tert-butyl hydroperoxide

Carcinogenicity

Description of key information

 A GLP compliant OECD guideline study has been completed to investigate the carcinogenic potential of TBHP following whole body inhalation.  Neoplastic nasal lesions were observed  only at the highest concentration tested, which also resulted in severe nasal irritation/corrosion/necrosis and was observed to exceed the Maximum Tolerated Dose as defined in OECD guidance documents. At the low and mid dose, there was no evidence of neoplastic lesions, whilst still observing nasal irritationa dn remodelling at the mid dose group.

Toxicokinetic data demonstrate very low or absent systemic bioavailability of TBHP, limiting the potential for exposure of internal tissues. Carcinogenicity studies on tertiary butyl alcohol (TBA) do not indicate the occurrence of tumours of expected relevance to humans, and TBA is not genotoxic. Therefore, the primary concern over the carcinogenicity of TBHP relates to its reactivity with biological membranes at the site of first contact. TBHP is genotoxic and mutagenic in bacterial and mammalian cells in vitro while results following intraperitoneal injection in vivo indicate it will be mutagenic at the sites of first contact in somatic cells. However, inhalation of TBHP vapour for 5 days resulted in no evidence of DNA damage in a Comet Assay in lung tissue in rats, indicating that TBHP does not penetrate in unreacted form into the lung. In another Comet Assay following inhalation of TBHP vapour for 3 days, no evidence of DNA damage was found in the nasal epithelial tissue. The results from this study indicate that site of contact genotoxicity does not occur. Although it does not address the carcinogenicity hazard, the results from this study provide supporting evidence that there may be limited risk of site of contact carcinogenicity via a genotoxic mechanism, when a human relevant route of exposure was employed.

The manufacturers submitting REACH registration for TBHP do not support consumer uses of the material, and workplace exposures are within acceptable risk assessment parameters, are closely controlled and monitored. Therefore, there is no widespread or dispersive use of TBHP, and no frequent occupational exposures of concern.

Key value for chemical safety assessment

Carcinogenicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

carcinogenicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 March 2016 - 14 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

not applicable

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

A 70% solution of 1,1-Ditmethyl ethyl hydroperoxide (also known as tertiary-butyl hydroperoide, TBHP) in water was tested. The 70% solution is generated in order to enhance the stability of the product.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The Wistar Han - Crl: WI has been used in a previous 5 day, 28 day, and 90 day study as well as in the 8-week Dose Range Finding Study

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation: 7 to 8 weeks at age of exposure initiation
- Weight at study initiation: (Expected) Males 140 - 260g; Females 110 - 200g
- Fasting period before study:
- Housing: Animals will be housed 2 per cage in solid bottom cages
- Diet (e.g. ad libitum): ad libitum expect during inhalation exposure periods
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 10 days

DETAILS OF FOOD AND WATER QUALITY: Reverse Osmosis treated water; PMI Nutrition International, LLC Certified Rodent LabDiet 5002

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 50 +/- 20
- Air changes (per hr): 10 room air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 14 March 2016 To: 12-19 March 2018

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 2.0 cu. meter stainless steel and glass whole doy exposure chambers
- Method of holding animals in test chamber: wire-mesh chambers
- Source and rate of air: breathing quality in-house compressed air source and/or from a HEPA- and charcoal filtered supply air source
- Method of conditioning air: HEPA - and Charcoal-filtered, temperature and humidity controlled supply air source
- System of generating particulates/aerosols: No aerosols were generated
- Temperature, humidity, pressure in air chamber: 21.4 - 21.7C; 50.3 - 57.7% humidity; slight negative pressure
- Air flow rate: 400 to 500 LPM
- Air change rate: 12 to 15 air changes per hour
- Method of particle size determination: Presence of aerosols was evaluated using a Casella CEL-712 Microdust Pro; no aerosol content detected.
- Treatment of exhaust air:

TEST ATMOSPHERE
- Brief description of analytical method used: Analyzed exposure concentrations of TBHP were determined at approximately 45-minute intervals using a gas chromatograph.
- Samples taken from breathing zone: yes

VEHICLE: Air

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were collected from the approximate animal-breathing zone of the chamber via 1/8-inch Teflon® tubing. Under the control of the WINH system, sampling and analyses was performed as follows. The program controlled an external multi-position valve (Model No. E16, Valco Instruments Co., Inc; Houston, TX) that permitted sequential sampling from the exposure room and each chamber. Gas sampling injection onto the chromatography column occurred via an internal gas sampling valve with a sample loop, the chromatograph was displayed and the area under each sample peak was calculated and stored. The WINH system then acquired the stored peak area data and used an ln-quadratic equation based on the GC calibration curve to calculate the concentration, in ppm, using the sum of the 2 stored peak areas.

Equipment
GC: Hewlett Packard 5890 Series II
GC Detector: Flame Ionization (FID)
GC Column: Agilent J & W, DB-5, 30 m X 0.530 mm ID, 1.5-micron film thickness
Integrator: Hewlett Packard Model 3396A

GC Gases Pressure Approximate Flow Rate
(psig) (mL/minute)
Carrier - Helium: 15 32 ± 1
Fuel - Hydrogen: 18 30 ± 3
Air: 34 292 ± 10

GC Temperatures
Column (Oven): 40
Detector: 250

Integrator Run Parameters
Chart Zero Offset: 0
Chart Attenuation: 1
Chart Speed : 1.0 cm/minute
Peak Area Rejection Value: 0
Peak Threshold: 1, 2
Peak Width: 0.04
Retention Time 1: Approximately 0.6 minutes
Retention Time 2: Approximately 1.7 minutes
Time Table Events: 0.000 Intg # = 9 (integration off)
0.500 Intg # = 9 (integration on)

Duration of treatment / exposure:

Filtered-air and TBHP vapor atmospheres will be administered as 6-hour, whole-body exposures

Frequency of treatment:

5-day per week basis for approximately 104 weeks.

Post exposure period:

None

Dose / conc.:

4 ppm

Dose / conc.:

15 ppm

Dose / conc.:

60 ppm

No. of animals per sex per dose:

50 males & 50 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The exposure levels were selected based on the results of 13-week and 8-week inhalation toxicity studies (WIL-14073, Kirkpatrick, 2014 and WIL-14078, Kirkpatrick, 2016). In the 13-week study, animals were exposed nose only 6 hours/day, 5 days/week for 13 weeks at target TBHP vapor concentrations of 2, 6 and 18 ppm. In the 8-week study, animals were exposed 6 hours/day, 5 days/week for 8 weeks at target TBHP vapor concentrations of 10, 20 and 60 ppm. Both whole-body and nose-only exposure groups were tested in WIL-14078, but only the whole-body group results will be considered here, as they have been determined to be more relevant (please see repeated dose toxicity, inhalation study for more details on this study). The high concentration of 60 ppm was selected based on the absence of overt toxicity and the 11% body weight decrement in the 60 ppm whole-body group, the maximum tolerated dose (MTD) in the 8-week study. A low concentration of 4 ppm was selected to provide a concentration at which hyperplasia of the transitional epithelium was not found in the 13-week toxicity study (= 6 ppm.). A mid-level concentration of 15 ppm was selected since this level provides appropriate spacing between the low and high concentrations and is expected to produce significant effects in the nasal cavity.

The objective of this study was to evaluate the potential toxicologic and carcinogenic effects of tertiary-butyl hydroperoxide (TBHP) when administered as a vapor via whole-body inhalation to Wistar Han rats for up to 24 months (104 weeks). The inhalation exposure schedule was 6 hours per day on a 5 day per week basis.

The test substance, tertiary-butyl hydroperoxide, was shipped and used as a 70% aqueous solution to limit physical hazards caused by the high reactivity and instability of the compound. TBHP is used in the chemical industry as a source of active oxygen (i.e., a strong oxidizing agent) in numerous synthetic processes.

The study design was as follows:

Experimental Design
Group Number Treatment Exposure Concentrationa (ppm) Number of Animals
Males Females
1 Filtered Air Control 0 50 50
2 TBHP 4 50 50
3 TBHP 15 50 50
4 TBHP 60 50 50

Animals were administered filtered air (control) or test substance atmospheres via whole-body inhalation exposures for 6 hours a day on a 5-day per week basis for 24 months (104 weeks) with target concentrations of 4, 15, and 60 ppm and a clean air control group.

The following parameters and end points were evaluated in this study: clinical signs, palpable masses, body weights, body weight gains, food consumption, gross necropsy findings, organ weights, and histopathologic examinations (non-neoplastic and neoplastic changes).

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily on exposure days, once per day on non-exposure days

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
• One week (± 2 days) prior to randomization
• On the day of randomization
• Study day -1 (prior to exposure)
• Weekly (±2 days) during the study period
• On the day of the scheduled necropsy

BODY WEIGHT: Yes
- Time schedule for examinations:
• One week (±2 days) prior to randomization
• On the day of randomization
• Study day -1 (prior to exposure)
• Once weekly (±2 days) for the first 14 weeks of the exposure period
• Once every 2 weeks (±2 days) from the 15th week of the exposure period for the remainder of the study
• On the day prior to the first day of the scheduled necropsy (non-fasted)
• On the day of the scheduled necropsy (non-fasted)

Sacrifice and pathology:

Unscheduled Deaths
A necropsy was conducted for main study animals that died on study, and specified tissues were saved.
If necessary for humane reasons, main study animals were euthanized as per Testing Facility SOPs. A detailed physical examination was conducted, a terminal body weight was recorded, and a sample for potential evaluation of clinical pathology parameters was obtained if possible. These animals underwent necropsy, and specified tissues were retained.

Scheduled Euthanasia
Main study animals surviving until the scheduled euthanasia were weighed and anesthetized by isoflurane inhalation and euthanized by exsanguination. Animals were not fasted before the scheduled necropsy.

Necropsy
Main study animals were subjected to a complete necropsy examination, which included evaluation of all external surfaces and orifices; the cranial cavity and external surfaces of the brain; thoracic, abdominal, and pelvic cavities with their associated organs and tissues; and palpable masses.
A veterinary pathologist, or other suitably qualified person, was available.

Organ Weights
The organs identified in the organ weight table were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals found dead or in extremis. Paired organs were weighed together. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated.

Representative samples of the tissues identified below in the Tissue Collection and Preservation list were collected from all animals and preserved in 10% neutral buffered formalin or relevant fixative. Samples were prepared with H&E staining and subjected to a pathological evalution by a board-certified verterinary pathologist. In addition, gross lesions, tissue masses, and target tissues (eyes, mandibular lymph node, lungs, and nasal tissues [all levels]) were examined microscopically from all animals in the low- and mid-dose groups at the terminal necropsy

Statistics:

Each mean was presented with the standard deviation (S.D.) and/or the number of animals or cages (N) used to calculate the mean. Statistical analyses were not conducted if the number of animals was 2 or less. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means and standard deviations on the summary and individual tables may differ slightly. Therefore, the use of reported individual values to calculate subsequent parameters or means will, in some instances, yield minor variations from those listed in the report data tables.

All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex.
Body weight, body weight change, food consumption, and organ weight data were subjected to a parametric one way ANOVA to determine intergroup differences. If the ANOVA revealed statistically significant (p < 0.05) intergroup variance, Dunnett’s test was used to compare the test substance treated groups to the control group. Palpable mass data were analyzed by comparing the control groups to the other groups (by sex) as follows. The number of animals with masses and the number of animals with palpable masses were analyzed using a two tailed Fisher’s Exact Test.

Statistical evaluation of survival data and tumor incidence was conducted by BioSTAT Consultants, Inc., Portage, MI, using SAS® version 9.2 software.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance related clinical observations. All clinical observations in the test substance treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose related manner, and/or were common findings for laboratory rats of this age and strain.

See attachment 1 for detailed summary tables of clinical signs.

Mortality:

mortality observed, treatment-related

Description (incidence):

50 males and 50 females were assigned to four different concentrations; 0 ppm, 4 ppm, 15 ppm, or 60 ppm TBHP for the duration of the study.

Survival of male Wistar-Han rats in the TBHP 2 year carcinogenicity study
Week 0 ppm 4 ppm 15 ppm 60 ppm
0 50 50 50 50
26 48 50 46 49
52 43 48 45 46
78 39 46 42 41
91 37 44 33 34
104 28 42 25 29

For males the following summary of the mortality can also be noted

Disposition of Male Rats
Disposition 0 ppm 4 ppm 15 ppm 60 ppm
Found Dead 6 2 8 4
Sent to Necropsy in Extremis 16 6 17 17
Scheduled Euthanasia 28 42 25 29

Survival of female Wistar-Han rats in the TBHP 2-year carcinogenicity study
Week 0 ppm 4 ppm 15 ppm 60 ppm
0 50 50 50 50
26 50 50 49 49
52 49 50 48 49
78 41 44 45 46
91 34 40 42 44
104 29 31 34 38

For females the following summary of the mortality can also be noted

Disposition of Female Rats
Disposition 0 ppm 4 ppm 15 ppm 60 ppm
Found Dead 8 4 4 1
Sent to Necropsy in Extremis 13 15 12 11
Scheduled Euthanasia 29 31 34 38

See attachment 1 for detailed summary tables of mortality incidence. This is also further summarised in text tables 1, 2 and 10 in the section 'any other information on results incl. tables'.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Final Body Weight Male Rats
0 ppm 4 ppm) 15 ppm 60 ppm
Final Body Wt (g)
Mean 558 604 558 467
% Difference 8.2 0.0 -16.3

Female Final Body Weights
0 ppm 4 ppm 15 ppm 60 ppm
Final Body Wt (g)
Mean 337 349 339 297
% Difference 3.6 0.6 -11.9

Maximal difference in body weights was noted near week 80 when animals in extremis were sent for eutahnization.

Mean Cumulative Body Weight Gain Male Rats
0 ppm 4 ppm) 15 ppm 60 ppm
Weight Gain (g)
Mean 346 386 345 253
% Difference 11.6 -0.3 -26.9

Mean Cumulative Body Weight Gain Femal Rats
0 ppm 4 ppm 15 ppm 60 ppm
Weight Gain (g)
Mean 177 191 184 138
% Difference 7.9 4.0 -22.0

See attachment 1 for detailed summary tables of bodyweight and bodyweight gains. This is also further summarised in text tables 3 and 4 in the section 'any other information on results incl. tables'.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Test substance-related effects on food consumption were noted in the 60 ppm group males and females.
Test substance-related lower mean food consumption (occasionally significant compared to the control group) was noted consistently in the 60 ppm group males and sporadically in the 60 ppm group females in the first 14 weeks of exposure and sporadically throughout the remainder of the exposure period in the 60 ppm group males and females compared to the control group. In general, lower mean food consumption values were correlated with the lower mean body weights in the 60 ppm group males and females.


See attachment 1 for detailed summary tables of food consumption.

Ophthalmological findings:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related changes in the cornea were noted in a single 15 ppm group male (corneal ulceration and neutrophil inflammation) and 60 ppm group males (neutrophil inflammation, corneal epithelial hyperplasia, and neovascularization) and a single 60 ppm group female (corneal epithelial hyperplasia).

See attachment 2 for detailed summary tables of ophthalmological findings at necropsy and after microscopic examination. This is also further summarised in text tables 7 and 8 in the section 'any other information on results incl. tables'.

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Organ weight differences that were statistically significant when compared to the control group were considered to be a result of a test substance-related effect on final body weight. These weight changes in 60 ppm group males included lower absolute adrenal gland weight; higher brain, epididymides, lung, and heart weights relative to final body weight; lower liver weight (absolute and relative to brain weight); and lower spleen weight (absolute and relative to brain weight). In 60 ppm group females, these weight changes included higher brain, lung, and heart weights relative to final body weight.

See attachment 2 for detailed summary tables of organ weights both absolute and relative.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related white areas (increased incidence) were noted in the lungs of males and females at 60 ppm. White areas correlated with increased incidences of alveolar macrophages, fibroplasia, and/or granulomas in the lung. Test substance-related nasal masses (bone or subcutis) were noted in males at 60 ppm and correlated with squamous cell carcinoma in the nasal cavity.

Week 104
Test substance-related white areas in the lungs (increased incidence) were noted in males and females at 60 ppm and correlated with increased incidences of alveolar macrophages, fibroplasia, and/or granulomas in the lung.

See attachment 2 for detailed summary tables of gross pathological findings. This is also further summarised in text table 5 in the section 'any other information on results incl. tables'.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related microscopic findings were noted in the nasal cavity (nasal levels I through VI), lung, mandibular lymph node, and eye. At 60 ppm, test substance-related findings in nasal levels I through VI were considered adverse in males and females given the high incidence and severity grades that were frequently moderate and occasionally marked or severe; also at
60 ppm, findings in the lungs of males and females were adverse given the combination of findings, and eyes in males were considered adverse given the moderate findings (in unscheduled death animals). Nonadverse changes were noted in the lungs of males and mandibular lymph nodes. At 15 ppm, adverse test substance-related findings were noted in nasal levels I and II given the incidence and/or severity scores (occasionally up to moderate), and eyes (males only) given the ulceration; nonadverse findings were noted in the lungs at 15 ppm. At 4 ppm, nonadverse test substance-related findings were noted in the lung of males only.
Nasal Cavity: Test substance-related findings were noted in nasal levels I through VI in 60 ppm groups and in nasal levels I and II in 15 ppm groups. Test substance-related nasal findings were not observed in 4 ppm groups.
At 60 ppm, findings related to epithelial injury and/or repair (noted as degeneration/regeneration, squamous/transitional/respiratory/olfactory) were noted in all nasal levels and involved squamous, transitional, respiratory and olfactory epithelium. In addition, metaplastic and/or hyperplastic changes in the transitional, respiratory and olfactory epithelium were also noted (noted as metaplasia, squamous and metaplasia, respiratory; olfactory epithelium). These findings had the greatest incidence and severity in nasal levels II and III.
Degeneration/regeneration and/or necrosis were observed in nasal levels I through VI and involved squamous epithelium (level I), transitional epithelium (level II), respiratory epithelium (levels II through VI), and olfactory epithelium (levels III through VI).
Degeneration/regeneration was characterized by areas of attenuation (degeneration) and/or expansion and disorganization of the epithelium (regeneration). Necrosis was also observed in a subset of rats with degeneration/regeneration and was characterized by hypereosinophilia of the epithelium with nuclear pyknosis and was intact or sloughing from the underlying stroma. In nasal level I, the squamous epithelial changes were noted on the dorsal, medial, and/or ventral nasal cavity. In nasal level II, transitional epithelium changes were noted on the lateral wall, nasoturbinates, and maxilloturbinates. Respiratory epithelial degeneration/regeneration was observed on the nasal septum (levels II and III), ventral nasal cavity, and nasopharyngeal duct/nasopharynx (levels III, IV, V, and VI). Degeneration/regeneration of olfactory epithelium was observed in the dorsal meatus and/or ethmoid turbinates in nasal levels III through VI with the most extensive changes in nasal levels III. Degeneration/regeneration (respiratory and olfactory) in nasal level VI was noted in unscheduled death rats only. Another finding accompanying degeneration/regeneration and necrosis was adhesion. This was observed in a low number of 60 ppm group males and/or females and was characterized by adhesion of a turbinate to the nasal septum or lateral wall in levels II (males and females), IV (1 female), and VI (1 male).
Mixed cell inflammation was observed in all groups (including control) but generally accompanied regeneration/degeneration changes and was characterized by neutrophils, lymphocytes, and plasma cells in the underlying stroma. Mixed cell inflammation had a higher incidence in nasal levels I (males), II (males and females), III (males and females), and
IV (males and females). Inflammatory exudate was generally observed in all groups with a test substance-related higher incidence in nasal levels I through III (60 ppm group males and females) and IV through VI (60 ppm group males).

Hemorrhage was variably observed in all groups and all levels in the stroma but had a higher incidence in 60 ppm group males and/or females in levels II and III.
Squamous epithelial metaplasia was characterized by replacement of transitional or respiratory epithelium with stratified squamous epithelium and was observed in nasal levels II through V. The most extensive squamous epithelial metaplasia occurred in levels II and III and involved transitional (level II) and respiratory (III, IV, and V) epithelium. In level II, squamous metaplasia was observed along the lateral wall of the nasal cavity, the naso- and maxilloturbinates, and occasionally the nasal septum. In level III, squamous metaplasia was noted along the lateral wall of the nasal cavity and occasionally the nasal septum. In levels IV and V, it was observed in the nasopharyngeal duct/nasopharynx and only in low number of males.
Respiratory epithelial metaplasia was characterized by replacement of olfactory epithelium with cuboidal to columnar epithelium containing ciliated and/or non-ciliated cells. This was most often noted in the dorsal meatus of nasal levels III, IV, and V.
Mucous cell metaplasia was noted in nasal level II only in the transitional epithelium lining the lateral meatus and was generally noted in all groups (control included). Mucous cell metaplasia had a test substance-related higher incidence in 60 ppm group males and females. Mucous cell hyperplasia was noted in respiratory epithelium lining the nasal septum in levels II and III and ventral nasal cavity/nasopharyngeal duct/nasopharynx in levels IV, V, and VI. Mucous cell hyperplasia was noted in all groups (control included) but had a higher test substance-related incidence in nasal level II (males and females) and III (males and females).

At 15 ppm, test substance-related nasal findings were noted in levels I and II. These included degeneration/regeneration of squamous epithelium in level I (males and females), mixed cell inflammation in levels I and II (males), degeneration/regeneration of transitional epithelium in level II (males and females) and mucous cell hyperplasia in level II (males).
Lung: Test substance-related findings included alveolar macrophages, granulomas, mononuclear inflammation, and fibrosis (interstitial or pleural) in 60 ppm group males and females and pigmented macrophages in 4 ppm, 15 ppm, and 60 ppm group males. Lung findings were collectively considered adverse in the 60 ppm group males and females with increased incidences of alveolar macrophages, mononuclear inflammation, interstitial fibrosis, and/or pleural fibrosis. Pigmented macrophages in the 4 and 15 ppm group males were considered nonadverse as there were no accompanying findings.
Alveolar macrophages were characterized by aggregates of foamy macrophages in centriacinar or subpleural alveoli and were noted in all groups (control included) with a higher incidence in 60 ppm group males and females. Occasionally these macrophages formed 1 or more granulomas and these were noted with a higher incidence in 60 ppm groups. Mononuclear inflammation, characterized by lymphocytes within alveolar septae, were occasionally noted in areas of alveolar macrophages, and had a test substance-related higher incidence in 60 ppm group females only. Interstitial fibrosis or pleural fibrosis was noted in a low number of rats with a higher incidence in 60 ppm groups. Pigmented macrophages, characterized by macrophages containing grey/brown and/or golden brown pigment were noted in centriacinar or subpleural alveoli in all groups (control included) with a test substance-related higher incidence in 4, 15, and 60 ppm group males.
Lymph node, mandibular: Dilation, characterized by expansion of sinuses, was noted in all groups (control group included) with a test substance-related higher incidence in 60 ppm group males and females and was considered nonadverse given the low incidence and severity.
Eyes: Test substance-related changes in the cornea were noted in a single 15 ppm group male (corneal ulceration and neutrophil inflammation) and 60 ppm group males (neutrophil inflammation, corneal epithelial hyperplasia, and neovascularization) and a single 60 ppm group female (corneal epithelial hyperplasia). Eye findings were considered adverse.

There were no other test substance-related non-neoplastic histologic changes. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test substance. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations

See attachment 2 for detailed summary tables of non-neoplastic histopathological findings. This is also further summarised in text tables 7 and 8 in the section 'any other information on results incl. tables'.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related neoplasms (squamous cell carcinoma or chondroma) were noted in the nasal cavity of 60 ppm group males and females. The majority of the nasal squamous cell carcinomas (8 of 9 males and 4 of 5 females) were noted in unscheduled death rats. The other 2 squamous cell carcinomas in the males and females were not noted macroscopically. Nasal squamous cell carcinomas were observed along the lateral wall and typically expanded laterally into the surrounding soft tissue and/or into the nasal cavity distorting or obliterating nasal structures. Multiple nasal levels were involved with levels II, III, and IV most often affected. Squamous cell carcinoma extended into nasal level V in 3 males and nasal level VI in 1 male. In a single 60 ppm group male, multiple squamous cell carcinomas were observed and were noted bilaterally involving the lateral wall on 1 side and the nasoturbinate on the other side. A single 60 ppm group female had a chondroma in 1 ethmoturbinate in nasal level V. There were no other test substance-related neoplasms.

See attachment 2 for detailed summary tables of neoplastic histopathological findings and tumour incidence. This is also further summarised in text table 9 in the section 'any other information on results incl. tables'.

Relevance of carcinogenic effects / potential:

The relevance of the carcinogenic effects are suspect for human risk assessment. Tumours are only observed in the presence of severe and persistence nasal irritation/corrosion/necrosis. The intense remodeling required by the continuous insult to the nasal tissues, coupled with a dose that exceeds the Maximum Tolerated Dose leads to an interpretation that is of questionable relevance. When the MTD is not exceeded, and animals are not biologically compromised, the presence of the nasal irritation does not lead to the onset of neoplastic lesions.

Key result

Dose descriptor:

NOEC

Remarks:

carcinogenicity

Effect level:

ca. 15 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: neoplastic

Remarks on result:

other:

Remarks:

Nasal carcinogenicity was limited to the group exposed to the 60 ppm TBHP concentration that was higher than the maximum tolerated dose based on body weight and that resulted in excessive irritation injury to nasal tissues including respiratory, transitional, and olfactory epithelium.

Key result

Dose descriptor:

NOEC

Remarks:

systemic toxicity

Effect level:

ca. 15 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

ophthalmological examination

organ weights and organ / body weight ratios

Dose descriptor:

NOAEC

Effect level:

ca. 4 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: The NOAEC for non-neoplastic changes was 4 ppm, because non-neoplastic nasal findings were considered adverse at 15 and 60 ppm.

Text Table 1: Survival – Number and Percentage of Animals Surviving (N = 50/group)

	Males				Females
Concentration (ppm)	0	4	15	60	0	4	15	60
Week
104	28/50 56%	42/50 84%	25/50 50%	29/50 58%	29/50 58%	31/50 62%	34/50 68%	38/50 76%

Text Table 2: Test Substance-Related Causes of Death, Males and Females

Animal, Group, Sex	Study Day of Death	Selected Clinical Observations	Selected Necropsy Findings	Nasal Levels Involved (Nasal Squamous Cell Carcinoma)
4264, 4M	474	Rales, dried red material around nose, swollen facial area	Bone mass (nasal)	II, III, IV, V
4357, 4M	503	Labored respiration, swollen facial area	Bone mass (nasal)	II, III, IV, V, VI
4268, 4M	571	Red material around nose, swollen facial area	Subcutis mass (nasal)	I, II
4364, 4M	592	Rales and labored respiration	Bone mass (nasal)	II, III
4412, 4M	594	Swollen facial area	No observable nasal mass	II, III, IV, V
4413, 4M	679	Labored respiration, dried red material nose	Subcutis mass (nose)	II, III, IV
4698, 4F	395	Labored respiration	No observable nasal mass	I, II, III
4685, 4F	641	Labored respiration, rales, gasping, red material around nose	No observable nasal mass	II
4523, 4F	716	Labored respiration, rales, red material around nose	No observable nasal mass	II, III
M = Male; F = Female

Text Table 3: Summary of Mean Body Weight for Selected Weeks

Concentration (ppm):	Males				Females
	0	4	15	60	0	4	15	60
Body Weight (g)
Week 13	415	422	414	350**	242	242	242	228**
%Difference	-	1.7%	-0.2%	-15.7%	-	0.0%	0.0%	-5.8%
Week 26	469	477	465	388**	261	261	259	239**
%Difference	-	1.7%	-0.9%	-17.3%	-	0.0%	-0.8%	-8.4%
Week 40	502	513	503	412**	279	277	276	247**
%Difference	-	2.2%	0.2%	-17.9%	-	-0.7%	-1.1%	-11.5%
Week 52	532	551	536	434**	294	293	290	257**
%Difference	-	3.6%	0.8%	-18.4%	-	-0.3%	-1.4%	-12.6%
Week 66	550	578	554	448**	310	309	300	267**
%Difference	-	5.1%	0.7%	-18.5%	-	-0.3%	-3.2%	-13.9%
Week 80	573	593	570	456**	328	329	320	278**
%Difference	-	3.5%	-0.5%	-20.4%	-	0.3%	-2.4%	-15.2%
Week 104	558	604*	558	467**	337	349	339	297**
%Difference	-	8.2%	0.0%	-16.3%	-	3.6%	0.6%	-11.9%
* = p<0.05 and ** = p<0.01 when compared to the control group using Dunnett's test.

Text Table 4: Summary of Mean Cumulative Body Weight Gain for Selected Intervals

Concentration (ppm):	Males				Females
	0	4	15	60	0	4	15	60
Body Weight (g)
Weeks 0–13	199	204	196	133**	82	84	85	70**
%Difference	-	2.5%	-1.5%	-33.2%	-	2.4%	3.7%	-14.6%
Weeks 0–26	255	259	247	171**	101	103	103	81**
%Difference	-	1.6%	-3.1%	-32.9%	-	2.0%	2.0%	-19.8%
Weeks 0–40	288	295	285	196**	119	118	120	89**
%Difference	-	2.4%	-1.0%	-31.9%	-	-0.8%	0.8%	-25.2%
Weeks 0–52	318	333	318	217**	134	135	134	99**
%Difference	-	4.7%	0.0%	-31.8%	-	0.7%	0.0%	-26.1%
Weeks 0–66	337	360	336	230**	149	150	144	108**
%Difference	-	6.8%	-0.3%	-31.8%	-	0.7%	-3.4%	-27.5%
Weeks 0–80	360	375	352	240**	168	171	165	120**
%Difference	-	4.2%	-2.2%	-33.3%	-	1.8%	-1.8%	-28.6%
Weeks 0–104	346	386*	345	253**	177	191	184	138**
%Difference	-	11.6%	-0.3%	-26.9%	-	7.9%	4.0%	-22.0%
* = p<0.05 and ** = p<0.01 when compared to the control group using Dunnett's test.

Text Table 5: Summary of Gross Pathology Findings – Unscheduled deaths

	Males				Females
Group	1	2	3	4	1	2	3	4
Dose (ppm)	0	4	15	60	0	4	15	60
No. Animals Examined	22	8	25	21	21	19	16	12
Lung (No. Examined)	(22)	(8)	(25)	(21)	(21)	(19)	(16)	(12)
Area(s), white	3	0	5	8	2	2	2	5
% incidence	13.6%	0%	20.0%	38.1%	9.5%	10.5%	12.5%	41.7%
Bone/Subcutis (nasal only) (No. Examined)	(22)	(8)	(25)	(21)	(21)	(19)	(16)	(12)
Mass	0	0	0	3/2	0	0	0	0
% incidence	%0	0%	0%	14.3/ 9.5%	0%	0%	(%	0%
Bold– test substance-related

Text Table 6: Summary of Gross Pathology Findings – Week 104

	Males				Females
Group	1	2	3	4	1	2	3	4
Dose (ppm)	0	4	15	60	0	4	15	60
No. Animals Examined	28	42	25	29	29	31	34	38
Lung (No. Examined)	(28)	(42)	(25)	(29)	(29)	(31)	(34)	(38)
Areas(s), white	1	6	3	17	4	5	6	25
% incidence	3.6%	14.3%	12.0%	58.6%	13.8%	16.1%	17.6%	65.8%
Bold– test substance-related

Text Table 7: Incidence of Selected Histopathologic Findings, Week 104 Scheduled Necropsy

Concentration (ppm):	Males				Females
	0	4	15	60	0	4	15	60
Nasal Level Ia	28	42	25	29	29	31	34	38
Hyperkeratosis, minimal	0 (0%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)	3 (7.9%)
Degeneration/regeneration,squamous epithelium	6 (21.4%)	10 (23.8%)	15 (60%)	12 (41.4%)	1 (3.4%)	10 (32.2%)	22 (64.7%)	10 (26.3%)
Minimal	4	3	8	8	1	5	9	9
Mild	2	7	3	4	0	5	12	1
Moderate	0	0	4	0	0	0	1	0
Nasal Level IIa	28	42	25	29	29	31	34	38
Adhesion, present	0 (0%)	0 (0%)	0 (0%)	5 (17.2%)	0 (0%)	0 (0%)	0 (0%)	6 (15.8%)
Degeneration/regeneration, respiratory epithelium	0 (0%)	2 (4.8%)	0 (0%)	29 (100%)	0 (0%)	0 (0%)	0 (0%)	36 (94.7%)
Minimal	-	1	-	0	-	-	-	4
Mild	-	1	-	18	-	-	-	15
Moderate	-	0	-	10	-	-	-	17
Marked	-	0	-	1	-	-	-	0
Degeneration/regeneration, transitional epithelium	5 (17.9%)	15 (35.7%)	25 (100%)	28 (96.6%)	5 (17.2%)	11 (35.5%)	23 (67.6%)	35 (92.1%)
Minimal	5	14	12	1	4	11	9	2
Mild	0	1	13	14	1	0	14	13
Moderate	0	0	0	11	0	0	0	16
Marked	0	0	0	2	0	0	0	4
Exudate, inflammatory	1 (3.6%)	3 (7.1%)	0 (0%)	15 (51.7%)	0 (0%)	0 (0%)	0 (0%)	14 (36.8%)
Minimal	1	2	-	9	-	-	-	12
Mild	0	1	-	6	-	-	-	2
Hemorrhage	0 (0%)	3 (7.1%)	0 (0%)	10 (34.5%)	2 (6.9%)	0 (0%)	1 (2.9%)	10 (26.3%)
Minimal	-	2	-	4	2	-	1	6
Mild	-	1	-	6	0	-	0	4
Hyperplasia, mucous cell	15 (53.6%)	21 (50%)	22 (88%)	26 (89.7%)	16 (55.2%)	20 (64.5%)	19 (55.9%)	37 (97.4%)
Minimal	15	18	16	8	12	17	15	15
Mild	0	3	6	17	4	3	4	19
Moderate	0	0	0	1	0	0	0	3
Inflammation, mixed cell	4 (14.3%)	6 (14.3%)	15 (60%)	29 (100%)	3 (10.3%)	5 (16.1%)	10 (29.4%)	37 (97.4%)
Minimal	4	4	14	0	3	5	8	0
Mild	0	2	1	18	0	0	2	18
Moderate	0	0	0	11	0	0	0	19
Metaplasia, mucous cell	12 (42.9%)	9 (21.4%)	14 (56%)	20 (69%)	19 (65.5%)	14 (45.2%)	15 (44.1%)	33 (86.8%)
Minimal	9	7	7	10	16	10	7	13
Mild	3	2	6	10	3	3	8	16
Moderate	0	0	1	0	0	1	0	4
Metaplasia, squamous cell	0 (0%)	0 (0%)	0 (0%)	27 (93.1%)	1 (3.4%)	0 (0%)	0 (0%)	38 (100%)
Minimal	-	-	-	1	0	0	0	3
Mild	-	-	-	15	1	0	0	19
Moderate	-	-	-	11	0	0	0	15
Marked	-	-	-	0	0	0	0	1
Necrosis, respiratory epithelium	0 (0%)	1 (2.4%)	0 (0%)	2 (6.9%)	0 (0%)	0 (0%)	0 (0%)	6 (15.8%)
Minimal	-	1	-	0	-	-	-	2
Mild	-	0	-	2	-	-	-	2
Moderate	-	0	-	0	-	-	-	2
Necrosis, transitional epithelium	0 (0%)	0 (0%)	0 (0%)	2 (6.9%)	0 (0%)	0 (0%)	0 (0%)	6 (15.8%)
Minimal	-	-	-	1	-	-	-	1
Mild	-	-	-	1	-	-	-	2
Moderate	-	-	-	0	-	-	-	3
Nasal Level IIIa	28	42	25	29	29	31	34	38
Degeneration/regeneration,olfactory epithelium	0 (0%)	0 (0%)	0 (0%)	25 (86.2%)	0 (0%)	0 (0%)	0 (0%)	32 (84.2 %)
Minimal	-	-	-	1	-	-	-	5
Mild	-	-	-	15	-	-	-	15
Moderate	-	-	-	9	-	-	-	11
Marked	-	-	-	0	-	-	-	1
Degeneration/regeneration, respiratory epithelium	0 (0%)	2 (4.8%)	0 (0%)	24 (82.8%)	0 (0%)	0 (0%)	0 (0%)	33 (86.8%)
Minimal	-	0	-	9	-	-	-	10
Mild	-	2	-	11	-	-	-	19
Moderate	-	0	-	3	-	-	-	4
Marked	-	0	-	1	-	-	-	0
Exudate, inflammatory	0 (0%)	2 (4.8%)	0 (0%)	9 (31%)	0 (0%)	0 (0%)	0 (0%)	6 (15.8%)
Minimal	-	1	-	5	-	-	-	5
Mild	-	1	-	3	-	-	-	1
Moderate	-	0	-	1	-	-	-	0
Hemorrhage	0 (0%)	2 (4.8%)	0 (0%)	6 (20.7%)	1 (3.4%)	0 (0%)	1 (2.9%)	4 (10.5%)
Minimal	-	0	-	3	1	-	1	3
Mild	-	2	-	3	0	-	0	1
Hyperplasia, mucous cell	6 (21.4%)	10 (23.8%)	8 (32%)	19 (65.5%)	9 (31%)	3 (9.7%)	8 (23.5%)	30 (78.9%)
Minimal	6	7	5	13	9	3	5	17
Mild	0	3	2	5	0	0	2	11
Moderate	0	0	1	1	0	0	1	2
Inflammation, mixed cell	0 (0%)	1 (2.4%)	1 (4%)	15 (51.7%)	0 (0%)	0 (0%)	1 (2.9%)	31 (81.6%)
Minimal	-	0	1	4	-	-	1	21
Mild	-	0	0	10	-	-	0	10
Moderate	-	1	0	1	-	-	0	0
Metaplasia, respiratory epithelium; olfactory epithelium	0 (0%)	0 (0%)	0 (0%)	15 (51.7%)	0 (0%)	0 (0%)	0 (0%)	13 (34.2%)
Minimal	-	-	-	0	-	-	-	1
Mild	-	-	-	5	-	-	-	5
Moderate	-	-	-	10	-	-	-	7
Metaplasia, squamous cell	0 (0%)	0 (0%)	0 (0%)	7 (24.1%)	0 (0%)	0 (0%)	0 (0%)	14 (36.8%)
Minimal	-	-	-	4	-	-	-	5
Mild	-	-	-	3	-	-	-	8
Severe	-	-	-	0	-	-	-	1
Mineralization	0 (0%)	0 (0%)	0 (0%)	1 (3.4%)	0 (0%)	0 (0%)	0 (0%)	1 (2.6%)
Minimal	-	-	-	0	-	-	-	1
Mild	-	-	-	1	-	-	-	0
Necrosis, respiratory epithelium, mild	0 (0%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)	2 (5.3%)
Nasal Level IVa	28	42	25	29	29	31	34	38
Degeneration/regeneration, olfactory epithelium	0 (0%)	0 (0%)	0 (0%)	13 (44.8%)	1 (3.4%)	0 (0%)	0 (0%)	22 (57.9%)
Minimal	-	-	-	4	1	-	-	10
Mild	-	-	-	8	0	-	-	12
Moderate	-	-	-	1	0	-	-	0
Degeneration/regeneration,respiratory epithelium, minimal	0 (0%)	0 (0%)	0 (0%)	2 (6.9%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)
Exudate, inflammatory	0 (0%)	0 (0%)	0 (0%)	3 (10.3%)	0 (0%)	0 (0%)	0 (0%)	1 (2.6%)
Minimal	-	-	-	0	-	-	-	1
Mild	-	-	-	3	-	-	-	0
Hemorrhage	2 (7.1%)	0 (0%)	1 (4%)	8 (27.6%)	4 (13.8%)	0 (0%)	1 (2.9%)	5 (13.2%)
Minimal	1	-	0	2	3	-	0	5
Mild	1	-	1	6	1	-	1	0
Hyperplasia, mucous cell	2 (7.1%)	16 (38.1%)	8 (32%)	7 (24.1%)	5 (17.2%)	4 (12.9%)	5 (14.7%)	17 (44.7%)
Minimal	2	13	4	7	5	4	3	16
Mild	0	3	4	0	0	0	1	1
Moderate	0	0	0	0	0	0	1	0
Metaplasia, respiratory epithelium; olfactory epithelium	0 (0%)	0 (0%)	0 (0%)	3 (10.3%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)
Minimal	-	-	-	0	-	-	-	-
Mild	-	-	-	2	-	-	-	-
Moderate	-	-	-	0	-	-	-	-
Marked	-	-	-	1	-	-	-	-
Metaplasia, squamous cell	0 (0%)	0 (0%)	0 (0%)	2 (6.9%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)
Minimal	-	-	-	1	-	-	-	-
Mild	-	-	-	1	-	-	-	-
Nasal Level Va	28	42	25	29	29	31	34	38
Degeneration/regeneration,olfactory epithelium	0 (0%)	0 (0%)	0 (0%)	4 (13.8%)	0 (0%)	0 (0%)	0 (0%)	5 (13.2%)
Minimal	-	-	-	3	-	-	-	4
Mild	-	-	-	1	-	-	-	1
Degeneration/regeneration,respiratory epithelium, minimal	0 (0%)	0 (0%)	0 (0%)	1 (3.4%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)
Exudate, inflammatory	0 (0%)	0 (0%)	0 (0%)	1 (3.4%)	0 (0%)	0 (0%)	0 (0%)	1 (2.6%)
Minimal	-	-	-	0	-	-	-	1
Mild	-	-	-	1	-	-	-	0
Hyperplasia, mucous cell	5 (17.9%)	5 (11.9%)	6 (24%)	10 (34.5%)	5 (17.2%)	5 (16.1%)	4 (11.8%)	14 (36.8%)
Minimal	5	4	4	10	5	5	3	13
Mild	0	1	2	0	0	0	1	1
Metaplasia, respiratory epithelium; olfactory epithelium	0 (0%)	0 (0%)	0 (0%)	2 (6.9%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)
Mild	-	-	-	1	-	-	-	-
Marked	-	-	-	1	-	-	-	-
Nasal Level VIa	28	42	25	29	29	31	34	38
Exudate, inflammatory, minimal	0 (0%)	0 (0%)	0 (0%)	1 (3.4%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)
Hyperplasia, mucous cell	7 (25%)	15 (35.7%)	9 (36%)	14 (48.3%)	8 (27.6%)	5 (16.1%)	8 (23.5%)	15 (39.5%)
Minimal	7	13	7	13	8	5	7	15
Mild	0	2	2	1	0	0	1	0
Lunga	28	42	25	29	29	30	34	38
Fibrosis, interstitial	0 (0%)	3 (7.1%)	1 (4.0%)	5 (17.2%)	1 (3.4%)	2 (6.7%)	3 (8.8%)	8 (21.1%)
Minimal	-	2	1	4	1	2	3	5
Mild	-	1	0	1	0	0	0	3
Fibrosis, pleural	0 (0%)	0 (0%)	0 (0%)	4 (13.8%)	0 (0%)	0 (0%)	0 (0%)	3 (7.9%)
Minimal	-	-	-	1	-	-	-	2
Mild	-	-	-	3	-	-	-	1
Granuloma	3 (10.7%)	1 (2.4%)	2 (8.0%)	8 (27.6%)	3 (10.3%)	2 (6.7%)	2 (5.9%)	11 (28.9%)
Minimal	3	1	2	7	3	2	1	7
Mild	0	0	0	1	0	0	1	4
Inflammation, mononuclear cell	7 (25%)	12 (28.6%)	5 (20%)	12 (41.4%)	4 (13.8%)	7 (23.3%)	6 (17.6%)	22 (57.9%)
Minimal	7	11	3	12	4	6	6	18
Mild	0	1	2	0	0	1	0	4
Macrophages, alveolar	16 (57.1%)	22 (52.4%)	12 (48%)	25 (86.2%)	18 (62.1%)	20 (66.7%)	18 (52.9%)	36 (94.7%)
Minimal	15	17	6	16	17	17	14	21
Mild	1	4	6	9	1	3	4	13
Moderate	0	1	0	0	0	0	0	2
Macrophages, pigmented	2 (7.1%)	6 (14.3%)	7 (28%)	9 (31%)	5 (17.2%)	5 (16.1)	7 (20.6%)	3 (7.9%)
Minimal	2	6	3	8	4	4	6	3
Mild	0	0	4	1	1	1	1	0
Mandibular Lymph Nodea	28	42	25	29	29	31	34	38
Dilation	4 (14.3%)	4 (9.5%)	3 (12%)	10 (34.5%)	0 (0%)	1 (3.2%)	2 (5.9%)	19 (50%)
Minimal	2	1	1	7	-	1	1	9
Mild	2	3	2	3	-	0	0	9
Moderate	0	0	0	0	-	0	1	1
Eyea	28	42	25	29	29	31	34	38
Hyperplasia, corneal epithelium	0 (0%)	0 (0%)	0 (0%)	2 (6.9%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)
Minimal	-	-	-	1	-	-	-	-
Mild	-	-	-	1	-	-	-	-
Neovascularization	0 (0%)	0 (0%)	0 (0%)	2 (6.9%)	0 (0%)	0 (0%)	0 (0%)	0 (0%)
Minimal	-	-	-	1	-	-	-	-
Mild	-	-	-	1	-	-	-	-

aNumber of tissues examined from each group.

- = No noteworthy findings. (% incidence)

Bold– test substance-related

Text table 8: Incidence of Selected Histopathologic Findings, All Animals-Scheduled and Unscheduled Necropsies

 

Concentration (ppm):	Males				Females
	0	4	15	60	0	4	15	60
Nasal Level Ia	50	50	50	50	50	50	50	50
Exudate, inflammatory	2	0	1	8	1	1	1	7
Minimal	2	-	1	4	1	1	1	5
Mild	0	-	0	4	0	0	0	2
Hyperkeratosis	0	0	0	0	0	0	1	6
Minimal	-	-	-	-	-	-	0	5
Mild	-	-	-	-	-	-	1	0
Moderate	-	-	-	-	-	-	0	1
Degeneration/regeneration, squamous epithelium	7	10	21	22	6	13	29	17
Minimal	4	3	11	14	6	6	13	11
Mild	3	7	6	7	0	7	15	4
Moderate	0	0	4	0	0	0	1	1
Marked	0	0	0	1	0	0	0	1
Inflammation, mixed cell	4	7	18	15	5	9	19	8
Minimal	3	6	15	13	5	4	15	5
Mild	1	1	3	2	0	5	4	2
Moderate	0	0	0	0	0	0	0	1
Nasal Level IIa	50	50	50	50	50	50	50	50
Adhesion, present	0	0	0	8	0	0	0	9
Degeneration/regeneration, respiratory epithelium	0	2	0	48	0	1	0	45
Minimal	-	1	-	4	-	0	-	6
Mild	-	1	-	28	-	1	-	22
Moderate	-	0	-	15	-	0	-	17
Marked	-	0	-	1	-	0	-	0
Degeneration/regeneration, transitional epithelium	6	15	44	42	7	16	35	44
Minimal	6	14	25	6	6	16	15	4
Mild	0	1	19	17	1	0	20	19
Moderate	0	0	0	15	0	0	0	16
Marked	0	0	0	4	0	0	0	5
Exudate, inflammatory	3	3	2	27	1	3	1	21
Minimal	2	2	2	15	1	1	0	16
Mild	1	1	0	10	0	2	1	4
Moderate	0	0	0	2	0	0	0	1
Hemorrhage	1	3	0	11	2	0	1	10
Minimal	1	2	-	5	2	-	1	6
Mild	0	1	-	6	0	-	0	4
Hyperplasia, mucous cell	22	22	33	32	28	33	27	41
Minimal	22	19	27	10	24	25	23	18
Mild	0	3	6	20	4	8	4	20
Moderate	0	0	0	2	0	0	0	3
Inflammation, mixed cell	7	6	27	49	4	9	16	49
Minimal	5	4	25	1	4	7	12	1
Mild	2	2	2	30	0	1	3	26
Moderate	0	0	0	18	0	1	1	21
Marked	0	0	0	0	0	0	0	1
Metaplasia, mucous cell	17	9	20	30	26	20	24	41
Minimal	14	7	13	15	21	15	13	16
Mild	3	2	6	13	5	4	11	21
Moderate	0	0	1	2	0	1	0	4
Metaplasia, squamous cell	0	0	0	45	1	0	0	48
Minimal	-	-	-	4	0	-	-	4
Mild	-	-	-	24	1	-	-	24
Moderate	-	-	-	16	0	-	-	17
Marked	-	-	-	1	0	-	-	2
Severe	-	-	-	0	0	-	-	1
Necrosis, respiratory epithelium	0	1	0	9	0	0	1	8
Minimal	-	1	-	0	-	-	0	2
Mild	-	0	-	7	-	-	1	3
Moderate	-	0	-	2	-	-	0	3
Necrosis, transitional epithelium	1	0	1	11	0	0	0	12
Minimal	1	-	1	1	-	-	-	3
Mild	0	-	0	7	-	-	-	3
Moderate	0	-	0	3	-	-	-	6
Septal perforation, present	0	0	0	1	0	0	0	1
Nasal Level IIIa	50	50	50	50	50	50	50	50
Degeneration/regeneration, olfactory epithelium	0	0	1	39	0	0	0	41
Minimal	-	-	1	3	-	-	-	6
Mild	-	-	0	22	-	-	-	18
Moderate	-	-	0	14	-	-	-	16
Marked	-	-	0	0	-	-	-	1
Degeneration/regeneration, respiratory epithelium	0	2	0	39	0	1	0	45
Minimal	-	0	-	11	-	0	-	13
Mild	-	2	-	23	-	1	-	27
Moderate	-	0	-	4	-	0	-	5
Marked	-	0	-	1	-	0	-	0
Exudate, inflammatory	1	2	2	19	0	2	1	10
Minimal	0	1	1	8	-	0	1	6
Mild	1	1	1	8	-	2	0	3
Moderate	0	0	0	3	-	0	0	1
Hemorrhage	1	2	1	9	1	0	1	5
Minimal	1	0	0	5	1	0	1	3
Mild	0	2	1	4	0	0	0	2
Hyperplasia, mucous cell	12	13	10	29	17	14	12	39
Minimal	12	10	7	15	17	12	8	22
Mild	0	3	2	12	0	2	3	15
Moderate	0	0	1	2	0	0	1	2
Inflammation, mixed cell	0	1	4	32	1	1	5	41
Minimal	-	0	4	8	1	0	4	24
Mild	-	0	0	21	0	0	1	16
Moderate	-	1	0	3	0	1	0	1
Metaplasia, respiratory epithelium; olfactory epithelium	0	0	0	26	0	0	0	18
Minimal	-	-	-	1	-	-	-	2
Mild	-	-	-	10	-	-	-	5
Moderate	-	-	-	15	-	-	-	11
Metaplasia, squamous cell	0	0	0	12	0	0	0	19
Minimal	-	-	-	6	-	-	-	5
Mild	-	-	-	6	-	-	-	12
Moderate	-	-	-	0	-	-	-	1
Severe	-	-	-	0	-	-	-	1
Mineralization	0	0	0	3	0	0	0	3
Minimal	-	-	-	0	-	-	-	2
Mild	-	-	-	3	-	-	-	1
Necrosis, olfactory epithelium, mild	0	0	0	3	0	0	0	2
Necrosis, respiratory epithelium	0	0	0	7	0	0	1	3
Minimal	-	-	-	2	-	-	0	0
Mild	-	-	-	4	-	-	1	3
Moderate	-	-	-	1	-	-	0	0
Nasal Level IVa	50	50	50	50	50	50	50	50
Adhesion, present	0	0	0	0	0	0	0	1
Degeneration/regeneration, olfactory epithelium	0	0	0	27	1	0	0	32
Minimal	-	-	-	6	1	-	-	13
Mild	-	-	-	19	0	-	-	19
Moderate	-	-	-	2	0	-	-	0
Degeneration/regeneration, respiratory epithelium	0	0	0	7	0	1	0	0
Minimal	-	-	-	5	-	0	-	-
Mild	-	-	-	2	-	0	-	-
Moderate	-	-	-	0	-	1	-	-
Exudate, inflammatory	1	0	2	10	1	2	1	3
Minimal	0	-	1	1	1	0	1	3
Mild	0	-	1	5	0	1	0	0
Moderate	1	-	0	3	0	1	0	0
Marked	0	-	0	1	0	0	0	0
Inflammation, mixed cell	0	4	1	13	0	3	0	3
Minimal	-	2	1	6	-	1	-	3
Mild	-	2	0	5	-	1	-	0
Moderate	-	0	0	2	-	1	-	0
Metaplasia, respiratory epithelium; olfactory epithelium	0	0	0	8	0	0	0	2
Minimal	-	-	-	1	-	-	-	1
Mild	-	-	-	5	-	-	-	1
Moderate	-	-	-	1	-	-	-	0
Marked	-	-	-	1	-	-	-	0
Metaplasia, squamous cell	0	0	0	3	0	0	0	0
Minimal	-	-	-	1	-	-	-	-
Mild	-	-	-	2	-	-	-	-
Mineralization	0	0	0	3	0	0	0	0
Minimal	-	-	-	2	-	-	-	-
Mild	-	-	-	1	-	-	-	-
Necrosis, olfactory epithelium	0	0	0	2	0	0	0	1
Mild	-	-	-	1	-	-	-	1
Moderate	-	-	-	1	-	-	-	0
Necrosis, respiratory epithelium	0	0	0	3	0	0	0	0
Minimal	-	-	-	1	-	-	-	-
Mild	-	-	-	2	-	-	-	-
Nasal Level Va	50	50	50	50	50	50	50	50
Degeneration/regeneration, olfactory epithelium	0	0	0	11	0	0	0	9
Minimal	-	-	-	4	-	-	-	5
Mild	-	-	-	6	-	-	-	4
Moderate	-	-	-	1	-	-	-	0
Degeneration/regeneration, respiratory epithelium	0	0	0	3	0	0	0	0
Minimal	-	-	-	1	-	-	-	-
Mild	-	-	-	2	-	-	-	-
Exudate, inflammatory	1	0	2	10	0	1	0	1
Minimal	0	-	2	2	-	0	-	1
Mild	1	-	0	7	-	1	-	0
Moderate	0	-	0	1	-	0	-	0
Metaplasia, respiratory epithelium; olfactory epithelium	0	0	0	4	0	0	0	0
Mild	-	-	-	3	-	-	-	-
Marked	-	-	-	1	-	-	-	-
Metaplasia, squamous cell, mild	0	0	0	1	0	0	0	0
Mineralization	0	0	0	2	0	0	0	0
Mild	-	-	-	1	-	-	-	-
Moderate	-	-	-	1	-	-	-	-
Necrosis, olfactory epithelium	0	0	0	2	0	0	0	1
Mild	-	-	-	1	-	-	-	0
Moderate	-	-	-	1	-	-	-	1
Nasal Level VIa	50	50	50	50	50	50	50	50
Adhesion, present	0	0	0	1	0	0	0	0
Degeneration/regeneration, olfactory epithelium, mild	0	0	0	3	0	0	0	0
Degeneration/regeneration, respiratory epithelium, minimal	0	0	0	1	0	0	0	0
Exudate, inflammatory	1	0	2	7	0	1	0	1
Minimal	0	-	1	4	-	1	-	1
Mild	1	-	1	3	-	0	-	0
Necrosis, olfactory epithelium	0	0	0	2	0	0	0	1
Mild	-	-	-	1	-	-	-	1
Moderate	-	-	-	1	-	-	-	0
Lunga	50	50	50	50	50	50	50	50
Fibrosis, interstitial	1	3	1	5	2	4	3	9
Minimal	1	2	1	4	2	4	3	6
Mild	0	1	0	1	0	0	0	3
Fibrosis, pleural	0	0	1	4	0	0	0	3
Minimal	-	-	0	1	-	-	-	2
Mild	-	-	1	3	-	-	-	1
Granuloma	7	1	4	13	3	2	2	12
Minimal	6	1	4	10	3	2	1	8
Mild	1	0	0	3	0	0	1	4
Inflammation, mononuclear cell	7	13	6	16	6	9	6	26
Minimal	7	12	4	16	6	8	6	22
Mild	0	1	2	0	0	1	0	4
Macrophages, alveolar	21	26	22	34	24	27	23	44
Minimal	19	21	11	23	21	20	18	26
Mild	2	4	11	11	3	7	5	16
Moderate	0	1	0	0	0	0	0	2
Macrophages, pigmented	3	8	10	11	5	6	8	4
Minimal	2	8	6	9	4	5	6	3
Mild	1	0	4	2	1	1	2	1
Mandibular Lymph Nodea	49	50	50	50	50	49	50	50
Dilation	5	4	6	11	0	1	2	21
Minimal	2	1	3	7	-	1	1	11
Mild	2	3	3	4	-	0	0	9
Moderate	1	0	0	0	-	0	1	1
Eyea	50	50	50	50	50	50	50	50
Hyperplasia, corneal epithelium	0	0	0	4	0	0	0	1
Minimal	-	-	-	2	-	-	-	1
Mild	-	-	-	1	-	-	-	0
Moderate	-	-	-	1	-	-	-	0
Inflammation, neutrophil	0	0	1	3	0	0	0	0
Mild	-	-	0	2	-	-	-	-
Moderate	-	-	1	1	-	-	-	-
Neovascularization	0	0	0	2	0	0	0	0
Minimal	-	-	-	1	-	-	-	-
Mild	-	-	-	1	-	-	-	-
Ulceration, corneal, minimal	0	0	1	0	0	0	0	0

aNumber of tissues examined from each group.

- = No noteworthy findings.

Bold– test substance-related.

Text Table 9: Nasal Neoplastic Findings (6 levels combined) – Number of Animals with Findings, All Animals Combined

	Males				Females
Exposure Concentration (ppm):	0	4	15	60	0	4	15	60
Nasal^a	50	50	50	50	50	50	50	50
#M Carcinoma, squamous cell	0	0	0	8	0	0	0	5
Nasal Level II	-	-	-	5* p=0.0318	-	-	-	5* p=0.0372
Nasal Level III	-	-	-	3	-	-	-	0
#M Carcinoma, squamous cell, multiple	0	0	0	1	0	0	0	0
#S Carcinoma, squamous cell, multiple; Nasal level IV	0	0	0	1	0	0	0	0
#S Carcinoma, squamous cell; Nasal level II	0	0	0	4	0	0	0	2
#S Carcinoma, squamous cell; Nasal level III	0	0	0	3	0	0	0	0
#B Chondroma	0	0	0	0	0	0	0	1
^a– number of tissues examined. P-Values for peto analysis: Listed under individual treatment Group: 1-sided pairwise comparison of 0 ppm with treatment group. Statistical Significance: Rare tumor – p < 0.025 (trend), p < 0.05 (pairwise); Common tumor – p < 0.005 (trend), p < 0.01 (pairwise). * Statistically significant at the defined significance level. #B – benign; #M – malignant, #S – metastatic.

Text Table 10: Kaplan-Meier Estimates of Survival

		Kaplan-Meier Estimates (%) and P-values
Sex	Week	0 ppm	4 ppm	15 ppm	60 ppm	Trend
M	50	88	96	90	92
	80	80	92	84	82
	End of Study	57	84	50	58
	p-value (a)	-	0.0036*	0.4167	0.9984	0.2550
F	50	100	100	96	98
	80	76	88	88	92
	End of Study	58	62	68	76
	p-value (a)	-	0.5638	0.2749	0.0451*	0.0370*
(a): p-value for comparisons using 0 ppm group * Statistically significant

Conclusions:

In this study animals were exposed to three concentrations of TBHP vapour via whole body inhalation. At the highest concentration, animals were exposed to concentrations of TBHP that exceeded the Maximum Tolerated Dose, as evidenced by early deaths and significant decreases in body weight. These animals also experienced severe nasal irritation/corrosion/necrosis and developed neoplastic nasal lesions. Animals in the mid dose group which did not exceed the MTD yet also experienced severe nasal irritation and remodelling did not develop neoplastic nasal lesions.

This study entry, summary and conclusion is based on the QA audited draft report of the study; however no major substantive changes are expected for the final report.

Executive summary:

The objective of this study was to evaluate the potential toxicologic and carcinogenic effects of tertiary-butyl hydroperoxide (TBHP) when administered as a vapor via whole-body inhalation to Wistar Han rats for up to 24 months (104 weeks). The inhalation exposure schedule was 6 hours per day on a 5-day per week basis.

The test substance, tertiary-butyl hydroperoxide, was shipped and used as a 70% aqueous solution to limit physical hazards caused by the high reactivity and instability of the compound. TBHP is used in the chemical industry as a source of active oxygen (i.e., a strong oxidizing agent) in numerous synthetic processes.

The study design was as follows: 50 males and 50 females were exposed to concentrations of 0 ppm (filtered air), 4 ppm, 15 ppm, or 60 ppm TBHP. Animals were administered filtered air (control) or test substance atmospheres via whole-body inhalation exposures for 6 hours a day on a 5-day per week basis for 24 months (104 weeks). Mean analyzed exposure concentration (as grand mean ± SD for all exposures) in the 4, 15, and 60 ppm groups was 4.0 ± 0.21, 15.0 ± 0.45, and 60.5 ± 2.22 ppm, respectively.

 

The following parameters and end points were evaluated in this study: clinical signs, palpable masses, body weights, body weight gains, food consumption, gross necropsy findings, organ weights, and histopathologic examinations (non-neoplastic and neoplastic changes).

 

Statistically significant lower survival was not associated with test substance exposure at 4, 15, or 60 ppm. In fact there was a statistically significant increase in survival (males) for the 4 ppm group when compared to the 0 ppm control group (p=0.0036). The survival in males at concentrations of 0 ppm,4 ppm,15 ppm, and 60 ppm were 28/50, 42/50, 25/50, and 29/50; whilst for females the survival was 29/50, 31/50, 34/50, and 38/50 respectively. There were no test substance-related effects on clinical observations or palpable masses. Test substance-related effects on organ weights resulted from lower final body weights in the 60 ppm group males.

Test substance-related lower body weight was noted in the 60 ppm group males and females. Negative test substance-related effects on body weight and cumulative body weight gain were observed for all intervals from Week 1 to Week 104 for males and females in this group. For Weeks 13, 26, 52, and 80, percent difference from control for body weight was -15.7%, -17.3%, -18.4%, and -20.4% for males and -5.8%, -8.4%, -12.6%, and -15.2% for females, respectively. For the 0–13, 0–26, 0–52, and 0–80 week intervals, percent difference from control for cumulative body weight gain was -33.2%, -32.9%, -31.8, and -33.3% for males and -14.6%, -19.8%, -26.1%, and -28.6% for females, respectively. Body weight changes were generally consistent with these lower body weights.

Test substance-related lower food consumption was noted consistently in the 60 ppm group males and sporadically in the 60 ppm group females in the first 14 weeks of exposure andsporadically throughout the remainder of the exposure period in the 60 ppm group males and females.

Test substance-related macroscopic findings included increased incidence of white areas in the lungs in the 60 ppm group males and females and nasal masses (bone or subcutis) in the 60 ppm group males.

 

Test substance-related causes of death were noted in males and females at 60 ppm. Nasal squamous cell carcinoma was observed in 6 males and 3 females that were euthanized in extremis and for which the carcinoma was considered to have the strongest causative relationship with the clinical condition of eachanimal. These animals were euthanized between Weeks 67and 97 for males and Weeks 56 and 102 for females.

 

Test substance-related nasal squamous cell carcinoma was observed for 9 of 50 males and 5 of 50 females in the 60 ppm group and a single nasal chondroma was observed in one 60 ppm group female (for all animals whether found dead or euthanized in extremis or at scheduled terminal necropsies).There was a statistically significant increase in the incidence of nasal level II tumor in males and females when comparing the 60 ppm group with the 0 ppm control group. Although the p-values for some trend tests, including nasal level III in males, indicated statistical significance, there were no nasal carcinomas in the 4 or 15 ppm groups. These results led to analysis of pairwise comparisons and provided additional support for the conclusion that the nasal squamous cell carcinoma was a test substance-dependent finding at 60 ppm only.

Test substance-related microscopic non-neoplastic findings included adverse changes in nasal levels I through VI, lungs, and eyes in the 60 ppm group males and females and adverse changes in nasal levels I and II and eyes (males only) in the 15 ppm group males and females. Test substance-related changes in nasal levels I through VI included degeneration/regeneration and/or necrosis of squamous, transitional, respiratory, and olfactory epithelium; squamous mucous, and/or respiratory metaplasia; mucous cell hyperplasia; mixed cell inflammation; inflammatory exudate; and/or adhesion. Test substance-related changes in the lung included alveolar macrophage, granulomas, interstitial and pleural fibrosis, and mononuclear cell inflammation only in the 60 ppm group males and females. Test substance-related changes in the eye included corneal ulceration, neutrophil inflammation, hyperplasia (60 ppm group only), and neovascularization (60 ppm group only) in the 15 and 60 ppm group males and corneal epithelium hyperplasia in a single 60 ppm group female.

 

Based on the results of this study, exposure of Crl:WI(Han) rats to TBHP via whole-body inhalation for 6 hours a day, 5 days a week for 104 weeks at target exposure concentrations of 4, 15, and 60 ppm resulted in systemic toxicity in the 60 ppm group as evidenced by early death and persistent lower body weight and body weight gain and lower food consumption. In this group, percent difference from control for body weight reached -20% in males and -15% in females. Based on body weights and cumulative body weight gains, the high concentration of 60 ppm clearly exceeded the maximum tolerated dose (MTD), defined as a dose resulting in a decrease of body weight by no more than 10% relative to controls (OECD Environment, Health and Safety Publications, Series on Testing and Assessment No. 35 and Series on Pesticides No. 14.Guidance Notes For Analysis and Evaluation of Chronic Toxicity and Carcinogenicity Studies, ENV/JM/MONO(2002)19, 2002.). The early large effects on body weight parameters and early and persistent effects on food consumption suggest the presence of chronic poor nutritional status in this group. Poor nutritional status can have negative effects on individual animal defenses against progression of disease, including neoplastic disease.

 

Inhalation exposures at TBHP concentrations up to 60 ppm had no negative effect on overall survival but resulted in test substance-related early deaths of animals with nasal squamous cell carcinomas at 60 ppm. The incidence of the animals euthanized in extremis at 0, 4, 15, and 60 ppm was 15, 6, 16 and 17 for the males and 13, 15, 11, and 11 for the females. Nine rats (6 males and 3 females) in the 60 ppm group were euthanized in extremis with clinical observation of rales, labored respiration, gaspings, and dried red material around the nose and/or swollen facial areas. The facial swelling was associated with bone or subcutis nasal masses which were noted in 5 of the early death animals (all males) with nasal tumors. However, nasal tumors, regardless of external evidence, extended into the nasal cavity, often obstructing it. Test substance-related nasal squamous cell carcinoma was observed for 9 of 50 males and 5 of 50 females in the 60 ppm group. Nasal squamous cell carcinoma in nasal level II was statistically significant in males and females based on pairwise comparisons versus control.

 

Nasal squamous cell carcinomas were only observed at the 60 ppm concentration, the only exposure level at which a broad range of adverse irritant/corrosive-dependent non-neoplastic nasal lesions were observed at high incidences and frequently with moderate severity grades. Combined male and female incidence rates for selected lesions in nasal level II were 93% for squamous cell metaplasia, 93% for degeneration/regeneration of the respiratory epithelium, 86% for degeneration/regeneration of the transitional epithelium, 98% for mixed cell inflammation, 73% for mucous cell hyperplasia, and 71% for mucous cell metaplasia, and in nasal level III were 80% for degeneration/regeneration of the olfactory epithelium, 84% for degeneration/regeneration of the respiratory epithelium, 73% for mixed cell inflammation, and 44% for metaplasia, respiratory epithelium, and olfactory epithelium.

Based on the OECD Guidance Notes for Analysis and Evaluation of Chronic Toxicity and Carcinogenicity Studies(cited above), five principles are to be considered in the selection of doses in chronic rodent bioassays that were originally published by The International Life Sciences Institute (ILSI) Risk Sciences Working Group on Dose Selection, Principle 5 states that doses should be selected so as to minimize or avoid adverse nutritional, physical, organoleptic, and irritant effects. Although it may be argued that responses observed at doses far in excess of levels experienced under real or potential exposure conditions fall within the bounds of the classic dose-response concept, there are valid scientific concerns that such doses introduced biases of considerable significance into the already difficult task of evaluating dose-response relationships in animals and the assessment of their relevance in the identification of human hazard and risk.

 

In the current study using the inhalation route of administration for a known irritant compound, it was not possible to minimize or avoid irritant effects. That is, to provide a valid assessment of chronic toxicity and carcinogenicity, it was necessary to use at least 1 exposure concentration that would produce significant irritant effects in the nasal tissues. However, based on the overall scope and high magnitude (incidence and severity) of nasal irritant/corrosive effects observed inthe60 ppm group, this exposure concentration resulted in excessive nasal irritation in particular in relation to the concerns regarding interpretation of results, as stated in ILSI Principle 5 for dose selection. For example, squamous cell metaplasia, with the presence of squamous epithelium in areas of nasal passages normally covered by functional respiratory or transitional epithelium, indicated a high degree of prolonged airway irritation and was found only in the 60 ppm group. Squamous cell metaplasia in nasal level II was not only observed in a very high proportion of the animals in this group (93%), but the severity grade was moderate in 33 animals and marked or severe in 4 animals.

 

Test substance-related lung lesions were observed in the 4 ppm group males (pigmented macrophages), 15 ppm group males (pigmented macrophages), 60 ppm group males and females (mononuclear cell inflammation alveolar macrophage, pigmented macrophages, granulomas, and interstitial and pleural fibrosis). Pigmented macrophages in the 4 and 15 ppm group males were considered non-adverse as there were no accompanying findings. There were no test substance-related non-neoplastic or neoplastic lesions outside of the respiratory tract, with the exception of non-neoplastic changes in the eye and mandibular lymph node. Test substance-related mandibular lymph node sinus dilation was noted in the 60 ppm group males and females. Test substance-related eye lesions were noted in a very low number of rats (one 15 ppm group male [corneal ulceration and neutrophil inflammation], 60 ppm group males [corneal epithelium hyperplasia, neovascularization, and neutrophil inflammation], and one 60 ppm group female [corneal epithelium hyperplasia]) and were considered adverse.The no-observed-effect concentration (NOEC) for carcinogenicity and the NOEC for systemic toxicity was 15 ppm. The no-observed-adverse effect concentration (NOAEC) for non-neoplastic changes was 4 ppm, because non-neoplastic nasal findings were considered adverse at 15 and 60 ppm. Nasal carcinogenicity was limited to the group exposed to the 60 ppm TBHP concentration that was higher than the maximum tolerated dose based on body weight and that resulted in excessive irritation/corrosive injury to nasal tissues including respiratory, transitional, and olfactory epithelium, general toxicity and early deaths.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

15

Study duration:

chronic

Species:

rat

System:

respiratory system: upper respiratory tract

Justification for classification or non-classification

TBHP is a non-genotoxic carcinogen (based on the latest available information) and therefore the requirement for classification as a Carcinogen Category 2 under Regulation (EC) No 1272/2008 is barely met.

Additional information

In the key study, nasal lesions were observed in animals that were exposed to concentrations that exceeded the maximum tolerated dose. At the MTD it has been observed that normal biological response to chemical insult may not be properly functioning making the interpretation of the results difficult. The presence of severe damage to the squamous epithelium with subsequent repair and remodelling at both the mid and high dose reveals the severe irritant/corrosive property of the test substance. Only at the highest concentration (60 ppm), when both the MTD has been exceeded and the severe nasal lesions were observed, was the presence of neoplastic nasal lesions observed. At the middle concentration (15 ppm), neoplastic lesions were not observed, and no adverse effects were observed at the lowest concentration (4 ppm) used in the study. 

In another study the tumour promoting properties of TBHP were investigated using mouse skin initiated with 4-nitroquinoline 1-oxide (4NQO; Hoshino et al., 1970). Treatment with TBHP alone lead to ulceration, erosion and hair-follicle hyperplasia at the treatment site but no skin tumours, while no local responses were present in mice given 4NQO alone. Treatment with 4NQO followed by TBHP resulted in 9 malignant and 4 benign skin tumours while 4NQO and tert butanol resulted in 1 malignant tumour only. The results indicate that one clearly toxic concentration of TBHP was capable of promoting the development of dermal tumours after induction by 4-nitroquinoline 1-oxide.

TBHP has also been tested in a guideline inhalation comet assay and was determined to be non-genotoxic. Older studies indicate that TBHP was genotoxic and mutagenic in bacterial and mammalian cells in vitro while results following intraperitoneal injection in vivo indicate it could be mutagenic at the sites of first contact in somatic cells. However the state of the art study conducted on the nasal lining tissues indicate that no site of contact mutagenicity was observed.

The lack of a genotoxic mode of action in the nasal tissue of the rat should be a key piece of information in determining the carcinogenic potential of this substance. The fact that the neoplastic nasal lesions were only observed when there was persistent and severe nasal damage coupled with concentrations that exceeded the Maximum Tolerated Dose, indicates that this substance is a non-genotoxic carcinogen with a clearly identifiable threshold. The evaluation of the neoplastic lesions in light of the compromised animal model needs to be weighed carefully going forward.