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Diss Factsheets

Administrative data

Endpoint:
repeated dose toxicity: oral, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
OECD Series on Testing and Assessment, 29.7.2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
tert-butylbenzene
EC Number:
202-632-4
EC Name:
tert-butylbenzene
Cas Number:
98-06-6
Molecular formula:
C10H14
IUPAC Name:
tert-butylbenzene
Test material form:
liquid
Details on test material:
- Name of test material: tert-butylbenzene
- IUPAC name: tert-Butylbenzene
- Molecular formula: C10H14
- Molecular weight: 134.22 g/mol
- Substance type: Organic
- Physical state: Liquid

Test animals

Species:
rat
Strain:
Wistar
Remarks:
rat
Details on species / strain selection:
Wistar rats, Species: Rattus spp., Strain: Wistar, outbreed
This rat strain was selected since extensive historical control data are available for Wistar rats.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Crl:WI Charles River Germany, Velaz, Czech Republic
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8-12 weeks
- Fasting period before study: no
- Housing: Each animal was marked with an ID number. Each cage was affixed with a cage card
containing pertinent animal and study information. The animals in cages were marked by a line on
the tail with an ink marker.
Number of animals in cage was according period of study:
.Pre-treatment period (14 days) – 5 animals per cage
.Pre-mating (14 days) – 5 animals per cage
.Mating (maximum 14 days) - 1 male/1 female per cage
.Gestation (approximately 22 days) - 1 female per cage
.Post-partum (13 days) - 1 female with offspring per cage
Satellite animals – 5 animals per cage during all the study
- Diet: ad libitum. A laboratory food ssniff (ssniff Spezialdiäten GmbH)
- Water: ad libitum, drinking water
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C;
- Humidity (%): 55 ± 10 %
- Air changes (per hr): with central air-conditioning
- Photoperiod (hrs dark / hrs light): 12-hour light / 12-hour dark cycle

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected since administration by gavage has been proven to be appropriate for the detection of a toxicological hazard.
Vehicle:
olive oil
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Doses of the test item were prepared daily. The required amount of the test item (according to the dose) was diluted in vehicle (olive oil).
- Mixing appropriate amounts with (Type of food): The test item suspended in vehicle was prepared in required amount and theoretical concentrations: Low dose: 25 mg/mL, Mid dose: 175 mg/mL and
High dose: 325 mg/mL.
VEHICLE
- Justification for use and choice of vehicle (if other than water): Test item is not soluble in water; olive oil was used. Olive oil is a standard vehicle according to OECD TG 422
- Concentration in vehicle: Low dose: 25 mg/mL, Mid dose: 175 mg/mL and High dose: 325 mg/mL
- Amount of vehicle (if gavage): Dose volume was 2.0 mL/kg of body weight and was adjusted according to the weight development of the animals.
- Lot/batch no.: L91087
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
>The quality of drinking water is periodical monitored and recorded.
>During dosing period, formulations prepared at concentrations of 25, 175 and 325 mg/mL in vehicle (olive oil) were analyzed to verify test item concentration and assess test item homogeneity.
>The content of test item in the vehicle was checked twice during the study. The first time at the end of the gestation period, second during the lactation period. An adequate quantity of each test item dosing formulation for dose concentrations of 25, 175 and 325 mg/mL were analyzed. The test item concentrations in analysed samples were within 94.6-103.4 % of the target concentrations.
Duration of treatment / exposure:
Females were treated during:
>14-day pre-mating,
>14-day mating (maximum)
>22-day gestation (approximately)
>13-day lactation
Males were treated during:
>14-day pre-mating
>throughout the pairing period for a total of 56 doses
The animals designated for post-treatment observation (5 animals per sex in control and high groups,
respectively) remained untreated for subsequent 14 days.
Frequency of treatment:
once daily, 7 days per week
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Low
Dose / conc.:
350 mg/kg bw/day (nominal)
Remarks:
Mid
Dose / conc.:
650 mg/kg bw/day (nominal)
Remarks:
High
No. of animals per sex per dose:
13 Females
10 Males
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected based on Dose Range Finding Assay (Non GLP
study, study code: 600484810).
- Fasting period before blood sampling for clinical biochemistry: Yes
Positive control:
No positive control conducted.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were checked for morbidity or mortality at twice daily. The health condition of the animals, behaviour, reaction of the animals to the applied item, their well-beings were
monitored and recorded in raw data.
- Cage side observations checked in table No. were included.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: General clinical observations were performed once a day, 2 hours after dosing

BODY WEIGHT: Yes
- Time schedule for examinations: Each adult animal was weighted on the first day of dosing, weekly during the treatment period and prior to the scheduled euthanasia.
Specifically:
>Female rats – dams were weighted on Days 1, 7, 14 and 20 during pregnancy, and on Day 1 (24 hours of parturition), Day 4 and Day 13 during lactation and on Day 14 prior to necropsy.
>Males in four experimental groups and two satellite groups were weighted weekly and on necropsy day.
>Females in satellite groups were weighted weekly and on necropsy day.
>Live pup weight was recorded within 24 hours of parturition, Day 4 and Day 13 post-partum.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Food consumption was recorded weekly for All rats. The food consumption was not observed during the mating period in females as well as males.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
Haematology: Yes
- Time schedule for collection of blood: Haematological examination was made in five males randomly selected from each group and all females. The blood collection in males was done at the end of the
mating period. The blood collection in females was done at prior to of euthanasia of the animals. In satellite animals 14-days after the last treatment.
- Animals fasted: Yes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Clinical chemistry examination was obtained from the selected five males and all females of each group.
Blood samples for T4 analysis were taken based on the following schedule:
- from at least two pups per litter on day 4 after birth
- from all dams and at least two pups per litter at termination on Day 13
- from all adult males, at termination
Pup blood was pooled by litter for thyroid hormone analyses. Samples intended for hormone determination were obtained at a comparable time of the day.
- Animals fasted: Yes
URINALYSIS: Yes
- Time schedule for collection of urine: The urinalysis was performed in five randomly selected males of each group during the last week of the treatment and in satellite animals using urine volume
collected from animals in metabolic cages during 6 hours.
PATHOLOGY EXAMINATION: Yes
- Time schedule for examinations: All test animals were subjected to gross necropsy at the end of dead.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Other examinations:
Furthermore observations and examinations concerning Organ weights, Sestrous cycle, male reproduction, female reproduction and delivery and litter/pups were conducted. The details on those determinations are provided in IUCLID section 7.8.1.
Statistics:
Means and standard deviations were calculated.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No statistically significant test item-related mortality, no significant behavioural changes, no significant clinical signs or any significant deviations from normal findings were recorded during the study either in adult exposed animals or in the offspring.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
During the study mortality of 3 males were recorded. Two males from Mid dose (ID 32 and 35) and 1 male from High dose satellite (ID 49). These males died on day 14 of the treatment. Death not connected with the test item treatment(note: it is result of incorrect administration of the test item (staff mistake)). All animals at all dosage levels survived to the scheduled necropsy without significant visible clinical signs.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
During the study no statistically significant differences in body weight among the treated groups of adult male rats and their control counterparts. Statistically significant differences between Control group and all doses groups from Day 1 of treatment in up to end of treatment in females were observed. Body weight of adult male as well as female rats accordingly increased in time in all experimental groups. The body weight of High dose-satellite males and females was similar in comparison with recovery Control animals during the whole study.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption of males and females of all dose groups (including satellite animals) was similar to the controls group during the whole study. Increased physiological demands in pregnant and lactating female rats were evident from individual mean consumption per day as well as from total food consumption per week.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No test substance related findings were observed.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Only statistically significant increase of prothrombin time (PT) in males of all doses groups and decrease of PT in females against Control were observed.
No changes in satellite male were observed. Decrease of concentration of haemoglobin (HGB) and monocytes (Mon) against Control in satellite females was noticed.
The changes were of small magnitude and was not considered as a toxicologically important. During the study, haematology parameters in both sexes were within or close to the historical control data for this species. No test item related effects on the haematology parameters were observed in this study.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The test item did not induce significant clinical findings.
Statistically significant higher concentration of Total protein (TP), urea, inorganic phosphorus (PHOS) and potassium (K) in males of High dose were observed when compared to the Control group. In satellite males were observed statistically significant increase of alanine aminotransferase (ALT), alka
line phosphatase (ALP) and Natrium (Na) against Control.
Females of High dose had statistically significant increase of Total cholesterol (CHOL), Total bilirubin and decrease of TP and chloride (Cl) against Control. In satellite females decrease of glucose and increase of K against Control were registered.
These changes were sporadic, without the test item dose dependent relationship; they were considered to be a result of intra-individual and inter-individual variability for this species or they have only statistical character. The average values of clinical chemistry parameters in all animals were within or slightly outside the serum historical control data. There were no findings in clinical chemistry parameters which could be definitively attributed to the treatment.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
In the urine of some animals, small amounts of protein, ketones and presence of leukocytes were observed. There are no differences between Control and the dose groups and these findings can be considered close to normal (8). No test item related effect was observed.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
The test item did not induce significant behavioural deviations in the experimental animals.
Open Field Test: There were no behavioral differences between groups compared to control. No changes in the motor activity were present in the group of males and satellite females.
Tail flick Test: There is a significant difference between the Control and High dose groups in males.
This increase was reversible; no differences were registered in satellite males.
In comparison with Control group, the reaction time of females was not influenced by the administration of the test item.
Grip Strength Test: Significant difference between the Control group and Mid dose in males was observed. No differences were registered in females and satellite animals.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Significant increase relative weight of liver, kidney, right adrenal in males of High dose; increase relative weight of liver, left kidney and decrease relative weight of prostate in males of Mid dose were registered. Increase of relative weight of liver was registered in satellite males too.
Statistically significant change in relative weight of liver compared to the Control in females of High dose was observed. No significant differences were found in satellite females.
The higher relative weights of organs are considered adaptive and hence non-adverse.
Gross pathological findings:
no effects observed
Description (incidence and severity):
At the end of the study, the animals were subjected to gross pathology examination. During the necropsy, no macroscopic findings were finding.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
The etiology of follicular cysts in ovary (ID 101F/HD-561 Ov) remains a mystery, in part due to the difficulty of studying them during their formation (4). Follicular cysts may be associated with oestrogenic compounds or they may develop from preovulating follicles which fail to ovulate (9). Lesion is not related with the applicated substance.
Dilatation of distended acinus of prostate (ID10 M/C-400-Pr; ID 2M/C 261-Pr) and seminal vesicles (ID 39M/HD-125 Sv/A; ID42 M/HD-178 Sv/B; ID43 M/HD-184Sv/A) consists of distension of the gland with secretory material. This condition is usually associated with older age (15). Lesions are not related to the toxicological study.
Perivascular focus of chronic inflammatory cells (ID 4M/C 301-Li; ID 5M/C 334-Li; ID 1M/C-202-Li; ID 3M/C 268-Li) can be found in control animal livers (6).
Mucification of cervix (ID 51F/C 604-CU) is a common change in rodents (9).
Epithelial hyperplasia of vagina (ID 51F/C 606-V) occurs in a variety of settings, including control rats in persistent oestrus during reproductive senescence, and secondary to increase in endogenous estradiol production or administration of hormonally active xenobiotics (5).
Inflammatory prostatitis (ID 6M/C 372 Pr) can be bacterial or non-bacterial origin. Recent evidence suggests that reflux of urine into the prostate may lead to this inflammation (Takechi et al.,1999). Inflammatory lesions are frequently seen, particularly in the prostate either as an age related spontaneous lesion or as a result of urogenital infection (1, 17).
The presence of inflammatory cells in seminal vesicles (ID 8M/C 384-Sv/A; ID 8M/C 385-Sv/B; ID 6M/C 370 Sv/A; ID 2 M/C 259 Sv/A; ID 4M/C 325-Sv/A, ID 4 M/C 326-Sv/B) is very common as a background finding in rats and mice. It is often accompanied by denuded cells or cellular debris when intraluminal (2).
In this study observed changes are considered to be incidental findings or results of experimental manipulation other than administration of the test item. There were no test item - related alterations in the prevalence, severity or histological character of these incidental found lesions.
Histopathological findings: neoplastic:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
ca. 650 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: corpora lutea and implantations

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The study was designed to evaluate the potential toxic effect of the test item tert-butylbenzene when administered to rats for a minimum of 28 days and to evaluate the potential of the test item to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition, and early postnatal development. Moreover, the study might comment on system general toxicity due to evaluations of haematological parameters, clinical biochemistry analyses, urinalysis and histopathological examinations.
The study was performed in compliance with the OECD Test Guideline No 422, Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test, Adopted: 29 July, 2016.
The test item, tert-butylbenzene in the vehicle olive oil, was administered by stomach gavage once daily, 7 days per week, to three groups of Wistar rats at the doses of 50, 350 and 650 mg/kg. A concurrent vehicle control group received the vehicle (olive oil) in a comparable regimen and in the same volume 2 mL/kg. The females (13 per group) were screened for normal oestrous cycles in a 2-week pre-treatment period. Each group consisted of 10 males; all females were used for dosing and mating. After mating, 10 females, showing evidence copulation were used in each test group.
Males were administrated 14 daily doses prior to mating and continuing throughout the mating period, for a total of 28 doses. Females received 14 daily doses prior to mating and were dosed through mating, gestation and lactation (13 days).
Satellite animals (five males and five females from control group and high dose group) were not mated, administrated for a total 28 doses, males and 54 doses, females, and euthanized after two-week recovery period. In total, 50 males and 50 females were treated in the study.
Oestrous cycle was monitored two weeks before treatment start until sperm appeared in vaginal lavage during mating period as an evidence of copulation. Vaginal smear was also examined before necropsy on Day 13 post-partum.
All rats were inspected daily for any signs of toxicity. Clinical observations, body weight and food consumption were recorded weekly. Functional Observational Battery (Open field test, Tail flick test and Grip-strength test) were recorded for 5 males per group at the end of dosing, for 5 females per group during the last week of lactation shortly before scheduled kill and in satellite animals at the end of in-life phase.
Each adult animal was weighted on the first day of dosing, weekly during the treatment period and prior to the scheduled euthanasia. Specifically, female rats – dams were weighted on Days 1, 7, 14 and 20 during pregnancy, and within 24 hours of parturition, Days 4 and 13 after delivery.
The gestation length was recorded and calculated since Day 0 of pregnancy to the spontaneous parturition. Each litter was examined for number of pups, dead pups, live births and the presence of gross anomalies. The pups found dead/stillborn were subjected to necropsy, but autolysis of the bodies made it impossible to determine the causes of death. Live pups were counted and sexed, the litters were weighted throughout 24 hours after delivery, Day 4 and Day 13 post-partum.
The size of litter was adjusted on Day 4 after birth by eliminating extra pups selected randomly, to yield four pups per sex. The anogenital distance (AGD) was measured within 24 hours of parturition in all pups, the number of nipples per areolae were counted on postnatal Day 13 in male pups.
Clinical pathology evaluations (haematology, coagulation, and clinical chemistry) were performed in half males and all females and in satellite animals at the end of in-life phase.
The urinalysis was performed in five randomly selected males of each group during the last week of the treatment and in satellite males using urine volume collected from animals in metabolic cages during 6 hours.
For purpose of analysis of plasma thyroid hormone concentrations (T4) blood samples were taken from at least two surplus pups per litter on Day 4 after birth, from all females on Day 14 post-partum and shortly before scheduled euthanasia, at least two pups per litter at termination on Day 13, and from all adult males at day of necropsy and all females and males in satellite groups.
On scheduled necropsy day, the animals were examined macroscopically for abnormalities and pathological changes. Complete necropsies were conducted on all animals and satellite animals, and selected organs were weighed. Selected tissues were examined microscopically from 5 males and 5 females in the vehicle control and high-dose groups from main subgroup; organs of the reproductive system in all males and females in the vehicle control and high-dose groups.
During dosing period, formulations prepared at concentrations of 25, 175 and 325 mg/mL in vehicle (olive oil) were analyzed to verify test item concentration and assess test item homogeneity. The stability and homogeneity of the test item in the vehicle were determined by GC method. The content of the test item in the formulation was checked twice during the study. The test item concentrations in analysed samples were within 94.6-103.4 % of the target concentrations.
No statistically significant test item-related mortality, no significant behavioural changes, no significant clinical signs or any significant deviations from normal findings were recorded during the study either in adult exposed animals or in the offspring.
During the study no statistically significant differences in body weight among the treated groups of adult male rats and their control counterparts. Statistically significant differences between Control group and all doses groups from Day 1 of treatment in up to end of treatment in females were observed. Body weight of adult male as well as female rats accordingly increased in time in all experimental groups. The body weight of High dose-satellite males and females was similar in comparison with recovery Control animals during the whole study.
Food consumption of males and females of all dose groups (including satellite animals) was similar to the controls group during the whole study. Increased physiological demands in pregnant and lactating female rats were evident from individual mean consumption per day as well as from total food consumption per week.
Weight of mothers measured after parturition was significantly decreased in Mid and High group compared to controls. Number of corpora lutea and number of implantations was significantly affected as well in High group. Pup weight at Day 4 post-partum was significantly increased in Mid and High group, however this increase might be caused be lower number of pups in litter and more food (milk) available for nursing. Increased retention of areolae in Low and High group compared to controls was also observed. The test item had no biologically/toxicologically relevant effects on the other examined reproductive parameters.
There were no behavioural differences between groups compared to control. No changes in the motor activity were present in the group of males and satellite females.
Functional battery observation tests (tail flick test and grip strength test) conducted at the end of the dosing period and at the end of recovery period revealed no abnormalities in females; in males of the highest dose, increase of reaction time. This increase was reversible; no differences were registered in satellite males.
No toxicologically significant test item-related changes in haematology were noted. The clinical chemistry parameters measured in serum of male and female rats showed some statistically significant differences. These differences were minimal in nature and had no biological or toxicological significance. There were no test item-related effects on a thyroxine level in plasma of parental rats and pups. In the urine no significant changes against normal physiological conditions were detected.
No macroscopic findings were registered.
Significant increase relative weight of liver, kidney, right adrenal in males of High dose; increase relative weight of liver, left kidney and decrease relative weight of prostate in males of Mid dose were registered. Increase of relative weight of liver was registered in satellite males too. Statistically significant change in relative weight of liver compared to the Control in females of High dose was observed. No significant differences were found in satellite females. The higher relative weights of organs are considered adaptive and hence non-adverse.
In this study observed changes are considered to be incidental findings or results of experimental manipulation other than administration of the test item. There were no test item - related alterations in the prevalence, severity or histological character of these incidental found lesions.
Based on these results, the no-observed-adverse-effect-level (NOAEL) was 650 mg/kg for parental systemic toxicity.
Executive summary:

After consideration of the study results following conclusions were made regarding the test item tert-butylbenzene.

> The test item did not cause mortality of animals

> The test item did not induce significant clinical findings or behavioural deviations in the experimental animals

> The test item had no influence on the body weight of adult male and female rats

> The test item had not impacted the food consumption

> There were no behavioral differences between groups compared to control

> The test item had no influence on the locomotor activity, reaction time and grip strength

> The test item had no influence on monitored haematological, clinical chemistry parameters and did not cause changes in urine

> The test item affected number of corpora lutea and number of implantations in High dose (650 mg/kg)

> The test item did not significant influence on relative weight of organs

> The test item did not cause histopathological changes on examined organs

Based on these results, the no-observed-adverse-effect-level (NOAEL) was 650 mg/kg for parental systemic toxicity.