Bis(4-(1,1,3,3-tetramethylbutyl)phenyl)amine

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 January 2015 to Febuary 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

see results

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Bis(4-(1,1,3,3-tetramethylbutyl)phenyl)amine

EC Number:

239-816-9

EC Name:

Bis(4-(1,1,3,3-tetramethylbutyl)phenyl)amine

Cas Number:

15721-78-5

Molecular formula:

C28H43N

IUPAC Name:

4-(2,4,4-trimethylpentan-2-yl)-N-[4-(2,4,4-trimethylpentan-2-yl)phenyl]aniline

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Batch No.: not specified
- Purity: not specified

Specific details on test material used for the study:

- CAS RN: CAS RN 15721-78-5
- Physical State/Appearance: Off white powder
- Purity: 93.2%
- Batch Number: HY14KSV07
- Date Received: 11 October 2015

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Male and female Wistar Han™:RccHan™:WIST strain rats were used
- Eighty animals (forty males and forty females) were accepted into the study
- The animals were acclimatized for nine days
- At the start of treatment animals were approximately six to eight weeks old; the males weighed 190 to 239 g, the females weighed 147 to 183 g (all weighing within ±20% of the mean weight for either sex)
- Housed in groups of three or four by sex
- Solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding
- Free access to food and water
- Pelleted diet rodent feed
- Tap drinking water was supplied from polycarbonate bottles attached to the cage.
- Environmental enrichment: wooden chew blocks; cardboard fun tunnels
- The animals were housed in a single air-conditioned room within the laboratory
- Rate of air exchange was at least fifteen air changes per hour
- 12 hours continuous light and 12 hours darkness
- Environmental conditions were continuously monitored by a computerized system; target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively; there were no deviations from these targets
- The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups
- Cage distribution within the holding rack was also randomized
- Animals were uniquely identified by an ear punching system

Schedule
Experimental Starting Date: 26 January 2015
Experimental Completion Date: 16 July 2015
In-Life Phase: 20 February 2015 to 22 May 2015

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Wistar Han™:RccHan™:WIST

Vehicle:

arachis oil

Details on oral exposure:

The test item was prepared at the appropriate concentrations as a suspension in Arachis oil BP, by gavage using a stainless steel cannula attached to a graduated, disposable plastic syringe.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results show the formulations spanning the concentration ranges used in this study to be homogenously prepared and stable for up to twenty-five days when stored refrigerated (approximately 4°C) in the dark. Formulations were therefore prepared and used within the stability period, divided into daily aliquots and stored at approximately 4 ºC, in the dark, prior to use.

Samples of each test item formulation were collected and analyzed for concentration of bis(4-(1,1,3,3-tetramethylbutyl)phenyl)amine (CAS RN 15721-78-5) at Envigo Research Limited, Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within 95% to 105% of the nominal concentration and, thus, within the specified ±10% of the nominal content.

Frequency of treatment:

The test item was administered once daily, for 90 consecutive days.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals per sex per dose.

Control animals:

yes

yes, concurrent vehicle

Details on study design:

The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.

Examinations

Observations and examinations performed and frequency:

Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Hematology and blood chemistry were evaluated for all animals at the end of the study. Ophthalmoscopic examination was also performed on animals from all dose groups before the start of dosing and on control group and high dose animals during Week 12 of the treatment period.

Sacrifice and pathology:

All animals were subjected to gross necropsy examination, and histopathological evaluation of selected tissues from high dose and control animals was performed.

Statistics:

Statistical analysis was performed on the following parameters:
- Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.
- Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed as follows:
- Data transformations were performed, if appropriate, using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data showed non-homogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical signs at any dose level that were considered to be related to treatment with the test item.
Behavioral assessment scores across all treatment groups remained similar to the controls.
There were no treatment-related changes in functional performance at any dose level.
Sensory reactivity scores were comparable across all dose groups including controls.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths during the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was no detrimental effect of test item administration on body weight performance in animals of both sexes at any dose level.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no effect of treatment on food intake or food conversion efficiency in animals of either sex receiving the test item at any dose level.

Food efficiency:

no effects observed

Description (incidence and severity):

There was no effect of treatment on food intake or food conversion efficiency in animals of either sex receiving the test item at any dose level.

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

no effects observed

Description (incidence and severity):

Visual inspection of water bottles did not indicate any intergroup differences in water intake.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ophthalmoscopic examination of males and females from control and high dose groups during Week 12 of the study did not reveal any treatment-related differences.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hematology evaluations did not reveal any toxicologically significant effects in animals of either sex resulting from treatment with the test item.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Blood chemistry evaluations did not indicate any treatment-related effects in animals of both sexes resulting from test item administration.

Urinalysis findings:

not specified

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Behavioral assessment scores across all treatment groups remained similar to the controls.
There were no treatment-related changes in functional performance at any dose level.
Sensory reactivity scores were comparable across all dose groups including controls.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Minor macroscopic changes were noted which were considered not to be related to administration of the test item.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Normal variations in a number of background findings were noted but no consistent changes were noted which could be attributed to treatment after histopathology evaluation.

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Description (incidence and severity):

There were no intergroup differences considered to be of toxicological relevance.

Details on results:

There were no unscheduled deaths during the study. Throughout the dosing period, there were no clinical signs at any dose level that were considered to be related to treatment with the test item.

There were no changes in the behavioral parameters considered to be related to treatment with the test item at any dose level. There were no intergroup differences considered to be related to treatment with the test item at any dose level. Sensory reactivity scores across all treatment groups were comparable with controls.

There was no detrimental effect of treatment with the test item at any dose level on body weight development in animals of either sex. Throughout the treatment period, weekly food consumption and food conversion efficiency values for test item-treated males and females were generally similar to the respective controls. Visual inspection of water bottles did not reveal any overt intergroup differences. No treatment-related ocular changes were apparent in animals of both sexes from the high dose group in relation to the controls.

There were no hematological findings of toxicological significance at the end of the treatment period. There were no intergroup differences considered to be related to treatment with the test item.

Minor changes were noted at necropsy that were considered not to be related to administration of the test item. At 1000 mg/kg bw/day, group mean absolute and body weight-related kidneys and ovaries weights in females were marginally but statistically significantly lower than controls (p<0.05). All individual values from the high dose animals were within the historical control data ranges and in the absence of any relevant microscopic findings, these findings were considered to be of no toxicological importance. Normal variations in a number of background histopathological findings were noted but no consistent changes were noted which could be attributed to treatment after histopathology evaluation.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

food efficiency

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Deviation No.1

The Study Plan incorrectly indicated that assessment of pupillary and corneal blink reflex would be performed as part of the ophthalmic examination conducted during the study.  These assessments are not part of the routine ophthalmic examinations required by the regulatory guidelines and were not performed, although assessment of pupil and blink reflex was performed as part of the Functional Observational Battery conducted during the study.  This deviation from Study Plan was considered not to have had any impact on the scientific validity of the report.

Deviation No. 2

On 09 April 2015, (Days 49 and 48 of dosing for males and females, respectively), one hour post-dose clinical observations were performed with a delay of approximately one hour and 50 minutes.  On 20 April 2015, (Days 60 and 59 of dosing for males and females, respectively), one hour post-dose clinical observations were performed with a delay of approximately 25 minutes.  These delays occurred due to technician error.  These incidents, occurred twice over the duration of the treatment period.  Post-dose clinical observations on these days were performed and there were no treatment-related clinical signs for any of the animals throughout the dosing period.  These deviations from the Study Plan were therefore considered not to have affected the scientific integrity of the study or the results obtained.

Deviation No.3

According to the Study Plan, animals were to be provided with Rodent 2014C Teklad Global Certified Pelleted Diet.  Whilst recording dietary intake on 09 May 2015, food hoppers for the females were topped up with Rodent 2018C Teklad Global Certified Pelleted Diet, in error.  This went unnoticed at the time and food hoppers for these females were topped up with the correct diet on 16 May 2015. The initial error resulted in all females on the study receiving a mixture of the two diets over Weeks 12 and 13 of dosing.  From the amount of the diet residue left in the food hopper and the total amount given to the females on 09 May 2015, it is calculated that the diet mixture provided to the females on this day would have contained 25% to 41% of Rodent 2018C Teklad Global Certified Pelleted Diet and this percentage is considered likely to have gradually decreased over the two weeks.  The main differences between the 2014C and 2018C pelleted diets is the slightly higher protein and fat content in the 2018C (approximately 4% and 2%, respectively) resulting in marginally higher energy density for this diet.  As females from all dose groups, including controls, received a mixture of the two diets over the noted period and an evaluation of the relevant data did not identify any effects considered to be related to this mixing of the diets, this deviation from the Study Plan was considered not to have affected the scientific integrity of the study or the results obtained.

Deviation No.4

The Study Plan incorrectly stated that urinalysis parameters (urine volume and specific gravity) will be statistically analysed.  No urinalysis was scheduled to be performed during this study and this deviation from the Study Plan had no impact on the scientific validity of the report.

See attachment for Tables.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the oral (gavage) administration of bis(4-(1,1,3,3-tetramethylbutyl)phenyl)amine (CAS RN 15721-78-5) to male and female Wistar Han™:RccHan™:WIST strain rats at dose levels of up to 1000 mg/kg bw/day was well tolerated. There was no effect of treatment on body weight development and dietary intake in animals of either sex. Hematology, blood chemistry and microscopic examination of the selected tissues did not identify any findings of toxicological relevance and a dose level of 1000 mg/kg bw/day is considered to be the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity.

Executive summary:

The study was designed to investigate the systemic toxicity of the test item, by repeated oral administration via gavage to the Wistar Han™: RccHan™: WIST strain rat for a period of ninety consecutive days at dose levels of 100, 300 or 1000 mg/kg bw/day. The study was conducted to OECD 408 Test Guidelines under GLP conditions. A control group of ten males and ten females was dosed concurrently with vehicle alone (Arachis oil BP). The oral route was selected as the most appropriate route of exposure, based on the physical propertiesof the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.

 

The dose levels were chosen in consultation with the Study Monitor for the Sponsor and were based on the preliminary data from a 14 day dose range-finding study in the rat (Envigo Study Number 41403760). In this range-finding study, administration of the test item to animals of either sex at dose levels of 250, 500 or 1000 mg/kg bw/day was well tolerated. There was no detrimental effect of treatment with the test item on body weight development or food consumption in males. Females receiving the test item at all dose levels showed sporadic instances of slightly reduced group mean body weight gains which resulted in slightly lower overall weight gains, albeit without any dose-relationship. Food consumption levels in these females were also generally slightly lower than those of the control animals, but there was no dose-dependence. There were no macroscopic findings for any of the animals and taking into consideration the overall results, a dose level of 1000 mg/kg bw/day was considered to be suitable for investigation in the current study together with 100 and 300 mg/kg bw/day as the low and intermediate dose levels, respectively.

 

Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Hematology and blood chemistry were evaluated for all animals at the end of the study. Ophthalmoscopic examination was also performed on animals from all dose groups before the start of dosing and on control group and high dose animals during Week 12 of the treatment period.

All animals were subjected to gross necropsy examination, and histopathological evaluation of selected tissues from high dose and control animals was performed.

 

There were no unscheduled deaths during the study. Throughout the dosing period, there were no clinical signs at any dose level that were considered to be related to treatment with the test item.

 

There were no changes in the behavioral parameters considered to be related to treatment with the test item at any dose level. There were no intergroup differences considered to be related to treatment with the test item at any dose level. Sensory reactivity scores across all treatment groups were comparable with controls.

 

There was no detrimental effect of treatment with the test item at any dose level on body weight development in animals of either sex. Throughout the treatment period, weekly food consumption and food conversion efficiency values for test item-treated males and females were generally similar to the respective controls. Visual inspection of water bottles did not reveal any overt intergroup differences. No treatment-related ocular changes were apparent in animals of both sexes from the high dose group in relation to the controls.

 

There were no hematological findings of toxicological significance at the end of the treatment period. There were no intergroup differences considered to be related to treatment with the test item. 

 

Minor changes were noted at necropsy that were considered not to be related to administration of the test item. At 1000 mg/kg bw/day, group mean absolute and body weight-related kidneys and ovaries weights in females were marginally but statistically significantly lower than controls (p<0.05). All individual values from the high dose animals were within the historical control data ranges and in the absence of any relevant microscopic findings, these findings were considered to be of no toxicological importance. Normal variations in a number of background histopathological findings were noted but no consistent changes were noted which could be attributed to treatment after histopathology evaluation.

 

Under the conditions of this study, the oral (gavage) administration of bis(4-(1,1,3,3-tetramethylbutyl)phenyl)amine (CAS RN 15721-78-5) to male and female Wistar Han™:RccHan™:WIST strain rats at dose levels of up to 1000 mg/kg bw/day was well tolerated. There was no effect of treatment on body weight development and dietary intake in animals of either sex. Hematology, blood chemistry and microscopic examination of the selected tissues did not identify any findings of toxicological relevance and a dose level of 1000 mg/kg bw/day is considered to be the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity.