Name: OECD_SIDS / SUBSTANCE : 1,3,5-Tri-tert-butylbenzene / 1,3,5-tri-tert-butylbenzene /1460-02-2 Fri, 16 Dec 2022, 13:58:47+0900 /Legal entity owner: National Institute of Health Sciences
Printing date: 2022-12-16T13:58:47.156+09:00

iTable of Contents
0/0 ............................................................................................................................. 1
National Institute of Health Science ..................................................................... 2
1,3,5-Tri-tert-butylbenzene .................................................................................... 3
1 General information .................................................................................. 3
1.1 Identification ................................................................................. 3
Identification ............................................................................... 3
Identification ...................................................................... 3
1.10 Assessment approach (assessment entities) ................................. 4
Assessment approach (assessment entities) ................................ 4
7 Toxicological information ......................................................................... 5
7.5 Repeated dose toxicity ................................................................... 5
7.5.1 Repeated dose toxicity: oral ................................................. 5
Repeated dose toxicity: oral.001 ........................................... 5
7.6 Genetic toxicity ............................................................................ 13
7.6.1 Genetic toxicity in vitro ...................................................... 13
Genetic toxicity in vitro.001 ................................................ 13
Genetic toxicity in vitro.002 ................................................ 18
7.8 Toxicity to reproduction ............................................................... 22
7.8.1 Toxicity to reproduction ..................................................... 22
Toxicity to reproduction.001 ............................................... 22
References ................................................................................................................ 31
Reference Substances ...................................................................................... 31
1,3,5-tri-tert-butylbenzene ........................................................................... 31
Literatures ....................................................................................................... 33
A combined repeated dose/reproductive developmental toxicity study of 1,
3, 5-Tri-tert-butylbenzene by oral administration in rats. ............................... 33
In Vitro Chromosomal Aberration Test of on 1,3,5-Tri-tert-butylbenzene
Cultured Chinese Hamster Cells ................................................................. 34
Reverse Mutation Test of 1,3,5-Tri-tert-butylbenzene .................................... 35
Legal Entities ................................................................................................... 36
National Institute of Health Sciences .......................................................... 36ii

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Dossier submission type
Name
OECD SIDS
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Dossier subject
Dossier subject
1,3,5-Tri-tert-butylbenzene / 1,3,5-tri-tert-butylbenzene / 1460-02-2
Public nameSubmitting legal entity
National Institute of Health Science
Dossier creation date/time
Fri, 16 Dec 2022, 13:58:47+0900
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LEGAL_ENTITY: National Institute of Health
Science
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General information
Legal entity nameNational Institute of Health Science2

1,3,5-Tri-tert-butylbenzene
General information
Identification
Identification
SUBSTANCE: 1,3,5-Tri-tert-butylbenzene

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Substance name
1,3,5-Tri-tert-butylbenzene
Legal entity
National Institute of Health Sciences / Kawasaki / Japan

Identification of substance
Reference substance
1,3,5-tri-tert-butylbenzene / 1,3,5-tri-tert-butylbenzene / 1460-02-2 / 215-952-4
EC number

EC name215-952-4

EC Inventory

CAS number

CAS name

1460-02-2
IUPAC name
1,3,5-tri-tert-butylbenzene

Role in the supply chain
Manufacturer
false
Importer
false
Only representativefalse
Downstream user
false

3

Assessment approach (assessment entities)
FIXED_RECORD: Assessment approach
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Toxicological information
Repeated dose toxicity
Repeated dose toxicity: oral
ENDPOINT_STUDY_RECORD: Repeated dose toxicity: oral.001

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Administrative data
Endpoint
repeated dose toxicity: oral, otherType of information
experimental study
Adequacy of study
key study
Robust study summary
false
Used for classification
false
Used for SDS
false
Reliability
1 (reliable without restriction)
Rationale for reliability incl. deficiencies
guideline study
Reliability 1
Cross-reference
Reason / purpose for cross-reference
reference to same study
Related information
OECD / Toxicity to reproduction / Toxicity to reproduction.001 / 1,3,5-Tri-tert-butylbenzene / 1,3,5-tritert-butylbenzene / 1460-02-2

Data source
Reference
A combined repeated dose/reproductive developmental toxicity study of 1, 3, 5-Tri-tert-butylbenzene /Ministry of Health, Labor and Welfare, Japan / study report5Data accessdata published

Materials and methods
Test guideline
Qualifieraccording to guideline
Guideline
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction /Developmental Toxicity Screening Test)
Deviationsno
GLP compliance
yes
Limit test
no

Test material
Specific details on test material used for the study- Name of test material (as cited in study report): 1, 3, 5-Tri-tert-butylbenzene- Analytical purity: 98%- Storage condition of test material: at a cold (temperature 2-6°C) and dark place, with airtight stopper.
- Stability under test conditions: The stability of test material was identified by analysis of the remai
nder.

Test animals
Speciesratcommon rodent species
Strainother: Crl:CD(SD)Sexmale/female
Details on test animals or test system and environmental conditions
TEST ANIMALS- Source: Charles River Japan, Inc., Atsugi Breeding Center.
- Age at study initiation: 10 weeks old- Weight at study initiation: male 374 g (335-414 g), female 229 g (205-255 g)- Housing: Animals were individually housed in bracket-type metallic wire-mesh cages (265W × 426D ×200H mm), Dams were bred individually or with individual littermates in polycarbonate cages with flat
floors (265W x 426D x 200H mm) and standard bedding.
- Diet: Solid feed (MR stock: Nosan Corporation) was given ad libitum.
- Water: Tap water was given ad libitum.
- Acclimation period: 12 days
ENVIRONMENTAL CONDITIONS
6

- Temperature (°C): 22±3 (actual temperature: 22.4-24.4°C)- Humidity (%): 55±10% (actual humidity: 45-56%)- Air changes (per hr): >10- Photoperiod (hrs dark / hrs light): 12 hr dark/12 hr light (light: 7:00~19:00)

Administration / exposure
Route of administration
oral: gavage
Vehicleolive oil
Details on oral exposure- Amount of vehicle (if gavage): 5 mL/kg- Dosing volume: 5 mL/kg
Analytical verification of doses or concentrations
yes
Duration of treatment / exposure(P) Males: 42 days including 14 days pre-mating(P)Females: 42-52 days including 14 days pre-mating, mating and gestation periods and the days until
day 4 of lactation
Female (no mating, satellite group): 42 days
Frequency of treatment
Once/day, 7 days/week
Doses / concentrations
Dose / conc.
0mg/kg bw/day (actual dose received)

Dose / conc.
2

mg/kg bw/day (actual dose received)

Dose / conc.
10

mg/kg bw/day (actual dose received)

Dose / conc.
50

mg/kg bw/day (actual dose received)

Dose / conc.
250

mg/kg bw/day (actual dose received)

No. of animals per sex per dose
Main group:12 animals/sex/dose7

Satellite (Recovery) group: 5 males/dose and 5 females/dose (0 and 250 mg/kg bw/day)Control animals
yes, concurrent vehicle
Details on study design- Dose selection rationale: Based on the results of a 14-day preliminary study, the highest dose of 250
mg/kg bw/day was selected as an expected obvious toxic dose, and the lowest dose of 2 mg/kg bw/
day was selected as an expected no toxic dose. The middle dose levels of 10 and 50 mg/kg bw/day
were selected.
- Rationale for animal assignment (if not random): Body weight-balanced randomization[14-day preliminary study]
A 14-day repeated dose oral toxicity test (Crl:CD(SD) rats, doses: 0 (olive oil), 10, 30, 100 or 300 mg/kg
bw/day). High value of albumin was observed in males at 30 mg/kg bw/day or more. High value of A/
G ratio was observed in male at 100 mg/kg bw/day or more, and high value of calcium was observedin females at 100 mg/kg bw/day or more. High value of total cholesterol was observed in males andfemales at 300 mg/kg/day. High value of ALT, ChE and inorganic phosphorus, low value of chlorine
were observed in males at 300 mg/kg bw/day. High value of sodium was observed in females at 300
mg/kg bw/day. Large liver was observed in males at 30 mg/kg bw/day or more, and in females at 100
mg/kg bw/day or more. Increase in liver weight was observed in males at 100 mg/kg bw/day or more, and in females at 300 mg/kg bw/day. Thickening of the forestomachial mucosa was observed infemales at 300 mg/kg bw/day.

Examinations
Observations and examinations performed and frequency
CAGE SIDE OBSERVATIONS: Yes- Time schedule: 3 times/day (am: before and after administration; pm) during the administration
period. 2 times/day (am and pm) during the recovery period.
DETAILED CLINICAL OBSERVATIONS: Yes- Time schedule: Once before the start of administration, and once every week by the end of the study
period.
BODY WEIGHT: Yes- Time schedule for examinations:
Males were weighed on Day 1, 7, 14, 21, 28, 35, and 42 of administration, and weighed on Day 7 and
14 of recovery.
Female satellite groups were weighted same frequencies to male recovery groups.
Females in the main groups were weighed on Day 1, 7 and 14 of administration and copulated
females were weighed on Day 0, 7, 14 and 20 of gestation, and days 0 and 4 of lactation.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):- Food consumption: Yes
Measurement of food consumption was conducted on all animals at the following frequencies:
Males in the main and recovery groups; on Day 1, 7, 14, 21, 28, 35, and 41 of administration, and on
Day 7 and 13 of recovery.
Female satellite groups were weighted same frequencies to male recovery groups.
Females in the main group; on Day 1, 7 and 14 of administration and copulated females were weighedon Day 0, 7, 14 and 20 of gestation, and days 0 and 3 of lactation.
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
8

- Time schedule for collection of blood: At the end of administration period, or at the end of recovery
period in both sexes- Anaesthetic used for blood collection: ether- Animals fasted: Yes- How many animals: 5 animals/sex/group- Parameters examined included RBC, hemoglobin, hematocrit, MCV, MCH, MCHC, platelet, ret
iculocyte, PT, APTT, WBC and differential WBC.
CLINICAL CHEMISTRY: Yes- Time schedule for collection of blood: At the end of administration period, or at the end of recovery
period in both sexes- Animals fasted: Yes- How many animals: 5 animals/sex/group- Parameters examined included total protein, albumin, A/G ratio, total bilirubin, glucose, total cho
lesterol, triglyceride, phospholipid, AST, ALT, LDH, ChE, ALP, gamma-GTP, BUN, creatinine, Na, K, Cl,
Ca and IP.
URINALYSIS OF MALES: Yes- Time schedule for collection of urine (male only): On Day 37 of administration, and on Day 9 of recovery.
- Metabolism cages used for collection of urine: Yes- How many animals: 5 animals/group- Parameters examined included color, cloudy, urine volume, specific gravity, pH, protein, glucose, ketone body, bilirubin, occult blood and urobilinogen.
NEUROBEHAVIOURAL EXAMINATION: Yes- Time schedule for examinations: On week 6 of the administration period, and on week 2 of therecovery period- Dose groups that were examined: All dose groups (5 animals/sex/group)- Battery of functions tested: sensory activity (hearing reaction, eye sight reaction, sense of touch
reaction, pain reaction, pupil reflex, righting reflex), grip strength, motor activity
Sacrifice and pathology
GROSS PATHOLOGY: Yes
ORGAN WEIBHT: Yes [brain, thymus, heart, liver, kidney, adrenal gland, spleen, seminal vesicle, testis,
epididymis, pituitary, thyroid]
HISTOPATHOLOGY: Yes, [brain, pituitary, spinal cord, thyroid, parathyroid, heart, thymus, trachea, lung,
liver, kidney, adrenal, spleen, stomach, small intestine, large intestine, sciatic nerve, bone, bone marrow, lymph nodes (mesenteric and cervical lymph nodes), urinary bladder, testis, seminal vesicle, prostate, epididymis, mammary gland, ovary and uterus.]

Statistics
As for parametric data (grip strength, locomotor activity, body weight, body weight gain, food consu
mption, hematology and clinical chemistry data, organ weights, quantitative urinalysis data, numberof corpora lutea, number of implantation sites, number of pups born, number of pups alive, number ofstillborn), the values of means and standard deviations were calculated per group. When more than
three groups exist in the test group, Bartlett test for variance was done, and if the variance was ho
mogenous, ANOVA was applied. If the variance was not homogenous or data was non-parametric(differential WBC percentage, qualitative urinalysis data, stages of spermatogenesis, length of the
estrous cycle, implantation index, delivery index, live birth index, viability index,), Kruskal-Wallis rank s
um test was used. Consequently, if the result was significant, Dunnett multiple comparison or Dunnett typed method was used for detection of statistical significance against control group. When the nu
mber of the test group was two, F-test was used as for parametric data.
9

Then, student's t-test or Aspin-Welch's t-test was applied depending on the result of homogeneity ofvariance. While, as for non-parametric data, Man-Whitney's U-test was applied. Furthermore, as fo
r categorized data (incidence of abnormal findings in clinical observation, detailed observation, sensory functional examination, necropsy and histopathology, copulation index, fertility index, gestationindex), Fischer's exact test was used. In the histopathological examination findings, Mann-Whitney's
U test was used for graded data, and chi-squared test was used for sex ratio of pups. In any tests,
level of significance was set at 5%.

Results and discussion
Results of examinations
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
effects observed, treatment-related
Clinical biochemistry findings
effects observed, treatment-related
Urinalysis findings
no effects observed
Behaviour (functional findings)
no effects observed
Organ weight findings including organ / body weight ratios
effects observed, treatment-related
Gross pathological findings
no effects observed
Histopathological findings: non-neoplastic
effects observed, treatment-related
Histopathological findings: neoplastic
not examined
Details on results
CLINICAL SIGNS AND MORTALITY:
10

Mortality: There was no death.
Clinical signs: There were no effects related to the test substance in any groups at the dosing andrecovery periods.
DETAILED CLINICAL OBSERVATIONS, MANIPULATIVE TEST, GRIP STRENGTH TEST AND LOCOMOTOR ACTIVITY MEASUREMENT: There were no changes related to the test substance in any groups
at the dosing and recovery periods.
BODY WEIGHT:
There were no changes related to the test substance in any groups at the dosing and recovery per
iods.
FOOD CONSUMPTION: There were no changes related to the test substance in any groups at the dosing and recovery periods.
HAEMATOLOGY:[At the end of dosing period]: Decrease in MCHC was observed in females at 250 mg/kg bw/day.
[At the end of recovery period]: Decrease in MCHC was observed in females at 250 mg/kg bw/day.
CLINICAL CHEMISTRY:[At the end of dosing period]: Increase in total protein, albumin were observed in males and females at
250 mg/kg bw/day. Increase in ALT and cholinesterase activity, decrease in glucose were observedin males at 250 mg/kg bw/day. Decrease in triglyceride were observed in females at 250 mg/kg bw/day.
[At the end of recovery period]: Increase in ALT activity were observed in males at 250 mg/kg bw/day.
URINALYSES OF MALES: There were no changes related to the test substance in any groups at thedosing and recovery periods.
ORGAN WEIGHTS:[At the end of dosing period]: The relative and absolute weights of the liver increased at 10 mg/kg
bw/day and greater doses in females and at 50 mg/kg bw/day and greater doses in males. Increased
relative and absolute weights of the kidney in females of the 250 mg/kg bw/day group were also observed.
[At the end of recovery period]: Increased relative and absolute weights of the kidney were observed inmales and females at 250 mg/kg bw/day.
GROSS PATHOLOGY: There were no changes related to the test substance in any groups at the endof dosing and recovery periods.
HISTOPATHOLOGY: NON-NEOPLASTIC:[At the end of dosing period]: Hypertrophy of centrilobular hepatocytes were observed at 10 mg/kg
bw/day and greater in females and at 50 mg/kg bw/day and greater in males. Dilatation of the distal/collecting tubules and the hyperplasia of the collecting tubular epithelium in the kidney were observedin females at 250 mg/kg bw/day.
[At the end of recovery period]: Hypertrophy of centrilobular hepatocytes were observed in males andfemales at 250 mg/kg bw/day.

Effect levels
Key result
true
Dose descriptor
NOAEL
Effect level2

mg/kg bw/day (actual dose received)11Based on
test mat.
Sexmale/female
Basis for effect level
histopathology: non-neoplastic
Hypertrophy of centrilobular hepatocytes were observed at 10 mg/kg bw/day in femalesorgan weights and organ / body weight ratios
The relative and absolute weights of the liver increased at 10 mg/kg bw/day in females

Any other information on results incl. tables
Figures and Tables (in English) are available in the following full report of the study.
http://dra4.nihs.go.jp/mhlw_data/home/pdf/PDF1460-02-2d.pdf

Applicant's summary and conclusion
Executive summary
A combined repeated oral-dose toxicity study with a reproduction/developmental toxicity screening
test performed in accordance with OECD TG 422. Male and female rats (12 animals/sex/dose) wereadministered 1, 3, 5-tri-tert-butylbenzene at 0 (vehicle: olive oil), 2, 10, 50, and 250 mg/kg bw/day. The
males were dosed for 42 days, including a 14-day pre-mating period and a subsequent mating period.
The females were dosed for 42–52 days, including 14-day pre-mating, mating, and gestation periods,
and until lactation day 4. Out of the 12 males, 5 were dosed at 0 and 250 mg/kg bw/day and were treatedas a recovery group. Five additional females at 0 and 250 mg/kg bw/day were assigned to a satellitegroup and were dosed 1, 3, 5-tritert-butylbenzene for 42 days without mating, and they were examined
after a 14-day recovery period.
No effects were found on clinical signs, FOB, body weight, food consumption, or urinalysis. Increase intotal protein and albumin were observed in males and females at 250 mg/kg bw/day. Increase in ALT
and cholinesterase activity, decrease in glucose were observed in males at 250 mg/kg bw/day. Decreasein triglyceride were observed in females at 250 mg/kg bw/day. The relative and absolute weights of theliver increased at 10 mg/kg bw/day and greater doses in females and at 50 mg/kg bw/day and greater
doses in males. Increased relative and absolute weights of the kidney in females of the 250 mg/kg
bw/day group were also observed. Histopathological examination showed hypertrophy of centrilobular
hepatocytes at 10 mg/kg bw/day and greater in females and at 50 mg/kg bw/day and greater in males;
dilatation of the distal/collecting tubules and the hyperplasia of the collecting tubular epithelium in thekidney were observed in females at 250 mg/kg bw/day. These changes were no longer found after therecovery period. The effects of 1, 3, 5-tritert-butylbenzene on the liver at 10 mg/kg bw/day, led to a
determination of the NOAEL for repeated-dose toxicity at 2 mg/kg bw/day in rats.

12

Genetic toxicity
Genetic toxicity in vitro
ENDPOINT_STUDY_RECORD: Genetic toxicity in vitro.001

UUID: 9fa33584-aa8a-4916-830f-7118b93ec78d
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Author:Date: 2019-09-03T16:21:24.000+09:00
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Administrative data
Endpoint
in vitro gene mutation study in bacteria
Type of information
experimental study
Adequacy of study
key study
Robust study summary
true
Used for classification
false
Used for SDS
false
Reliability
1 (reliable without restriction)
Rationale for reliability incl. deficiencies
guideline study
Reliability 1

Data source
Reference
Reverse Mutation Test of 1,3,5-Tri-tert-butylbenzene / study report
Data accessdata published

Materials and methods
Test guideline
Qualifieraccording to guideline
Guideline
OECD Guideline 471 (Bacterial Reverse Mutation Assay)13

in vitro gene mutation study in bacteria
GLP compliance
yesType of assay
bacterial reverse mutation assay
in vitro gene mutation study in bacteria

Test material
Specific details on test material used for the study- Lot No.: 08627MD (Sigma-Aldrich corporation)- Purity: >99.2%- Solubility: soluble in acetone and insoluble in water and DMSO.
- Physical state: white powder- Storage condition of test material: room temperature (16-27 degree C)

MethodSpecies / strain
Species / strain / cell typeS. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2bacteria
Metabolic activation
with and without
Metabolic activation system
S9 mix: Rat liver, induced with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose
Dosage of each strain with or without S9-S9 mix: 0, 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500, 5000 μg /plate+S9 mix: 0, 313, 625, 1250, 2500, 5000 μg /plate
Maximum concentration was established based on the result of the preliminary test at concentration
up to 5000 ug/plate.
The growth inhibition was observed without S9 mix at 313 μg/plate and higher in TA100, TA98, andTA1537, and at 1250 μg/plate and higher in TA1535.
Vehicle / solventacetoneControls
Untreated negative controlsnoNegative solvent / vehicle controls
yes
True negative controlsno14

Positive controls
yes
Positive control substancesodium azide
without S9 mix (TA 1535)
benzo(a)pyrene
with S9 mix (TA100, TA98, TA1537)
other: without S9 mix:2-(2-Furyl)-3-(5-nitro -2-furyl)acrylamide (TA100, TA98, WP2uvrA), without S9
mix: ICR-191 (TA1537) with S9 mix: 2-aminoanthracene (TA1535, WP2uvrA)
Details on test system and experimental conditions
METHOD OF APPLICATION: Preincubation
DURATION- Preincubation period: 20 min at 37°C
- Exposure duration:48 hrs
NUMBER OF PLATES: 3
NUMBER OF REPLICATIONS: 2
DETERMINATION OF CYTOTOXICITY- Method: other: growth inhibition
Evaluation criteria
A chemical was judged to be mutagenic when the mean number of revertant colonies per plateincreased more than twice that of the negative control and when the dose-related and reproducible increase was observed.
Statistics
not used

Results and discussion
Test results
Key result
trueSpecies / strain
S. typhimurium TA 1535
bacteria
Metabolic activation
with and without
Genotoxicity
negative
Cytotoxicity / choice of top concentrations
cytotoxicity Without S9mix: 1250 μg/plate
Vehicle controls validity
valid
Positive controls validity
valid
Key result
trueSpecies / strain
S. typhimurium TA 1537
15

bacteria
Metabolic activation
with and without
Genotoxicity
negative
Cytotoxicity / choice of top concentrations
cytotoxicity without S9 mix' >=156 μg/plate
Vehicle controls validity
valid
Positive controls validity
valid
Key result
trueSpecies / strain
S. typhimurium TA 98
bacteria
Metabolic activation
with and without
Genotoxicity
negative
Cytotoxicity / choice of top concentrations
cytotoxicity without S9 mix' >=156 μg/plate
Vehicle controls validity
valid
Positive controls validity
valid
Key result
trueSpecies / strain
S. typhimurium TA 100
bacteria
Metabolic activation
with and without
Genotoxicity
negative
Cytotoxicity / choice of top concentrations
cytotoxicity Without S9mix: at 313 μg/plate
Vehicle controls validity
valid
Positive controls validity
valid

16

Key result
trueSpecies / strain
E. coli WP2 uvr A pKM 101
bacteria
Metabolic activation
with and without
Genotoxicity
negative
Cytotoxicity / choice of top concentrations
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity
valid
Positive controls validityvalid

Any other information on results incl. tables
Figures and Tables (in Japanese) are available in the following full report of the study. http://dra4.nihs.go.jp/mhlw_data/home/pdf/PDF1460 -02 -2e.pdf
Tables (in English) are attachted to this document. Please download the export file to see the Tables.

Overall remarks, attachments
Overall remarks
Genotoxic effects:
With metabolic activation: Negative
Without metabolic activation: Negative

Applicant's summary and conclusion
Executive summary
In a bacterial reverse mutation assay using S. typhimuriumTA100, TA1535, TA98, and TA1537, and E.
coli WP2uvrA/pKM101 (OECD TG 471), negative results were obtained for 1,3,5-Tri-tert-butylbenzene
with or without metabolic activation.

17

ENDPOINT_STUDY_RECORD: Genetic toxicity in vitro.002

UUID: afa7813a-fa19-4a8e-b863-dd5258e2c4a9
Dossier UUID:
Author:Date: 2019-02-18T11:14:59.000+09:00
Remarks:

Administrative data
Endpoint
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information
experimental study
Adequacy of study
key study
Robust study summary
true
Used for classification
false
Used for SDS
false
Reliability
1 (reliable without restriction)
Rationale for reliability incl. deficiencies
guideline study
Reliability 1

Data source
Reference
In Vitro Chromosomal Aberration Test of on 1,3,5-Tri-tert-butylbenzene Cultured Chinese Hamster Cell /Ministry of Health, Labour and Welfare (MHLW), Japan / study report
Data accessdata published

Materials and methods
Test guideline
Qualifieraccording to guideline
Guideline
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)in vitro cytogenicity / chromosome aberration study in mammalian cells

18

Qualifieraccording to guideline
Guideline
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
genetic toxicity in vitro, other
GLP compliance
yesType of assay
in vitro mammalian chromosome aberration testin vitro cytogenicity / chromosome aberration study in mammalian cells

Test material
Specific details on test material used for the study- Lot No.: 08627MD (Wako Pure Chemical Corporation)- Purity: >99.2%- Solubility: soluble in acetone and insoluble in water, DMSO and ethanol.
- Physical state: white powder- Storage condition of test material: at room temperature (15-25 degree C)

MethodSpecies / strain
Species / strain / cell typeother:
Details on mammalian cell type (if applicable)
Chinese hamster lung(CHL/IU) cell
Metabolic activation
with and without
Metabolic activation system
S9 mix: Rat liver, induced with phenobarbital and 5,6- benzoflavone
Test concentrations with justification for top dose
0, 156.3, 312.5, 625.0, 1250, 2500 μg/mL (short-term, and continuous first test)
0, 4.883, 93766, 19.53, 39.06, 78.13 μg/mL (continuous, second test)
Cell-growth inhibition test was conducted up to the limited concentration of 2500 μg/mL (10 mM)
Short-term -S9mix: Concentration of 50% cell-growth inhibition was 62.9 μg/mL
Short-term +S9mix: Concentration of 50% cell-growth inhibition was 57.6 μg/mL
Continuous: Concentration of 50% cell-growth inhibition was 36.9 μg/mL
Vehicle / solventacetoneControls
Untreated negative controlsno19

Negative solvent / vehicle controls
yes
True negative controlsnoPositive controls
yes
Positive control substancecyclophosphamide(with S9 mix)
mitomycin C(without S9 mix)
Details on test system and experimental conditions
METHOD OF APPLICATION: Exposure duration: [short-term treatment]:6 hrs + 18 hr, [continuous
treatment]: 24h
NUMBER OF CELLS EVALUATED: 200 cells /concentration (100 cells/plate x 2)
Evaluation criteria
Positive: total chromosomal aberrations increased >=10% and concentration dependent increase or
reproducibility was observed
Equivocal: total chromosomal aberrations increased >=5-10%
Negative: total chromosomal aberrations increased <5%
Statistics
Not used

Results and discussion
Test results
Key result
trueSpecies / strainother: Chinese hamster lung(CHL/IU) cells
Metabolic activation
with and without
Genotoxicity
negative
Cytotoxicity / choice of top concentrations
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations short term
treatment
Vehicle controls validity
valid
Positive controls validity
valid
Key result
true20Species / strainother: Chinese hamster lung(CHL/IU) cells
Metabolic activation
with and without
Genotoxicity
negative
Cytotoxicity / choice of top concentrations
cytotoxicity 78.13 μg/mL (24h)Vehicle controls validity
valid
Positive controls validityvalid

Any other information on results incl. tables
Figures and Tables (inEnglish) are available in the following full report of the study.
http://dra4.nihs.go.jp/mhlw_data/home/pdf/PDF1460-02-2f.pdf

Applicant's summary and conclusion
Conclusions
1,3,5-Tri-tert-butylbenzene did not induce structural aberrations for the short-term study with andwithout S9 mix. Positive, vehicle and negative control groups were valid.
Executive summary
In anin vitro chromosomal aberration test using CHL/IU cells (OECD TG 473), negative results wereobtained with or without metabolic activation for 1,3,5-tri-tert-butylbenzene.

21

Toxicity to reproduction
Toxicity to reproduction
ENDPOINT_STUDY_RECORD: Toxicity to reproduction.001

UUID: 08848368-20dc-42cf-8ef0-bca234fe7ef3
Dossier UUID:
Author:Date: 2022-12-16T13:57:46.187+09:00
Remarks:

Administrative data
Endpoint
reproductive toxicity, other A combined repeated dose/reproductive developmental toxicity study
Type of information
experimental study
Adequacy of study
key study
Robust study summary
false
Used for classification
false
Used for SDS
false
Reliability
1 (reliable without restriction)
Rationale for reliability incl. deficiencies
guideline study
Reliability 1
Cross-reference
Reason / purpose for cross-reference
reference to same study
Related information
OECD / Repeated dose toxicity: oral / Repeated dose toxicity: oral.001 / 1,3,5-Tri-tert-butylbenzene /1,3,5-tri-tert-butylbenzene / 1460-02-2

Data source
Reference
A combined repeated dose/reproductive developmental toxicity study of 1, 3, 5-Tri-tert-butylbenzene /Ministry of Health, Labor and Welfare, Japan / study report
Data accessdata published22

Materials and methods
Test guideline
Qualifieraccording to guideline
Guideline
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction /Developmental Toxicity Screening Test)
Deviationsno
GLP compliance
yes
Limit test
no

Test material
Specific details on test material used for the study- Name of test material (as cited in study report): 1,3,5-tri-tert-butylbenzene- Analytical purity: 98%- Storage condition of test material: at a cold (temperature 2-6°C) and dark place, with airtight stopper.
- Stability under test conditions: The stability of test material was identified by analysis of theremainder.

Test animals
Speciesrat
Strainother: Crl:CD(SD)Sexmale/female
Details on test animals or test system and environmental conditions
TEST ANIMALS- Source: Charles River Japan, Inc., Atsugi Breeding Center.
- Age at study initiation: 10 weeks old- Weight at study initiation: male 374 g (335-414 g), female 229 g (205-255 g)- Housing: Animals were individually housed in bracket-type metallic wire-mesh cages (265W × 426D
× 200H mm) , Dams were bred individually or with individual littermates in polycarbonate cages withflat floors (265W x 426D x 200H mm) and standard bedding.
- Diet: Solid feed (MR stock: Nosan Corporation) was given ad libitum.
- Water: Tap water was given ad libitum.
- Acclimation period: 12 days
ENVIRONMENTAL CONDITIONS- Temperature (°C): 22±3 (actual temperature: 22.4-24.4°C)- Humidity (%): 55±10% (actual humidity: 45-56%)- Air changes (per hr): >10- Photoperiod (hrs dark / hrs light): 12 hr dark/12 hr light (light: 7:00~19:00)
23

Administration / exposure
Route of administration
oral: gavage
Vehicleolive oil
Details on exposure- Amount of vehicle (if gavage): 5 mL/kg- Dosing volume: 5 mL/kg
Analytical verification of doses or concentrations
yes
Duration of treatment / exposure(P) Males: 42 days including 14 days pre-mating(P)Females: 42-54 days including 14 days premating, mating and gestation periods and the days until
day 4 of lactation
Female (no mating, satellite group): 42 days
Frequency of treatment
Once/day, 7 days/week
Doses / concentrations
Dose / conc.
0mg/kg bw/day (actual dose received)

Dose / conc.
2

mg/kg bw/day (actual dose received)

Dose / conc.
10

mg/kg bw/day (actual dose received)

Dose / conc.
50

mg/kg bw/day (actual dose received)

Dose / conc.
250

mg/kg bw/day (actual dose received)

No. of animals per sex per dose
Main group:12 animals/sex/dose
Satellite (Recovery) group: 5 males/dose and 5 females/dose (0 and 250 mg/kg bw/day)Control animals
yes, concurrent vehicle24

Details on study design- Dose selection rationale: Based on the results of a 14-day preliminary study, the highest dose of 250
mg/kg bw/day was selected as an expected obvious toxic dose, and the lowest dose of 2 mg/kg bw/
day was selected as an expected no toxic dose. The middle dose levels of 10 and 50 mg/kg bw/day
were selected.
- Rationale for animal assignment (if not random): Body weight-balanced randomization[14-day preliminary study]
A 14-day repeated dose oral toxicity test (Crl:CD(SD) rats, doses: 0 (olive oil), 10, 30, 100 or 300 mg/kg
bw/day). High value of albumin was observed in males at 30 mg/kg bw/day or more. High value of A/
G ratio was observed in male at 100 mg/kg bw/day or more, and high value of calcium was observedin females at 100 mg/kg bw/day or more. High value of total cholesterol was observed in males andfemales at 300 mg/kg/day. High value of ALT, ChE and inorganic phosphorus, low value of chlorine
were observed in males at 300 mg/kg bw/day. High value of sodium was observed in females at 300
mg/kg bw/day. Large liver was observed in males at 30 mg/kg bw/day or more, and in females at
100 mg/kg bw/day or more. Increase in liver weight was observed in males at 100 mg/kg bw/day or
more, and in females at 300 mg/kg bw/day. Thickening of the forestomachial mucosa was observedin females at 300 mg/kg bw/day.

Examinations
Parental animals: Observations and examinations
CAGE SIDE OBSERVATIONS: Yes- Time schedule: 3 times/day (am: before and after administration; pm) during the administration
period. 2 rimes/day (am and pm) during the recovery period.
DETAILED CLINICAL OBSERVATIONS: Yes- Time schedule: Once before the start of administration, and once every week by the end of the study
period.
BODY WEIGHT: Yes- Time schedule for examinations:
Males were weighed on Day 1, 7, 14, 21, 28, 35, and 42 of administration, and weighed on Day 7 and
14 of recovery.
Female satellite groups were weighted same frequencies to male recovery groups.
Females in the main groups were weighed on Day 1, 7 and 14 of administration and copulated
females were weighed on Day 0, 7, 14 and 20 of gestation, and days 0 and 4 of lactation.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):- Food consumption: Yes
Measurement of food consumption was conducted on all animals at the following frequencies:
Males in the main and recovery groups; on Day 1, 7, 14, 21, 28, 35, and 41 of administration, and on
Day 7 and 13 of recovery.
Female satellite groups were weighted same frequencies to male recovery groups.
Females in the main group; on Day 1, 7 and 14 of administration and copulated females were weighedon Day 0, 7, 14 and 20 of gestation, and days 0 and 3 of lactation.
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes- Time schedule for collection of blood: At the end of administration period, or at the end of recovery
period in both sexes- Anaesthetic used for blood collection: ether- Animals fasted: Yes- How many animals: 5 animals/sex/group- Parameters examined included RBC, hemoglobin, hematocrit, MCV, MCH, MCHC, platelet, ret
iculocyte, PT, APTT, WBC and differential WBC.
25

CLINICAL CHEMISTRY: Yes- Time schedule for collection of blood: At the end of administration period, or at the end of recovery
period in both sexes- Animals fasted: Yes- How many animals: 5 animals/sex/group- Parameters examined included total protein, albumin, A/G ratio, total bilirubin, glucose, total cho
lesterol, triglyceride, phospholipid, AST, ALT, LDH, ChE, ALP, gamma-GTP, BUN, creatinine, Na, K, Cl,
Ca and IP.
URINALYSIS OF MALES: Yes- Time schedule for collection of urine (male only): On Day 37 of administration, and on Day 9 of recovery.
- Metabolism cages used for collection of urine: Yes- How many animals: 5 animals/group- Parameters examined included color, cloudy, urine volume, specific gravity, pH, protein, glucose, ketone body, bilirubin, occult blood and urobilinogen.
NEUROBEHAVIOURAL EXAMINATION: Yes- Time schedule for examinations: On week 6 of the administration period, and on week 2 of therecovery period- Dose groups that were examined: All dose groups (5 animals/sex/group)- Battery of functions tested: sensory activity (hearing reaction, eye sight reaction, sense of touch
reaction, pain reaction, pupil reflex, righting reflex), grip strength, motor activity
Oestrous cyclicity (parental animals)
Vaginal smears were collected from all females in the main groups and microscopically examined
every day from the day after the start of administration until the day copulation was confirmed.
Sperm parameters (parental animals)
Parameters examined in all P male parental generations: testis weight, epididymis weight, histopatho
logical examinations for testes, epididymides, seminal vesicle including coagulating gland and ventral
prostate.
Litter observations
PARAMETERS EXAMINED: The following parameters were examined in F1 offspring: number and sexof pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physicalor behavioral abnormalities.
Postmortem examinations (parental animals)
METHOD OF SACRIFICED: All animals were sacrificed by exsanguination under ether anesthesia.
SACRIFICE: Male animals: On Day 42, Maternal animals: on Day 5 of lactation, and Male recovery andfemale satellite animals: on next Day 14 of recovery.
GROSS PATHOLOGY: Yes
ORGAN WEIBHT: Yes [brain, thymus, heart, liver, kidney, adrenal gland, spleen, seminal vesicle, testis,
epididymis, pituitary, thyroid]
HISTOPATHOLOGY: Yes, [brain, pituitary, spinal cord, thyroid, parathyroid, heart, thymus, trachea,
lung, liver, kidney, adrenal, spleen, stomach, small intestine, large intestine, sciatic nerve, bone, bonemarrow, lymph nodes (mesenteric and cervical lymph nodes), urinary bladder, testis, seminal vesicle,
prostate, epididymis, mammary gland, ovary and uterus.]
Postmortem examinations (offspring)
SACRIFICE
26

- The F1 offspring were sacrificed at 4 days of age.
GROSS NECROPSY- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, andabdominal viscera.
HISTOPATHOLOGY / ORGAN WEIGTHS- Not examined.
Statistics
As for parametric data (grip strength, locomotor activity, body weight, body weight gain, food consu
mption, hematology and clinical chemistry data, organ weights, quantitative urinalysis data, numberof corpora lutea, number of implantation sites, number of pups born, number of pups alive, number ofstillborn), the values of means and standard deviations were calculated per group. When more than
three groups exist in the test group, Bartlett test for variance was done, and if the variance was ho
mogenous, ANOVA was applied. If the variance was not homogenous or data was non-parametric(differential WBC percentage, qualitative urinalysis data, stages of spermatogenesis, length of the
estrous cycle, implantation index, delivery index, live birth index, viability index,), Kruskal-Wallis rank s
um test was used. Consequently, if the result was significant, Dunnett multiple comparison or Dunnett typed method was used for detection of statistical significance against control group. When the nu
mber of the test group was two, F-test was used as for parametric data.
Then, student's t-test or Aspin-Welch's t-test was applied depending on the result of homogeneity ofvariance. While, as for non-parametric data, Man-Whitney's U-test was applied. Furthermore, as fo
r categorized data (incidence of abnormal findings in clinical observation, detailed observation, sensory functional examination, necropsy and histopathology, copulation index, fertility index, gestationindex), Fischer's exact test was used. In the histopathological examination findings, Mann-Whitney's
U test was used for graded data, and chi-squared test was used for sex ratio of pups. In any tests,
level of significance was set at 5%.
Reproductive indices
Estrous cycle: Mean days from metaeatrus I (III) to next III.
Copulation index (%) = (No. of pairs with successful copulation/No. of pairs mated) × 100
Fertility index (%) = (No. of pregnant females/No. of pairs with successful copulation) × 100
Gestation index (%) = (No. of females with live pups/No. of pregnant females) × 100
Implantation index (%) = (No. of implantation sites/No. of corpora lutea) × 100
Delivery index (%) = (No. of pups born/No. of implantation sites) × 100
Live birth index (%) = (No. of live pups on day 0/No. of pups born) × 100
Sex ratio =Total number of male pups/Total number of female pups

Offspring viability indices
Viability index (%) = (No. of live pups on day 4/No. of live pups on day 0) × 100

Results and discussion
Results: P0 (first parental generation)
General toxicity (P0)
Clinical signs
no effects observed
Mortality
no mortality observed
27

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
effects observed, treatment-related
Description (incidence and severity)
See 7.5.1
Clinical biochemistry findings
effects observed, treatment-related
Description (incidence and severity)
See 7.5.1
Urinalysis findings
no effects observed
Behaviour (functional findings)
no effects observed
Immunological findings
not examined
Organ weight findings including organ / body weight ratios
effects observed, treatment-related
Description (incidence and severity)
See 7.5.1
Gross pathological findings
no effects observed
Neuropathological findings
not examined
Histopathological findings: non-neoplastic
effects observed, treatment-related
Description (incidence and severity)
See 7.5.1
Histopathological findings: neoplastic
not examined

Reproductive function / performance (P0)
Reproductive function: oestrous cycle
no effects observed28

Reproductive function: sperm measures
no effects observed
Reproductive performance
effects observed, treatment-related
Description (incidence and severity)
The numbers of pups alive were lower on PND 0 and 4 at 250 mg/kg bw/day; the delivery index andlive-birth index were lower than for the control. At the same dose level, the body weights of the live p
ups also decreased on PND 0 and 4

Effect levels (P0)
Key result
true
Dose descriptor
NOAEL
Effect level50

mg/kg bw/day (actual dose received)

Based ontest mat.
Sexmale/female
Basis for effect level
reproductive performance
The numbers of pups alive were lower on PND 0 and 4 at 250 mg/kg bw/day; the delivery index andlive-birth index were lower than for the control. At the same dose level, the body weights of the live p
ups also decreased on PND 0 and 4

Results: F1 generation
General toxicity (F1)Clinical signs
no effects observed
Mortality / viability
mortality observed, treatment-related
Description (incidence and severity)
The numbers of pups alive were lower on PND 0 and 4 at 250 mg/kg bw/day.
Body weight and weight changes
effects observed, treatment-related
Description (incidence and severity)
The body weights of the live pups decreased on PND 0 and 4 at 250 mg/kg bw/day.
Gross pathological findings
no effects observed

Effect levels (F1)29

Key result
true
Dose descriptor
NOAEL
Generation
F1
Effect level50

mg/kg bw/day (actual dose received)

Based ontest mat.
Sexmale/female
Basis for effect levelviability
The numbers of pups alive were lower on PND 0 and 4 at 250 mg/kg bw/day.
body weight and weight gain
The body weights of the live pups decreased on PND 0 and 4 at 250 mg/kg bw/day.

Any other information on results incl. tables
Figures and Tables (in English) are available in the following full report of the study.
http://dra4.nihs.go.jp/mhlw_data/home/pdf/PDF1460-02-2d.pdf

Applicant's summary and conclusion
Executive summary
In a repeated-oral-dose toxicity study and a reproduction/developmental toxicity screening test (OECDTG 422), as described above, the numbers of pups alive were lower on PND 0 and 4 at 250 mg/kg bw/
day; the delivery index and live-birth index were lower than for the control. At the same dose level, thebody weights of the live pups also decreased on PND 0 and 4. The developmental toxicity at 250 mg/
kg bw/day led to the conclusion that the NOAEL for the rat reproduction/developmental toxicity of 1, 3,5-tritert-butylbenzene should be determined at 50 mg/kg bw/day; at this point, parental general toxicity
was observed.

30

References
Reference Substances
REFERENCE_SUBSTANCE: 1,3,5-tri-tert-butylbenzene
UUID: ECB5-0252de27-51d6-49c9-b482-64c900eadbb2
Dossier UUID:
Author:Date: 2007-05-10T18:00:00.000+09:00
Remarks:
Reference substance name
1,3,5-tri-tert-butylbenzene
IUPAC name
1,3,5-tri-tert-butylbenzene

Inventory
Inventory number
Inventory name
1,3,5-tri-tert-butylbenzene
Inventory
EC Inventory
Inventory number
215-952-4
CAS number
1460-02-2
Molecular formula
C18H30Description
CAS number
1460-02-2

Synonyms
Synonyms
Identity
1,_3,_5-Tri-tert-butylbenzene

Molecular and structural information
Molecular formula
C18H3031

Molecular weight
246.4308
SMILES notation
CC(C)(C)c1cc(cc(c1)C(C)(C)C)C(C)(C)C
InChl
InChI=1/C18H30/c1-16(2,3)13-10-14(17(4,5)6)12-15(11-13)18(7,8)9/h10-12H,1-9H3Structural formula

Related substancesGroup / category information
USEPA Category: Neutral Organics

32

Literatures
LITERATURE: A combined repeated dose/reproductive
developmental toxicity study of 1, 3, 5-Tri-tertbutylbenzene by oral administration in rats.
UUID: 9ce9c808-fda7-488e-94bb-d2effb4b0496
Dossier UUID:
Author:Date: 2019-03-22T10:25:28.000+09:00
Remarks:

General information
Reference Typestudy report
Title
A combined repeated dose/reproductive developmental toxicity study of 1, 3, 5-Tri-tert-butylbenzene
by oral administration in rats.
Author
Ministry of Health, Labor and Welfare, Japan
Bibliographic source
Japan Existing Chemical Data Base (JCDB)
Testing facility
Research institute for animal science in biochemistry and toxicology (RIAS)
Report number
07-111

33

LITERATURE: In Vitro Chromosomal Aberration Test of
on 1,3,5-Tri-tert-butylbenzene Cultured Chinese Hamster
Cells
UUID: b5165e02-1336-48d7-b3c2-16582d1ccaad
Dossier UUID:
Author:Date: 2019-02-18T10:54:41.000+09:00
Remarks:

General information
Reference Typestudy report
Title
In Vitro Chromosomal Aberration Test of on 1,3,5-Tri-tert-butylbenzene Cultured Chinese Hamster Cells
Author
Ministry of Health, Labour and Welfare (MHLW), Japan
Bibliographic source
http://dra4.nihs.go.jp/mhlw_data/jsp/SearchPageENG.jsp
Testing facility
BoZo Research Center

34

LITERATURE: Reverse Mutation Test of 1,3,5-Tri-tertbutylbenzene
UUID: 0be57198-4f9b-463a-9a10-22a144172cde
Dossier UUID:
Author:Date: 2019-02-18T09:49:29.000+09:00
Remarks:

General information
Reference Typestudy report
Title
Reverse Mutation Test of 1,3,5-Tri-tert-butylbenzene
Bibliographic source
http://dra4.nihs.go.jp/mhlw_data/jsp/SearchPageENG.jsp
Testing facility
BoZo Research Center

35

Legal Entities
LEGAL_ENTITY: National Institute of Health Sciences
UUID: IUC4-b036ff75-0f3c-323b-b200-ed5f46cf5101
Dossier UUID:
Author:Date: 2022-11-07T15:49:29.000+09:00
Remarks:

General information
Legal entity nameNational Institute of Health Sciences
Remarks
Disclaimer: The contents in this document were created based on the MHLW (Ministry of Health,Labour and Welfare) peer reviewed study reports (in Japanese) in JECDB (Japan Existing Chemical
Database) at http://dra4.nihs.go.jp/mhlw_data/jsp/SearchPageENG.jsp. Authorship is in the Divisionof Risk Assessment, the National Institute of Health Sciences, and the contents do not reflect any official MHLW opinions or any other regulatory policies.

Address
Address 1
Tonomachi 3-25-26
Address 2
Kawasaki-ku
Postal code
210-9501
Town
KawasakiRegion / State
Kanagawa
CountryJapan
JP

Identifiers
Other IT system identifiers
IT system
LEO
ID
10767
IT systemIUCLID436

ID
16558402024DIV750

37