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Description of key information

Available studies:

  • 90-day Repeated dose oral toxicity study with 28-day recovery period: NOAEL=150 mg/kg bw/d (females);  NOAEL=50 mg/kg bw/d (males)
  •  28 days repeated dose Oral toxicity test with 14 -day recovery period:   NOAEL=500mg/kg bw/day (males/females)
  •   30 day feeding study with FR-1360: NOAEL= 300 mg/kg bw/day (females); 30 mg/kg bw/day (males)
  • 14 -Day repeated dose Oral toxicity test:  NOAEL= 1000 mg/kg bw/day (females); 300 mg/kg bw/day (males)                                       

    90-Day Repeated dose oral toxicity with 28-day recovery period: The daily oral gavage administration of Tribromoneopentyl alcohol for 90 days to Sprague Dawley rats at 50, 150 and 450 mg/kg bwt/day resulted in no mortalities up to the highest dose over the 90-day dosing period. The body weights, body weight gains and food consumption were not altered by the treatment at any of the doses tested in either sex. Neurobehavioural observations of all the tested groups were comparable to the vehicle control group. There were no treatment-related changes observed in haematology, coagulation parameters, thyroid hormone levels, urine parameters, sperm analysis and there were no gross pathological changes observed in at any of the doses tested. The clinical signs of perineum wet with urine was observed in both males and females at 150 and 450 mg/kg bwt/day. At 150 mg/kg bwt/day in males, a increase in creatinine correlated with increased eosinophilic droplets in tubular epithelium in kidneys and urinary bladder epithelial hyperplasia were considered as test item related changes. At 450 mg/kg bwt/day in males, a minimal increase in blood urea nitrogen and creatinine with morphological correlates in kidneys and urinary bladder was observed. In kidneys, increased eosinophilic droplets in tubular epithelium and one animal with papillary necrosis were noted. In all males and only one female, diffuse epithelial hyperplasia was present in urinary bladder. All these changes reversed at the end of 28 day recovery period. Based on the above findings, treatment did not cause any adverse effects at 50 mg/kg bwt/day in males and at 150 mg/kg bwt/day in females during the 90 days treatment period. Hence, 50 mg/kg bwt/day in males and 150 mg/kg bwt/day in females are considered to be No Observed Adverse Effect Level (NOAEL) for the test item Tribromoneopentyl Alcohol, under the test conditions and doses employed.

    28-days oral toxicity followed by 2 weeks recovery period: Oral gavage administration of FR-513 to Sprague-Dawley rats at doses of 30, 150 or 500 mg/kg/day for four weeks was well-tolerated and did not cause any adverse change. A test-article related response was evident in the liver (predominantly at ≥150 mg/kg/day) as indicated by increased organ weight and a correlative microscopic finding of slight minimal centrilobular hypertrophy. Some changes in blood chemistry (low sodium and high potassium concentrations in males at 500 mg/kg/day) or urine composition/output (increased urinary volume and total protein and glucose output in males at 500 mg/kg/day) occurred and a slight increase in kidney weight was evident in both sexes (predominantly at ≥150 mg/kg/day). None of these changes was considered adverse in nature, however, and the majority showed full or at least partial recovery. Consequently, the no-observed-adverse-effect level (NOAEL) was considered to be 500 mg/kg/day of FR-513.

    30-day dietary feeding study with FR-1360: Ingestion of up to 30 mg/kg/day of FR-1360 in the diet of rats for 30 days did not cause changes in the toxicological parameters evaluated (NOAEL = 30 mg/kg/day). At levels of 100 and 300 mg/kg/day, histologic changes in kidney and urinary bladder were noted in male rats. No changes were noted in any of the female rats in this study

    14-day Oral toxicity test: It is concluded that daily oral gavage administration of Trinol to CD rats at doses up to 1000 mg/kg/day in females and 300 mg/kg/day in males was well tolerated and these doses were considered to be the no-observed-adverse-effect-level (NOAEL). However,doses of 1000 mg/kg/day in males necessitated premature sacrifice of these animals on Day 4 and was considered to exceed the maximum tolerated dose.

     

     

                

Key value for chemical safety assessment

Toxic effect type:
concentration-driven

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Per ECHA decision on Compliance Check (Decision number: CCH-D-2114381478-36-01/F). See attachemnet.
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
“Repeated Dose 90-Day Oral Toxicity Study in Rodents” adopted on June 25, 2018.

Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
See COA attachment
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Justification for the selection of species: Rat is the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.

Source: Vivo Bio Tech Ltd.
Sy. #349/A, Pregnapur-502311,
Gajwel Mandal, Medak District, Telangana
India
Sex:
male/female
Details on test animals or test system and environmental conditions:
Age at the start of treatment : 5-6 weeks
Body weight range at the start of treatment: Males: 155.54 to 204.99g
Females: 126.13 to 169.98g
At the commencement of the treatment, the weight variation of rats used did not exceed ± 20% of the mean body weight in each sex and group.

After detailed clinical examination for good health and the suitability for the study, the rats were acclimatized for five days before start of the treatment.
During acclimatization period, all rats were observed once daily for clinical signs, morbidity and mortality. Only nulliparous and non-pregnant females were used
in the study.

Environmental Conditions
The rats were housed under standard laboratory conditions, air conditioned with adequate fresh air supply (12.6–12.8 air changes/hour). Environment: Temperature 19–25°C, relative humidity 49–68 %, with 12 hours light and 12 hours dark cycle.
The maximum and minimum temperature in the experimental room was recorded once daily. The relative humidity in the experimental room was calculated daily from dry and wet bulb temperature recordings.

Housing
Two rats per sex per cage were housed in solid floor standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with stainless steel top grill having facilities for providing pelleted food and drinking water in polycarbonate bottle with stainless steel sipper tubes. The last animal in the recovery groups was housed individually.
Polycarbonate rat huts were provided to the animals from first day of acclimatization as environmental enrichment objects in the cages that either provide shelter or exercising opportunities to minimize animal stress and promote overall well-being.

Bedding
Steam sterilized clean corn cob was used as bedding and changed along with the cage twice a week.

Diet and Water
Altromin Rat/Mice Maintenance diets manufactured by Altromin Spezialfutter GmbH & Co. KG, Im Seelenkamp 20, 32791 Lage, Germany, was provided ad libitum to the rats.

Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard® on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd., Mumbai - 400 001, India, was provided ad libitum to rats in polycarbonate bottles with stainless steel sipper tubes.
Route of administration:
oral: gavage
Details on route of administration:
Route of test item administration was through oral gavage. The oral route was chosen because it will provide an exaggerated model of possible exposure in humans. The oral route is also an acceptable and standard method for administering test substances in toxicology studies, and is a standard method as per the Guidelines (OECD 408).
Vehicle:
corn oil
Remarks:
The corn oil was used as the vehicle to prepare the dose formulations in the previously conducted toxicity studies with the test item by the Sponsor (Envigo Study Number: AFH0038). Hence, the same was selected as vehicle to prepare the dose formulations.
Details on oral exposure:
The dose formulations were administered orally by gavage to a specific group of rats once daily at approximately the same time (± 3 hours) each day for a period of 90 consecutive days. Similarly, the vehicle was administered to rats in the vehicle control and vehicle control recovery group once orally for 90 consecutive days.
The vehicle and the dose formulations were not administered to the rats in the recovery groups for four weeks (i.e. 28 Days) following the 90 day treatment period.
The dose formulation and the vehicle were administered at an equivolume of 5 mL/kg body weight. The dose volume was calculated for individual animal on the first day of the treatment period and was adjusted according to the most recent body weights recorded during the treatment period.

A table summarizing the Experimental Design, Group Allocation and Number of Rats is shown in the appropriate section.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability and homogeneity of the test item in the vehicle was established at 1 and 250 mg/mL under Eurofins Advinus Study No. G18488 (attached). Based on the results, the test item was found to be resuspendable and
stable in the vehicle up to 15 days when stored at room temperature.
For homogeneity and test item concentration analysis, prepared formulation samples were sampled on Day 1, during month 2 (Day 33) and month 3 (Day 74) of the treatment period and analysed in-house.
Two replicates were drawn from the top, middle and bottom layers of each preparation and in case of the control, a single replication sample was drawn from the middle layer.
The analysis was done as per the method validated under Eurofins Advinus Study No.: G18448.
Dose formulations were considered acceptable as the overall mean (calculated using all the 6 replicate values) of all the layers and mean of each layer was within ± 20% of the claimed concentration and the relative standard deviation (% RSD, calculated using all the 6 replicate values) of assay of top, middle and bottom layers were less than 20%.
Duration of treatment / exposure:
Tribromoneopentyl Alcohol was administered orally by gavage for 90 consecutive days and to assess the reversibility of any effects following 28 days recovery period.
Frequency of treatment:
once daily
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
Low dose (G2)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
mid dose (G3)
Dose / conc.:
450 mg/kg bw/day (actual dose received)
Remarks:
High dose (G4)
Dose / conc.:
450 mg/kg bw/day (actual dose received)
Remarks:
Recovery group (G4R)
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle control (G1)
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Recovery Vehicle control (G1R)
No. of animals per sex per dose:
Main groups: 10 males + 10 females
Recovery groups: 5 males + 5 females

Total number of rats: 100 (50 males + 50 females)



Control animals:
yes, concurrent vehicle
Details on study design:
The test item was suspended in vehicle (Corn Oil) and administered to rats at the graduated dose levels of 50, 150, and 450 mg/kg bwt/day for low dose (G2), mid dose (G3) and high dose (G4)/high dose recovery (G4R) group rats, respectively. The rats in the vehicle control group (G1)/vehicle control recovery (G1R) groups received vehicle alone. The dose volume administered was 5 mL/kg body weight. Each main group in the experiment comprised of 10 males and 10 female rats and recovery groups comprised of 5 males and 5 female rats.

Each rat in the experiment was observed for clinical signs, mortality and morbidity. Ophthalmological examination was carried out for all the rats prior to start of treatment, at the end of treatment for main groups and at the end of recovery period for recovery groups. The body weights and food consumption were measured during in-life phase of the experiment. Neurological examinations were conducted towards the end of treatment for main groups and towards the end of recovery period for recovery groups. The clinical laboratory investigations such as haematology, coagulation, clinical chemistry, hormone analysis and urine analysis were performed at termination.
All rats in the experiment were subjected to detailed necropsy and the organ weights and their ratio were derived as percentage of fasting body weights and brain weights. Histopathological examination was carried out on all the preserved organs and tissues of vehicle control (G1) and high dose (G4) group rats and on all gross lesions. Kidneys, urinary bladder and stomach were examined from the lower dose (G2 and G3) and recovery (G1R and G4R) groups.
Observations and examinations performed and frequency:
Each rat in the experiment was observed for clinical signs, mortality and morbidity. Ophthalmological examination was carried out for all the rats prior to start of
treatment, at the end of treatment for main groups and at the end of recovery period for recovery groups. The body weights and food consumption were measured during in-life phase of the experiment. Neurological examinations were conducted towards the end of treatment for main groups and towards the end of recovery period for recovery groups. The clinical laboratory investigations such as haematology, coagulation, clinical chemistry, hormone analysis and urine analysis were performed at termination.
Sacrifice and pathology:
All rats in the experiment were subjected to detailed necropsy and the organ weights and their ratio were derived as percentage of fasting body weights and brain weights. Histopathological examination was carried out on all the preserved organs and tissues of vehicle control (G1) and high dose (G4) group rats and on all gross lesions. Kidneys, urinary bladder and stomach were examined from the lower dose (G2 and G3) and recovery (G1R and G4R) groups.
Other examinations:
Sperm parameters
Terminal fasting body weight
T3, T4,TSH
Statistics:
STATISTICAL ANALYSIS
Data captured using Provantis™ for the parameters body weight, organ weights, haematology, coagulation, clinical chemistry and terminal fasting body weight were analyzed using Provantis™ built-in statistical tests.
Derived data like net body weight change, food consumption and organ weight ratios were also analyzed using built-in statistical tests.
The data from the treatment groups (G2, G3 & G4) were compared with the vehicle control group (G1).
In case of recovery groups, data was also analysed using the methods stated above. Comparison of means between treatment recovery group and control recovery groups were performed.
The statistical analysis of the experimental data which were not collected using ProvantisTM, was carried out using the validated package in Excel and also using licensed copies of SYSTAT Statistical package ver.12.0.
All quantitative variables like neurological observations (neuromuscular observation/body temperature/body weights) and T3, T4, TSH data were tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene’s test) within the group before performing a one-factor ANOVA modeling by treatment groups. Non-optimal (non-normal or heteroschedastic) data was transformed, before performing ANOVA. Comparison of means between treatment groups and control group was done using Dunnett’s test when the overall treatment, ‘F’ test found to be significant.
The data pertaining to males and female rats were evaluated separately.
All analyses and comparisons were evaluated at the p<0.05. Statistically significant differences (p<0.05), indicated by the aforementioned tests were designated by the following symbols throughout the report:
*: Significantly different from the control group at p<0.05 level.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No clinical signs of toxicity were observed at 50 mg/ kg bwt/day in either sex.
The treatment related clinical sign of perinium wet with urine was observed in both males and females at 450 mg/kg bwt/day from treatment Day 57. By the end of the treatment period, this clinical sign was observed in 9/10 males and 7/10 female rats of main group and 4/5 male and 3/5 female rats of recovery group. This sign was persisted in few high dose recovery animals during first week of the recovery period and all the recovery group animals became normal during subsequent week (week 2) of the recovery period.
The clinical sign of perinum wet with urine was also observed in one male and female rat on Day 91 at 150 mg/kg bwt/day.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
A significantly higher body weight gain was observed during Days 15-22 and 36-43 and Total % weight gain in main group males tested at 50 mg/kg bwt/day and significantly lower body weight gain was observed during Days 15-22 and 78-85 in recovery group males tested at 450 mg/kg bwt/day.
These significant differences were considered incidental and toxicologically not relevant as the mean body weights at these respective periods were comparable to vehicle the control group. Thus, the treatment had no effects on body weights at all the doses tested in males and females.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Males:
A significantly lower food consumption was observed during Days 36-43 at 50 mg/kg bwt/day, during Days 36-43 and 43-50 in recovery group tested at 450 mg/kg bwt/day.
A significantly higher food consumption was observed during Days 50-57, 71-78 at 450 mg/kg bwt/day and during Days 85-90 in the main groups tested at all the doses.
Females:
A significantly lower food consumption was observed during Days 15-22 and 36-43 at 50 mg/kg bwt/day, during Days 50-57 at 450 mg/kg bwt/day and overall mean food consumption during Days 1-90 at 450 mg/kg bwt/day recovery group.
The above significant increase/decrease in the food consumption observed at different intervals during treatment period were considered to be toxicologically not relevant because of isolated occurrence, lack of dose correlation and/or body weights were unaffected.
Thus, the treatment did not affect the body weights or food consumption in males and females either during treatment or recovery periods at the doses tested.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmological examination carried out prior to start of treatment, at the end of the treatment and recovery periods did not reveal any abnormalities in the eyes of the experimental rats.
Haematological findings:
no effects observed
Description (incidence and severity):
Haematology, Coagulation and Clinical Chemistry
There were no test item related changes in Haematology, PT and APTT in males and females.
An increase in Blood Urea Nitrogen at 450 mg/kg bwt/day and creatinine at ≥150 mg/kg/day in males was considered as test item related. This increase was associated with microscopic findings of increased eosinophilic droplets in the tubular epithelium consisting tubular casts in kidneys. These findings reversed at the end of recovery period.
All the other variations were considered as incidental findings as either the values were within the normal biological range or there was no dose relation.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
See description above.
Urinalysis findings:
no effects observed
Description (incidence and severity):
The urinalysis parameters were not affected by test item administration.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Home cage and Handling observations:
None of the evaluated parameters shows any abnormal findings except a treatment-related sign of perinium wet with urine in 6/10 males and 7/10 females at 450 mg/ kg bwt/day during handling observations.

Open field observations:
No treatment-related abnormalities were observed in any of the doses tested in both sexes.
Sensory observations: No treatment-related abnormalities were observed in any of the groups in both sexes.

Motor Activity: No treatment-related abnormalities were observed in any of the doses tested in both sexes except for few incidences as mentioned below:
Higher:
• Ambulatory time at interval 3 and total Ambulatory time in the main group males tested at 150 and 450 mg/kg bwt/day.
• Ambulatory Time at all intervals, Total Ambulatory time, Horizontal Counts at all intervals, Total Horizontal Counts, Ambulatory Count at interval 2 and interval 3 and Total Ambulatory Counts in the recovery group males tested at 450 mg/kg bwt/day.
• Ambulatory Time at interval 1 in the recovery group females tested at 450 mg/kg bwt/day.
Lower:
• Ambulatory time at interval 1 and Total Ambulatory time in the main group females tested at 450 mg/kg bwt/day.
The above observed statistical variations in the motor activity measurements are considered to be incidental as there were no changes observed in the home cage or open field observations. Further there were no clinical signs observed during daily clinical observations.
Neuromuscular observation:

Landing hind limb footsplay:
No treatment-related changes were observed at all the tested doses in both sexes except an incidental finding of reduced hindlimb footsplay in 450 mg/kg bwt/day recovery dose group males. This change was considered incidental as there were no associated changes in the muscle tone, gait, posture and hind limb grip strength.

Grip strength:
No treatment-related changes were observed at all the tested doses in both sexes except an incidence of higher forelimb grip strength in females treated at 450 mg/kg bwt/day, which was considered incidental and not related to the treatment. (Mean of G4 Females: 1087.30, Historical Control Data Low: 924.0 and High: 1139.0)

Physiological observation:
Body temperature: No significant changes were observed at all the tested doses in both sexes.

Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no test item related changes in the organ weights and their ratios to body weights and brain weights.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item related gross lesions at all the doses tested.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item related microscopic changes were noted in kidneys and urinary bladder in males.
In kidneys, increased eosinophilic droplets were noted in the tubular epithelium in the cortex of 6 males treated at 150 mg/kg bwt/day and all males treated at 450 mg/kg bwt/day but not in females. A single incidence of papillary necrosis was also observed at 450 mg/kg bwt/day males and considered as test item related. Diffuse epithelial hyperplasia was noted in urinary bladder of 6 males at 150 mg/kg bwt/day and all males and one female at 450 mg/kg bwt/day.
Both these changes reversed at the end of recovery period.

All the other microscopic changes noted were considered as spontaneous/back ground findings common for this age group rats.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Details on results:
• Oestrous cycle evaluation : The stage of oestrous cycle was recorded prior to necropsy in the treated groups only to facilitate interpretation of ovary and uterus organ weight and histopathology. No intergroup difference were observed in either parameter.

• Hormone Analysis : There were no test item related changes in T3, T4 and TSH.

• Sperm Parareters: There were no significant intergroup differences in any of the sperm parameters evaluated

• Terminal Fasting Body Weight Organ Weight: The terminal fasting body weights were not affected by test item administration.
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
gross pathology
Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
gross pathology
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
50 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
bladder
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Conclusions:
The daily oral gavage administration of Tribromoneopentyl alcohol for 90 days to Sprague Dawley rats at 50, 150 and 450 mg/kg bwt/day resulted in no mortalities up to the highest dose over the 90-day dosing period. The body weights, body weight gains and food consumption were not altered by the treatment at any of the doses tested in either sex. Neurobehavioural observations of all the tested groups were comparable to the vehicle control group. There were no treatment-related changes observed in haematology, coagulation parameters, thyroid hormone levels, urine parameters, sperm analysis and there were no gross pathological changes observed in at any of the doses tested.
The clinical signs of perineum wet with urine was observed in both males and females at 150 and 450 mg/kg bwt/day. At 150 mg/kg bwt/day in males, a increase in creatinine correlated with increased eosinophilic droplets in tubular epithelium in kidneys and urinary bladder epithelial hyperplasia were considered as test item related changes. At 450 mg/kg bwt/day in males, a minimal increase in blood urea nitrogen and creatinine with morphological correlates in kidneys and urinary bladder was observed. In kidneys, increased eosinophilic droplets in tubular epithelium and one animal with papillary necrosis were noted. In all males and only one female, diffuse epithelial hyperplasia was present in urinary bladder. All these changes reversed at the end of 28 day recovery period.

No Observed Adverse Effect Level (NOAEL): Based on the above findings, treatment did not cause any adverse effects at 50 mg/kg bwt/day in males and at 150 mg/kg bwt/day in females during the 90 days treatment period. Hence, 50 mg/kg bwt/day in males and 150 mg/kg bwt/day in females are considered to be No Observed Adverse Effect Level (NOAEL) for the test item Tribromoneopentyl Alcohol, under the test conditions and doses employed.
Executive summary:

The purpose of this repeat dose (90-day) oral toxicity study was to assess the systemic toxicity potential of the test item, Tribromoneopentyl Alcoholin Sprague-Dawley rats when administered orally by gavage for 90 consecutive daysand to assess the reversibilityof any effects following 28days recovery period. This study was also intended to provide the information on major toxic effects, target organs and an estimation of a No Observed Adverse Effect Level (NOAEL).

The test item was suspended in vehicle (Corn Oil) and administered to rats at the graduated dose levels of 50, 150, and 450 mg/kg bwt/day for low dose (G2), mid dose (G3) and high dose (G4)/high dose recovery (G4R) group rats, respectively. The rats in the vehicle control group (G1)/vehicle control recovery (G1R) groups received vehicle alone. The dose volume administered was 5 mL/kg body weight. Each main group in the experiment comprised of 10 males and 10 female rats and recovery groups comprised of 5 males and 5 female rats.

The identity of Tribromoneopentyl Alcohol was provided by the study Sponsor by a Certificate of Analysis. The stability and homogeneity of the test item in the vehicle was established at 1 and 250 mg/mL under Eurofins Advinus Study No. G18488. Based on the results, the test item was found to be resuspendable and stable in the vehicle up to 15 days when stored at room temperature. The dose formulations were analysed for homogeneity and test item concentration on Day 1 and during month 2 (Day 33) and month 3 (Day 74) of the treatment period. The results indicated that the dose formulations were considered acceptable as the overall mean (calculated using all the 6 replicate values) of all the layers and mean of each layer was ≤8% of the claimed concentration and the relative standard deviation (% RSD, calculated using all the 6 replicate values) of assay of top, middle and bottom layers was ≤ 2.037%, against the target limit of ±20%.

Each rat in the experiment was observed for clinical signs, mortality and morbidity. Ophthalmological examination was carried out for all the rats prior to start of treatment, at the end of treatment for main groups and at the end of recovery period for recovery groups. The body weights and food consumption were measured during in-life phase of the experiment. Neurological examinations were conducted towards the end of treatment for main groups and towards the end of recovery period for recovery groups. The clinical laboratory investigations such as haematology, coagulation, clinical chemistry, hormone analysis and urine analysis were performed at termination.

All rats in the experiment were subjected to detailed necropsy and the organ weights and their ratio were derived as percentage of fasting body weights and brain weights. Histopathological examination was carried out on all the preserved organs and tissues of vehicle control (G1) and high dose (G4) group rats and on all gross lesions. Kidneys, urinary bladder and stomach were examined from the lower dose (G2 and G3) and recovery (G1R and G4R) groups.

Under the experimental conditions employed, the following results were obtained:

·        Clinical Signs, Mortalities and Ophthalmological Examination:There were no clinical signs or mortality observed at 50 and 150 mg/kg bwt/day dose levels tested in both sexes except an incidence of perineum wet with urine in a male and female on Day 91. Treatment related clinical signs of perineum wet with urine was observed in both males and females at 450 mg/kg bwt/day and it could be due to the changes in epithelial cells in the urinary bladder.

·        Ophthalmological examination did not reveal any ocular abnormalities.

·        Neurological Findings:No treatment-related neurological abnormalities were observed at any of the doses tested

·        Body Weights, Body Weight Gains and Food Consumption:The body weights and food consumption were unaffected by the treatment at all the tested doses in both the sexes.The percentage difference of mean body weights in the treatment groups was within -6.1 to +11.8 from the respective control group during the treatment period.The percentage difference of average food consumption in the treatment groups was within -3.2 to +3.2 from the respective control group during treatment period.

·        Haematology, Coagulation, Clinical Chemistry, Hormone Analysis and Urine Parameters:There were no test item related changes in Haematology, PT and APTT in males and females.An increase in Blood Urea Nitrogen at 450 mg/kg bwt/day and creatinine at ≥150 mg/kg/day in males was considered as test item related. These increases were associated with microscopic findings of increased eosinophilic droplets in the tubular epithelium in kidneys. These findings reversed at the end of recovery period.

·        There were no test item related changes in T3, T4 and TSH.

·        The urinalysis parameters were not affected by test item administration.

·        Sperm Parameters: There were no significant intergroup differences in sperm motility, sperm morphology and sperm counts evaluated.

·        Terminal Fasting Body Weight, Organ Weights and Organ Weight Ratios:The terminal fasting body weights were not affected by test item administration. There were no test item related changes in the organ weights and their ratios to body weights and brain weights.

·        Gross and Histopathology:There were no test item related gross lesions at all the doses tested.An isolated incidence of dilated uterus one each in 50 and 450 mg /kg bwt/day dose group female was observed and considered as incidental finding and not related to test item administration.At 450 mg/kg bwt/day dosed males, increased eosinophilic droplets in tubular epithelium, tubular basophilia and papillary necrosis were noted in kidneys. In both males and females, diffuse epithelial hyperplasia was present in urinary bladder.At 150 mg/kg bwt/day, increased eosinophilic droplets in tubular epithelium in kidneys and urinary bladder epithelial hyperplasia were considered as test item related in males.

          All these changes reversed at the end of 28 day recovery period.

Based on the above findings, treatment did not cause any adverse effects at 50 mg/kg bwt/day in males and at 150 mg/kg bwt/day in females during the 90 days treatment period. Hence, 50 mg/kg bwt/day in males and 150 mg/kg bwt/day in females are considered to be No Observed Adverse Effect Level (NOAEL) for the test item Tribromoneopentyl Alcohol, under the test conditions and doses employed.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1/4/2015-28/8/2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: study conducted under GLP and OECD guidelines
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
OPPTS 870.3050, Repeated Dose 28-Day Oral Toxicity Study in Rodents, July 2000.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animals
Strain/Species Sprague-Dawley; Crl:CD(SD) rat.
Supplier Charles River (UK) Ltd.
Number of animals 33 males and 33 females.
Spare animals were removed from the study room after treatment commenced.
Duration of acclimatisation 13 days before commencement of treatment.
Age of the main study and recovery animals at start of treatment Approximately 7 to 8 weeks of age.
Weight range of the main study and recovery animals at the start of treatment Males: 201 to 246 g
Females: 154 to 188 g
Allocation and identification
Allocation Randomly allocated on arrival.
Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals. Each sex was allocated separately.
Identification of animals Each animal was assigned a number and identified uniquely within the study by a microchip inserted shortly after arrival.
Identification of cages Each cage label was colour-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupants.
2.4.2 Animal housing, diet and water supply
Environmental control
Rodent facility Restricted entry - to minimise entry of external biological and chemical agents and to minimise the transference of such agents between rooms.
Air supply Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity Monitored and maintained within the range of 19-23ºC and 40-70%.
There were no deviations from these ranges.
Lighting Artificial lighting, 12 hours light: 12 hours dark.
Electricity supply Public supply with automatic stand-by generators.
Animal accommodation
Cages Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals.
Cage distribution Males and females were blocked by sex and the cages constituting each group were dispersed in batteries so that possible environmental influences arising from their spatial distribution were equilibrated, as far as was practicable. The positions of the cage batteries in the room were changed weekly, following a rotation plan, to further minimise possible effects of spatial variations.
Number of animals per cage 5 of the same sex (main study and recovery) or 3 of the same sex (spares).
Bedding Wood based bedding which was changed at appropriate intervals each week.
Environmental enrichment
Aspen chew block Provided to each cage throughout the study and replaced when necessary.
Plastic shelter Provided to each cage throughout the study and replaced when necessary.
Diet supply
Diet Harlan Teklad 2014C Diet.
Availability Non-restricted (removed overnight before the commencement of clinical pathology investigations).
Water supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability Non-restricted (except during urine collection).
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
Route Oral, by gavage, using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Treated at Constant doses in mg/kg/day.
Volume dose 5 mL/kg body weight.
Individual dose volume Calculated from the most recently recorded scheduled body weight.
Control (Group 1) Vehicle at the same volume dose as the treated groups.
Frequency Once daily at approximately the same time each day.
Formulation Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical procedure was successfully validated for FR-513 in corn oil with respect to the specificity of chromatographic analysis, limit of detection and quantification, the linearity of detector response, repeatability, method accuracy and precision.
Homogeneity and stability of FR-513 in corn oil formulations (at nominal concentrations of 6 and 100 mg/mL) was confirmed during magnetic stirring for up to 2 hours, and on re suspension following storage at ambient temperature for 1 day and refrigeration for up to 10 days.
The mean concentrations of FR-513 in test formulations analysed from formulations prepared for use in Week 1 were between +4.3 and -1.5% of nominal concentrations for the intermediate (30 mg/mL) and high (100 mg/mL) dose concentrations, respectively, confirming accurate formulation. The low dose (6 mg/mL) preparation was -19.5% of nominal concentration which was just outside standard variance limits (-15 to +10% of nominal concentration). Reanalysis of the contingency samples confirmed the original low result but the cause was not established since the study records confirmed that this formulation had been accurately prepared. It was considered that this result had no adverse impact on the overall interpretation of study results or the integrity of the study. An additional sample from the low dose (6 mg/mL) formulation prepared for use in Week 3 was taken and the mean result from this sample was 99.8% of nominal concentration, confirming accurate formulation.
The mean concentrations of FR-513 in test formulations analysed from formulations prepared for use in Week 4 were between 6.3, 5.7 and 8.0% of nominal concentrations for the low (6 mg/mL), intermediate (30 mg/mL) and high (100 mg/mL) dose concentrations, respectively, confirming accurate formulation.
FR-513 was not detected in any of the control samples, demonstrating that no cross contamination had occurred.
Duration of treatment / exposure:
Minimum period 4 weeks followed by a 2 week recovery period

All main study animals were treated throughout the necropsy period; cessation of treatment for the recovery phase animals started on the first day of the study week closest to the start of necropsy of animals allocated to the main study. Serial observations were recorded at appropriate intervals.
Frequency of treatment:
Once daily at approximately the same time each day.
Remarks:
Doses / Concentrations:
30, 150 or 500 mg/kg/day of FR-513 (equivalent to 6, 30 and 100 mg/mL using a dose volume of 5 mL/kg body weight).
Basis:
nominal in diet
No. of animals per sex per dose:
5 males and 5 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
see in attached document (experimental procedure)
Positive control:
a control group which received the vehicle only
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No data
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment.
BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded one week before treatment commenced, on the day that treatment and recovery commenced, weekly throughout the treatment and recovery periods and before necropsy.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started and for each week throughout the treatment and recovery periods.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/week
FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Fluid intake was assessed by daily visual observation during treatment and recovery periods. Quantitative measurements were not performed.
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected after overnight withdrawal of food at the following occasions:
Occasion Animals

Day 29 (after the 28th dose) All main study animals
Recovery Day 15 All recovery animals
Blood sampling was performed on the morning after overnight collection for urine and animals were allowed access to water for a minimum of one hour before the sampling procedures.

- Anaesthetic used for blood collection: Yes (identity): Animals were held under light general anaesthesia induced by isoflurane
- Animals fasted: yes
- How many animals: all main study animals/ all recovery animals

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected after overnight withdrawal of food at the following occasions:
Occasion Animals

Day 29 (after the 28th dose) All main study animals
Recovery Day 15 All recovery phase animals

Blood sampling was performed on the morning after overnight collection for urine and animals were allowed access to water for a minimum of one hour before the sampling procedures.

- Animals fasted: yes
- How many animals: all main study animals/ all recovery animals

URINALYSIS: Yes
- Time schedule for collection of urine: Animals were placed in an individual metabolism cage, without food or water. Urine samples were collected overnight at the following occasions:
Occasion Animals

Day 29 All main study animals
Recovery Day 15 All recovery phase animals

- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see Macropathology table)
HISTOPATHOLOGY: Yes (see Histopathology table )
Statistics:
see attached document on statistical analysis
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
animals receiving 500 mg/kg/day appeared to drink a greater volume of water than the Controls, but not consistent. males appeared slightly more affected than females. Complete recovery was demonstrated for this finding.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
The haematology investigation performed at the end of the 4-week treatment period did not identify any clear treatment related findings.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
slightly low sodium concentration (0.98X Control) and slightly high potassium concentration (1.18X Control) in males given 500 mg/kg/day; both of these findings showed full recovery at the end of the 2-week recovery period.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
at the end of the 4-week treatment period revealed a dose-related increase in urinary volume and slightly high total protein and glucose outpout in males given ≥30 mg/kg/day. All of these findings showed full recovery at the end of the 2-week recovery
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
slightly high kidney weights for females given ≥30 mg/kg/day and males given ≥150 mg/kg/day. these findings showed full recovery with the exception of kidney weights which remained slightly high at the end of the recovery period for males given 500.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY: Frequent incidences of chin rubbing (animals seen to be pushing their muzzle through cage bedding) and/or salivation (sometimes reported as excessive) occurred from Week 2 with all females and the majority of males given 500 mg/kg/day being affected at some point during the treatment phase. Single incidences of chin rubbing and excessive salivation occurred on Days 11 and 16, respectively, in one female given 150 mg/kg/day. In all cases, these signs were seen after completion of dosing but had resolved by 1-2 hours thereafter.
Females receiving 500 mg/kg/day displayed transient unsteady gait approximately 20 minutes after completion of dosing the group on Days 27 and 28 of treatment and one high dose female (No. 54) appeared to be less active than the other females within the group (on Day 28) at the same time. These were transient signs which had resolved by 1 to 2 hours after dosing.
The general appearance and behaviour of the animals was otherwise unaffected by treatment and no animals died.

BODY WEIGHT AND WEIGHT GAIN:There was no effect of treatment on body weight.

FOOD CONSUMPTION AND COMPOUND INTAKE : There was no effect of treatment on food consumption.

WATER CONSUMPTION AND COMPOUND INTAKE : A visual assessment of water intake revealed that animals receiving 500 mg/kg/day appeared to drink a greater volume of water than the Controls during all treatment weeks. The effect was not consistently seen on a daily basis but the highest incidences occurred in the last week of treatment (Week 4) and, overall, males appeared slightly more affected than females. Complete recovery was demonstrated for this finding.

HAEMATOLOGY: The haematology investigation performed at the end of the 4-week treatment period did not identify any clear treatment related findings.
Mean activated partial thromboplastin times were slightly shorter (down to 0.84X Control) for females given ≥150 mg/kg/day but this finding had a doubtful relationship to treatment given that the differences lacked dose-response and no similar finding was evident in males. No similar trend was evident at the end of the 2-week recovery period.
All other inter-group differences from Controls, including those which attained statistical significance, were generally minor, confined to one sex, lacked dose-relationship or, in the case of those seen at the end of the recovery period, were not affected at the end of the treatment period and were therefore attributed to normal biological variation. Such differences seen at the end of the treatment period included the slightly high basophil counts in males given 500 mg/kg/day (where the higher mean value was due to an elevated value in one animal (No. 12)) and the slightly low mean cell haemoglobin and mean cell haemoglobin concentration in females given 500 mg/kg/day (where all individual values were within the background range; 90-percentile range 18.3 to 20.9 pg for mean cell haemoglobin and 33.0 to 37.7 g/dL for mean cell haemoglobin concentration; n=305 in both cases). Such differences seen at the end of the recovery period included the slightly high large unstained cell and platelet counts seen for females which had previously been given 500 mg/kg/day; these changes were dismissed given that there had been no effect on these parameters at the end of the treatment period.

CLINICAL CHEMISTRY: The biochemical examination of the blood plasma at the end of the 4-week treatment period revealed slightly low sodium concentration (0.98X Control) and slightly high potassium concentration (1.18X Control) in males given 500 mg/kg/day; both of these findings showed full recovery at the end of the 2-week recovery period.
All other inter-group differences from Controls, including those which attained statistical significance, were generally minor, confined to one sex, lacked dose-relationship or, in the case of those seen at the end of the recovery period, were not affected at the end of the treatment period and were therefore attributed to normal biological variation. Such differences included, but were not limited to, slightly low alanaine amino-transferase activities seen at the end of the treatment period in males given ≥150 mg/kg/day (which lacked dose-relationship) and slightly low calcium, protein and albumin concentrations seen at the end of the recovery period in females which had previously been given 500 mg/kg/day (where no effect on these parameters was evident at the end of the treatment period).

URINALYSIS: Urinalysis performed at the end of the 4-week treatment period revealed a dose-related increase (up to 2.3X Control) in urinary volume (with an associated decrease in specific gravity down to 0.98X Control) and slightly high total protein and glucose output (up to 2.5 and 1.37X Control, respectively but without dose-relationship) in males given ≥30 mg/kg/day. All of these findings showed full recovery at the end of the 2-week recovery period.
Trace or large amounts of blood pigment were detected in the urine at the end of the treatment period in one male (No. 8, where the presence of intact red blood cells was also evident following microscopic examination of the urine) which received 150 mg/kg/day and two males (Nos. 14 and 15) which received 500 mg/kg/day but no similar findings were evident amongst treated groups of females.

NEUROBEHAVIOUR:
Sensory reactivity and grip strength
Sensory reactivity responses and grip strength were unaffected by treatment.

Motor activity
There was no effect of treatment on motor activity.
The mean low and high beam break scores (cage floor and rearing activity, respectively) at the 36 minute interval were statistically significantly low for females receiving 500 mg/kg/day but the mean scores for the concurrent control females were unusually high at that time-point with mean scores being higher than the maximum historical control ranges. Consequently, this finding was considered due to atypical control values rather than a test substance-related effect.

ORGAN WEIGHTS: Analysis of organ weights for animals killed after 4-weeks of treatment revealed slightly high adjusted liver weights for males given ≥30 mg/kg/day (up to 1.11X Control) and females given ≥150 mg/kg/day (up to 1.18X Control), but no clear dose-response was evident in either sex. At the end of the of the 2-week recovery period, adjusted liver weights were lower than controls for females previously given 500 mg/kg/day and remained marginally high for males previously given this dose level but, in the latter case, the magnitude of change (1.05X Control) was less than that evident at the end of the treatment period. This indicated that complete or partial recovery had occurred.
Absolute and adjusted kidney weights were slightly high at the end of the treatment period for females given ≥30 mg/kg/day (but without dose-relationship) and males given ≥150 mg/kg/day and remained marginally high at the end of the 2-week recovery period in males previously given 500 mg/kg/day. None of these differences attained statistical significance, however, and, in all cases, the magnitude of change from controls was slight (<1.10X).
All other inter-group differences from Controls, including those which attained statistical significance, were generally minor, confined to one sex, lacked dose-relationship or, in the case of those seen at the end of the recovery period, were not affected at the end of the treatment period and were therefore considered attributed to normal biological variation. Such differences included, but were not limited to, the slightly low absolute and adjusted prostate/seminal vesicle weights and pituitary weights seen at the end of the recovery period for males and females, respectively, which had previously been given 500 mg/kg/day (but where no effect on these organs was evident at the end of the treatment period).

GROSS PATHOLOGY: no effect

HISTOPATHOLOGY: NON-NEOPLASTIC: no effect


HISTORICAL CONTROL DATA: available

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified
Conclusions:
It is concluded that oral gavage administration of FR-513 to Sprague-Dawley rats at doses of 30, 150 or 500 mg/kg/day for four weeks was well-tolerated and did not cause any adverse change. A test-article related response was evident in the liver (predominantly at ≥150 mg/kg/day) as indicated by increased organ weight and a correlative microscopic finding of slight minimal centrilobular hypertrophy. Some changes in blood chemistry (low sodium and high potassium concentrations in males at 500 mg/kg/day) or urine composition/output (increased urinary volume and total protein and glucose output in males at 500 mg/kg/day) occurred and a slight increase in kidney weight was evident in both sexes (predominantly at ≥150 mg/kg/day). None of these changes was considered adverse in nature, however, and the majority showed full or at least partial recovery. Consequently, the no-observed-adverse-effect level (NOAEL) was considered to be 500 mg/kg/day of FR-513.
Executive summary:

Summary

The objective of this study was to assess the systemic toxic potential of FR-513 administered daily by oral gavage for four weeks in Sprague-Dawley rats. The potential for any treatment related changes to recover was assessed in a subsequent 2-week recovery period. Three groups, each comprising five males and five females, received doses of 30, 150 or 500 mg/kg/day of FR-513 (equivalent to 6, 30 and 100 mg/mL using a dose volume of 5 mL/kg body weight). A similarly constituted Control group received the vehicle (corn oil) at the same volume‑dose. An additional five males and five females were assigned to each of the control and high dose groups. These animals were treated for four weeks, followed by a two week period without treatment to assess the potential for any treatment-related changes to recover.

During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, body weight, food consumption, water consumption (by visual assessment), oestrous cycles, haematology (peripheral blood), blood chemistry, urinalysis, organ weight, sperm analysis, macropathology and histopathology investigations were undertaken.

Results

FR-513 was well tolerated and did not result in any deaths or other adverse toxicity. Oestrus cycles and sperm were considered unaffected by treatment and there were no clear treatment-related macroscopic findings.

Transient incidences of chin rubbing and salivation occurred shortly after dosing at 500 mg/kg/day and were considered to be associated with non-palatability of the test substance formulations. Single incidences of these signs also occurred in one female at 150 mg/kg/day. Unsteady gait was noted shortly after dosing on two occasions (Days 27 and 28) towards the end of the treatment period in females given 500 mg/kg/day and one female at this dose level appeared less active at the same time on Day 28. Both of these signs had resolved by 1-2 hours after dosing and the extensive sensory reactivity response, grip strength and motor activity assessments did not reveal any clear treatment-related effects. All test substance-related signs observed in this study showed complete recovery.

Findings which were considered to be indicative of an effect on water balance or renal function included higher than control water intake in both sexes given 500 mg/kg/day;high (up to 2.3X) urinary volume (with an associated decrease in specific gravity down to 0.98X) and slightly high total protein and glucose output (up to 2.5 and 1.37X, respectively) in males given ≥30 mg/kg/day;slightly low plasma sodium (0.98X) and slightly high plasma potassium (1.18X) for males given 500 mg/kg/day; slightly high(<1.10X)kidney weights for females given≥30 mg/kg/day and males given ≥150 mg/kg/day. All of these findings showed full recovery with the exception of kidney weights which remained slightly high at the end of the recovery period for males previously given 500 mg/kg/day. There was no evidence of any adverse renal pathology;an increased incidence of minimal tubular basophilia was observed at the end of the treatment period in males given 500 mg/kg/day but this finding had an uncertain relationship to treatment.

Minimal centrilobular hypertrophy of the liver was seen in females given 150 mg/kg/day and both sexes given 500 mg/kg/day, correlating with slight increases in liver weights in females (at≥150 mg/kg/day) and males (at 500 mg/kg/day). The slight increases in liver weights seen in males at the lower dose levels (≤150 mg/kg/day) had no histopathological correlate. These findings showed complete or partial recovery (in the case of hypertrophy and increased liver weight, respectively) at the end of the recovery period.

The haematology investigation performed at the end of the 4-week treatment period did not identify any clear treatment related findings; mean activated partial thromboplastin times were slightly shorter (0.84X) for females given ≥150 mg/kg/day but this finding had a doubtful relationship to treatment and was not evident at the end of the recovery period.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Due to the preliminary nature of this study no specific regulations or guidelines were applicable.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline required
Guideline:
other: This is a preliminary study that was conducted as a Range finding trial prior to 28 days repeated dose oral toxicity study.
Principles of method if other than guideline:
see attached document
GLP compliance:
no
Remarks:
This is a preliminary study that was conducted as a Range finding trial prior to 28 days repeated dose oral toxicity study.
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
Crl:CD(SD) rats (a total of 25 male and 25 female) were received from Charles River (UK).
The rats were ordered at 29 to 35 days of age and within a weight range of 118 to 145 g for
males and 108 to 135 g for females.
On arrival, the animals were removed from the transit boxes and allocated to study cages.
Using the sequence of cages in the battery, one animal at a time was placed in each cage with
the procedure being repeated until each cage held the appropriate number of animals. Each
sex was allocated separately.
The cages constituting each group were blocked together by sex and the groups were
dispersed in batteries so that possible environmental influences arising from their spatial
distribution were equilibrated, as far as was practicable. Additionally, batteries of cages were
rotated around the room at weekly intervals to further minimise possible spatial variations.
Each animal was assigned a number and identified uniquely within the study by a tail tattoo.
Each cage label was colour-coded according to group and was numbered uniquely with cage
and study number, as well as the identity of the occupants.
Before the start of treatment two females with bodyweights at the extreme of the weight
range were replaced with spare animals of suitable weight from the same batch.
The animals were allowed to acclimatise to the conditions described below for 12 days before
treatment commenced. For those animals selected for this study, their age at the start of
treatment was 41 to 47 days and their bodyweights were in the range of 209 to 272 g for
males and 161 to 197 g for females.
The spare animals were removed from the study room after treatment commenced.
Animals were housed inside a barriered rodent facility (Building 8, Room 0802). Before the
study the room was cleaned and disinfected.
Each animal room was kept at positive pressure with respect to the outside by its own supply
of filtered fresh air, which was passed to atmosphere and not re-circulated. The temperature
and relative humidity controls were maintained within the range of 19 to 23°C and 40 to 70%
respectively and monitored continuously. There were no deviations from these ranges during
the study. Artificial lighting was controlled to give a cycle of 12 hours continuous light and
12 hours continuous dark per 24 hours.
Alarms were activated if there was any failure of the ventilation system, or temperature limits
were exceeded. A stand-by electricity supply was available to be automatically brought into
operation should the public supply fail.
The animals were housed five of one sex per cage. The cages were made of a polycarbonate
body with a stainless steel mesh lid. Wood based material was used as bedding and was
sterilised by autoclaving and changed at appropriate intervals each week. Cages, food
hoppers and water bottles were changed at appropriate intervals.
The animals were allowed free access to a standard rodent diet (Rat and Mouse No. 1
Maintenance Diet). This diet contained no added antibiotic or other chemotherapeutic or
prophylactic agent.
Potable water taken from the public supply was freely available via polycarbonate bottles
fitted with sipper tubes.
Each cage of animals was provided with an Aspen chew block for environmental enrichment.
Chew blocks were provided throughout the study and were replaced when necessary. Each
cage of animals was provided with a plastic shelter for environmental enrichment, which was
replaced at the same time as the cages.
Each batch of diet was analysed routinely by the supplier for various nutritional components
and chemical and microbiological contaminants. Supplier’s analytical certificates were
scrutinised and approved before any batch of diet was released for use. The quality of the
water supply is governed by regulations published by the Department for Environment, Food
and Rural Affairs. Certificates of analysis were received routinely from the water supplier
and the suppliers of the bedding and Aspen chew blocks. Since the results of these various
analyses did not provide evidence of contamination that might have prejudiced the study,
they are not presented.
No other specific contaminants that were likely to have been present in the bedding, chew
blocks, diet or water were analysed, as none that may have interfered with or prejudiced the
outcome of the study was known.
Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
ml/kg
Details on oral exposure:
Animals received the test substance or vehicle control formulations orally at a volume-dose
of 5 mL/kg bodyweight, using a suitably graduated syringe and a rubber catheter inserted via
the mouth into the stomach.
All animals were dosed once each day at approximately the same time each day. Group 4
males were dosed for four consecutive days, whilst the remaining animals were dosed for
14 consecutive days. The volume administered to each animal was calculated from the most
recently recorded bodyweight.
A daily record of the weight of each formulation dispensed and the amount remaining after
dosing was made. The balance of these two weights was compared with the predicted usage
as a check that the doses had been administered correctly. The weight/mL for the
formulations on this study in corn oil was 0.920, 0.930, 0.948 and 1.015 for Groups 1, 2, 3
and 4, respectively. Huntingdon Life Sciences Standard Operating Procedures state that
“For formulations with a weight/mL (specific gravity) below 0.95g or above 1.05g the
following adjustment will be required: Expected Total Volume Dosed x Weight/mL
= Expected Weight used.” It was considered necessary for this adjustment to be made when
calculating the expected weight used for Groups 1, 2 and 3.
In addition, the normal acceptable deviations from acceptable volume were extended due to
the viscous nature of the corn oil formulations and low animal numbers to be dosed, which
may have resulted in a larger volume of wastage than a less viscous formulation. The
acceptable limits of dosing overage were 20% over and 1% under. On a few occasions these
limits were exceeded, however, this increase in usage was considered not to affect the
integrity of the study because it was most likely associated with wastage seen when wiping
the catheter prior to intubation.
Formulations were stirred using a magnetic stirrer before and throughout the dosing
procedure.
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
The test substance, Trinol, was administered over a period of 14 consecutive days. The
necropsy procedures were completed on Day 15. Males receiving 1000 mg/kg/day were
killed early on Day 4 of treatment.
Frequency of treatment:
Animals received the test substance or vehicle control formulations orally at a volume-dose
of 5 mL/kg bodyweight, using a suitably graduated syringe and a rubber catheter inserted via
the mouth into the stomach.
All animals were dosed once each day at approximately the same time each day. Group 4
males were dosed for four consecutive days, whilst the remaining animals were dosed for
14 consecutive days.
Remarks:
Doses / Concentrations:
The doses used in this study (0, 100, 300 and 1000 mg/kg/day). Concentrations used in this study (0, 20, 60, 200 mg/ml)
Basis:
actual ingested
No. of animals per sex per dose:
5 male and 5 female per dose
Control animals:
yes
Details on study design:
see attached document
Observations and examinations performed and frequency:
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to
treatment. Cages were inspected daily for evidence of ill-health amongst the occupants. Any
deviation from normal was recorded at the time in respect of nature and severity, date and
time of onset, duration and progress of the observed condition, as appropriate.
Daily detailed observations were recorded at the following times in relation to dose
administration:
Immediately before dosing
Immediately after dosing on return of the animal to its cage
On completion of dosing of each group
Between one and two hours after completion of dosing of all groups
As late as possible in the working day
In addition, a more detailed weekly physical examination was performed on each animal to
monitor general health.
During the acclimatisation period, observations of the animals and their cages were recorded
at least once per day.
Sacrifice and pathology:

Animals were killed by carbon dioxide asphyxiation. The sequence in which the animals
were killed after completion of treatment was selected to allow satisfactory inter-group
comparison.

All animals were subject to a detailed necropsy.
After a review of the history of each animal, a macroscopic examination of the tissues was
performed. All external features and orifices were examined visually. After ventral mid-line
incision, the thoracic and abdominal cavities and their viscera were exposed and examined in
situ. Any abnormal position, morphology or interaction was recorded. If observations
indicated possible neurotoxic action the cranial roof was removed to allow observation of the
brain, pituitary gland and cranial nerves.
The requisite organs were weighed and external and cut surfaces of the organs and tissues
were examined as appropriate. Any abnormality in the appearance or size of any organ and
tissue was recorded and abnormalities were preserved in appropriate fixative (10% Neutral
Buffered Formalin).

The following organs, taken from each animal were dissected free of adjacent fat and other
contiguous tissue and the weights recorded:
Liver
Kidneys
Spleen
Bilateral organs were weighed together. Organ weights were also adjusted for terminal
bodyweight, using the weight recorded before necropsy.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All males receiving 1000 mg/kg/day were killed for welfare reasons on Day 4 of treatment.
Mortality:
mortality observed, treatment-related
Description (incidence):
All males receiving 1000 mg/kg/day were killed for welfare reasons on Day 4 of treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Bodyweight for males receiving 1000 mg/kg/day was affected to varying degrees prior to their early termination, with some animals losing weight whilst others appeared unaffected.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was considered to be unaffected by treatment.
Food efficiency:
not specified
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
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The organ weight variations were considered to be within background weight ranges and therefore considered to be unaffected by treatment.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Enlargement of the liver with associated dark areas was seen in one female receiving 1000 mg/kg/day.
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
Mortality:
All males receiving 1000 mg/kg/day were killed for welfare reasons on Day 4 of treatment.
Last in-life signs included, abnormal gait, unresponsive, underactive, flat posture, prostrate
posture and high levels of urine staining in all males at this dosage. Macroscopic
examination revealed abnormal contents and pallor of the jejunum in three of the five
animals, but there were no other consistent macroscopic observations recorded for these
animals.
Signs:
In addition to the post dosing and clinical observations seen in the males killed prematurely,
post dose salivation and chin rubbing was observed on occasion in the majority of animals at
all dose levels. However, this is a commonly observed finding in animals where the testmaterial
is administered by oral gavage and as such is considered to be of no toxicological
significance.
The clinical observations not associated with dosing revealed three out of five females
receiving 1000 mg/kg/day to show urine staining during the treatment period (Day 4, 11 and
15), with this sign also observed at macroscopic examination.
Bodyweight
Bodyweight and bodyweight gains for those animals surviving the treatment period were
considered to be unaffected by treatment.
Bodyweight for males receiving 1000 mg/kg/day was affected to varying degrees prior to
their early termination, with some animals losing weight whilst others appeared unaffected.
3.4 Food consumption

Food consumption was considered to be unaffected by treatment.
Organ weights
The organ weight variations were considered to be within background weight ranges and
therefore considered to be unaffected by treatment.
Huntingdon Life Sciences
Macropathology
The macroscopic examination performed after 14 days of treatment revealed the following
intergroup differences:
Liver
Enlargement of the liver with associated dark areas was seen in one female receiving
1000 mg/kg/day.
The nature and incidence of all other findings were consistent with the commonly seen
background of macroscopic changes.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Females well tolerated doses up to 1000 mg/kg bw/day
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Males well tolerated doses up to 300 mg/kg bw/day
Critical effects observed:
not specified
Conclusions:
It is concluded that daily oral gavage administration of Trinol to CD rats at
doses up to 1000 mg/kg/day in females and 300 mg/kg/day in males was well tolerated and
these doses are considered to be the no-observed-adverse-effect-level (NOAEL). However,
doses of 1000 mg/kg/day in males necessitated premature sacrifice of these animals on Day 4
and was considered to exceed the maximum tolerated dose.
Executive summary:

The systemic toxic potential of Trinol (a flame retardant), to Crl:CD(SD) rats by oral

administration was assessed over a period of 14 days. Three groups, each comprising five

male and five female rats received Trinol at doses of 100, 300 or 1000 mg/kg/day. A

similarly constituted Control group received the vehicle, corn oil, at the same volume-dose.

The males assigned to the high dose group (1000 mg/kg/day) were killed early, on Day 4 of

treatment, due to the signs observed.

During the study, clinical condition, bodyweight, food consumption, water consumption

(visual assessment only), organ weight and macropathology investigations were undertaken.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1973
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: A non GLP study, conducted at Halogen Research Lab.
Qualifier:
no guideline available
Principles of method if other than guideline:
Male and female rats were randomly groupd by sex and fed diets containg sufficient FR-1360 to provide the following doses 0 (control), 10, 30, 100 and 300 mg/kg bw for 30 days. On day 24 hematologic evaluations and urinalysis were conducted on the male and female rats from the control grous and groups receiving 300 mg/kg/bw of FR-1360. At the termination of the test biochemical examination were conducted. At necropsy a complete gross pathology examination was conducted.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
Specific pathogen free (SPF) derived rats
6-7 weeks of age
All rats were housed individually in wire bottomed cages with food and water available at all times.

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
Food and water was available for the rats at all times. The test diets were prepared weekly from a premix of the test material and ground laboratory chow.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
not available
Duration of treatment / exposure:
30 days
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0, 10, 30, 100 and 300 mg/kg bw /day
Basis:
nominal in diet
No. of animals per sex per dose:
5/sex/dose
Control animals:
yes
Details on study design:
See attached (experimental procedure)
Positive control:
no
Observations and examinations performed and frequency:
On day 24 hematologic evaluations were conducted (packed cell volume, hemoglobin, total erythrocyte count, total and differential leukocyte counts) and urinalysis (specific gravity, pH, sugar, proteins, occult blood and bilirubin) were conducted on the male and female rats from the control groups and groups receiving 300 mg/kg/day of FR-1360. At the termination of the test, blood samples were collected from all the rats for determination of serum urea nitrogen, alkaline phosphatase and glutamic pyruvic transaminase.
Sacrifice and pathology:
At necropsy, a complete gross pathological examination was conducted and the weights of heart, liver, kidney, testes and brain were recorded. Samples of the following tissues from all rats were fixed in 10% buffered formalin: Thyriod, trachea, para-thyroid, lung, heart, liver, kidney, adrenal gland, spleen, pancreas, stomach, small intestine, large intestine, urinary bladder, reproductive organs, skeletal muscle, brain spinal cord, eye, pitutiary gland, thymus, aorta and mesenteric and mediastinal lymph nodes. Sections were prepared and stained with hematoxylin and eosin for histopathological evaluation. All of the tissues from the control rats and those receiving the highest dose were examined microscopically. Histologic examination of tissues from rats on lower doses was limited to liver, kidney and urinary bladder. Additional sections of kidney from the control and high dose groups were stained according to the periodic acid-Schiff process.
Statistics:
Statistical differences from control were checked using Analysis of variance and Dunnett's test
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
conducted on male rats
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
decease Serum Glutamic Pyruvic transaminase (SGPT) (300 and 100 mg/kg/day)and increase in Blood Urea Nitrogen in males (300 mg/kg/day)
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
heart (g/100g) in Males (100 mg/kg/day); 100 and 300 mg/kg/day if expressed in gr)
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
hyperplasia of transitional epithelium of the urinary bladder (100 and 300 mg/kg/day;males); eosinophilic clumping of the cytoplasm and darkening of nuclei in cortical tubular epithelial cells. Regenerative changes in the large epithelial tubular cells
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes in kidneys and urinary bladders (male at 300 and 100 mg/kg/day
Histopathological findings: neoplastic:
not examined
Details on results:
The only effects observed that were considered related to treatment were:
1. an increase in serum urea nitrogen content in male rats receiving 300 mg/kg/day FR-1360 in their diet., and (2) renal tubular damage and generalized hyperplasia of the mucosal lining of urinary bladders of male rats reciving 300 and 100 mg/kg/day of FR-1360 in their diet.
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Critical effects observed:
not specified
Conclusions:
Based on the results above, the ingestion of up to 30 mg/kg/day of FR-1360 in the diet of rats for 30 days did not cause changes in the toxicological parameters evaluated. At levels of 100 and 300 mg/kg/day, histologic changes in kidney and urinary bladder were noted in male rats. No changes were noted in any of the female rats in this study.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subchronic
Experimental exposure time per week (hours/week):
13
Species:
rat
Quality of whole database:
K-1
System:
urinary
Organ:
bladder
kidney

Additional information

Justification for classification or non-classification

Based on the information gained, the test substance Tribromoneopentyl glycol does not need to be classified for repeated dose toxicity according to Directive 67/548/EEC or Regulation (EC) No 1272/2008 and the CLP.