated on

the following day. Overall body weight gains in males receiving 150 or 50 mg/kg bw/day

were lower than controls in a dose-related manner whereas an overall group mean body

weight loss was noted for females treated with 150 mg/kg bw/day. Body weight

development in females from the 50 mg/kg bw/day was generally comparable with controls.

Food Consumption

Whilst males from the 250 mg/kg bw/day dose group showed a marked reduction in food

intake at the start of dosing (Days 1 to 4), the corresponding value in females from this dose

group was similar to controls. At 150 mg/kg bw/day, dietary intake for males generally

remained lower than controls throughout the dosing phase with females from this dose group

also showing a similar effect during the first week of dosing. Food consumption for animals

of either sex treated with 50 mg/kg bw/day was generally comparable with controls.

Water Consumption

Gravimetric measurement identified higher water consumption for animals of either sex

treated with 250 mg/kg bw/day in relation to controls. Sporadic instances of slightly higher

water intake were also observed in both males and females receiving 150 mg/kg bw/day

whilst the corresponding values in the 50 mg/kg bw/day dose group were generally

comparable with controls.

Necropsy

At necropsy, macroscopic findings in the male found dead on Day 6 (treated with

250 mg/kg bw/day) were confined to dark liver, sloughing of the stomach, thinning of the

non-glandular region of the stomach and gaseous distension of the stomach and small

intestine with the latter also filled with yellow fluid. The surviving animals from this dose

group, also killed on the same day, showed dark or reddened patches in the stomach with one

male and one female also showing thickened or pale (with dark patches) glandular region in

the stomach, respectively. At 150 mg/kg bw/day, 2/3 males were observed with pale and

thickened glandular region in the stomach. There were no macroscopic abnormalities in the

remaining dose groups.

Conclusion

Administration of (μ(5-amino-1,3,3-trimethylcycloihexylamine-N,N')hexafluorodiboron to

Wistar Han™:RccHan™:WIST strain rats at a dose level of 250 mg/kg bw/day resulted in

the premature death of one male on Day 6 (before dosing) relative to the start of dosing and

this dose level was deemed to be excessive for repeat dosing.

Treatment at 150 mg/kg bw/day was associated with reduced overall body weight gain in

males and an overall body weight loss in females. The dietary intake for animals of either

sex was marginally lower than controls (limited to the first week of dosing in females) with

these animals also showing an overall increase in water consumption. Macroscopic findings

at necropsy were confined to two males exhibiting pale/thickened glandular region in the

stomach.

Taking into account the overall results from this study and in consultation with the Sponsor, a

high dose level of 100 mg/kg bw/day was considered to be suitable for use in a subsequent

OECD 422 study (Envigo Study Number: VN75RW). Furthermore, dose levels of 25 and 50

mg/kg bw/day were considered to be appropriate as the low and intermediate doses,

respectively.

Endpoint:
repeated dose toxicity: oral, other
Remarks:
OECD 422 Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
This study was conducted between 30 November 2016 and 29 May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
28 July 201522 March 1996
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
haematology
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
haematology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Key result
Critical effects observed:
no
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
25 mg/kg bw/day
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

Additional information

Justification for classification or non-classification

Conclusive information available, not sufficient for classification.