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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant study following modified OECD TG 412 protocol focussing on changes in upper respiratory tract.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Principles of method if other than guideline:
Wistar rats were exposed head-nose only to 0, 2, 6 or 18 ppm TBHP vapor (0, 7.4, 22.2 or 66.7 mg/m3) for 6 hr/d, 5 d/wk for 4 weeks. Data collected include an assessment of histopathological alterations and cell proliferation (BrdU incorporation) in nasal epithelial tissue
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt fűr Umwelt Wasserwirtschaft und Gewerbeaufsicht, 21 October 2005
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Aqueous solution containing ca. 70%wt TBHP (CAS No. 75-91-2)
- Lot/batch no. S-102008-236674

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories GmbH, Sulzfeld, Germany
- Age at study initiation: approx. 7 wk
- Weight at study initiation: approx. 240-250 g
- Housing: group housed (6/cage)
- Diet (e.g. ad libitum): stock laboratory pelleted diet (Provimi Kliba SA, Basel, Switzerland) ad libitum
- Water (e.g. ad libitum): tap-water ad libitum

ENVIRONMENT
- Temperature: 20-24 degrees C
- Humidity: 30-70%
- Light/dark cycle: 12 light / 12 hr dark

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
nose/head only
Vehicle:
other: conditioned air
Remarks on MMAD:
MMAD / GSD: not applicable (vapor exposure)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: INA 20 stainless steel aerodynamic exposure system (BASF SE), internal volume 55 l
- Method of holding animals in test chamber: animals restrained in glass exposure tubes with snouts extending into centre of chamber
- Source and rate of air: conditioned filtered compressed air, 6 m3/hr
- Method of conditioning air: charcoal filter, warmed to 22 +/- 2 degrees C and 50% +/- 20% humidity
- System of generating particulates/aerosols:

TEST ATMOSPHERE
- Brief description of analytical method used: head space gas chromatography (18 and 6 ppm exposure groups) or on-line IR spectrometry (2 ppm group)
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Infrared-spectroscopy:
The achieved exposure concentration for the intermediate (6 ppm) and high (18 ppm) treatment groups was quantified 14 times each day using on-line calibrated infrared spectrometer (Nicolet Avatar 370).

Gas chromatography:
GC was used to quantify exposure concentrations in the low treatment group (2 ppm; below limit of quantification of the on-line infrared method). Chamber air was drawn through 3 collection vessels connected in series and filled with deionzied water. The contents of vessels 1 and 2 were pooled and analyzed at approx. 2 hr intervals (3 analyses per day) using GC-FID (Hewlett Packard gas chromatograph with fused silica DB1 column). The contents of the third vessel were analyzed at the end of the day and used to check of the absorptive efficiency of vessels 1 and 2.

Analyses were performed by the GKA Competence Center Analytics, BASF SE, Ludwigshafen, Germany in compliance with the principles of Good Laboratory Practice.
Duration of treatment / exposure:
6hr/d
Frequency of treatment:
daily, 5 d/wk
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 2, 6, 18 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 7.4, 22.2, 66.7 mg/m3
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 1.94(0.16), 6.1(0.3), 18.0(0.5) mg/m3 (mean and SD)
Basis:
analytical conc.
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The exposure concentrations based on results from a preliminary range finding study in which groups of 5 male Wistar rats were exposed nose only to a dynamic inhalation atmosphere of 5, 25 and 100 ppm TBHP vapour for 6 hours on 5 consecutive days; a concurrent control group of 10 male animals was exposed to conditioned air. Due to lethality (2 deaths) and severe clinical signs (pronounced respiratory irritation) on the first two exposure days, the high concentration was decreased to 50 ppm for the remainder of the study. Histopathological examination revealed severe irritation/corrosion of the upper respiratory tract (degeneration of olfactory and respiratory epithelia, necrosis and inflammation) and a 13% (p<0.05) reduction in body weight in the 100/50 ppm group with slight to moderate irritation/corrosion (location and effects as for 100/50 ppm group) and a 6% reduction in body weight (p<0.05) in the 25 ppm group. No treatment-related histopathology was found in the lung even at concentrations severely affecting the upper airways, thus clearly illustrating the upper airways as the critical target tissue. There were no adverse effects in the study at 5 ppm.
Positive control:
not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- twice daily, mornings and afternoon

DETAILED CLINICAL OBSERVATIONS: Yes
- before, during and after exposure

BODY WEIGHT: Yes
- at start of acclimatization; at the start of exposure period (day 0) and on days 7, 14, 21 and 27

FOOD CONSUMPTION: Yes
- on study days 5, 12, 19 and 26

OPHTHALMOSCOPIC EXAMINATION: Yes
- all animals examined pre-treatment; control and high dose animals at study end

HAEMATOLOGY: Yes
- on the morning of necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Parameters: leukocyte count; erythrocyte count; haemoglobin concentration; haematocrit; mean corpuscular volume; mean corpuscular haemoglobin; mean corpuscular haemoglobin concentration; platelet count; differential blood count; reticulocytes; prothrombin time.

CLINICAL CHEMISTRY: Yes
- on the morning of necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Parameters: alanine aminotransferase; aspartate aminotransferase; alkaline phosphatase; gamma-glutamyl transferase; sodium; potassium; chloride; inorganic phosphate; calcium; magnesium; urea; creatinine; glucose; total bilirubin; total protein; albumin; globulins; triglycerides; cholesterol.
Sacrifice and pathology:
Animals were sacrificed (Narcoren anesthesia / exsanguinations) and subject to gross necropsy.

Organ weight data were collected for: adrenal gland, brain, epididymides; heart; kidneys; liver; lungs; spleen; testes; thymus; thyroid.

The following organs/tissues were preserved in 4% formaldehyde, processed (4-6 um sections) and examined by light microscopy:
- all treatment groups: nasal cavity (level 1-4);
- control and high dose groups only: larynx (level 1-3); mediastinal lymph nodes; traecheobronchial lymph nodes; trachea (longitudinal with carina); lung (5 lobes); liver; kidneys

S-phase response
Animals were administered 5-bromo-2´-deoxyuridine (BrdU; 20 mg/ml) via a subcutaneously implanted Alzet minipump commencing 3 d prior to necropsy. Tissue sections (4 um thick) were cut at level 1 of the nasal cavity (as defined by Young (1981) Fund Appl Toxicol 1: 309 – 312) and S-phase response characterised in those regions showing site-specific alterations following conventional light microscopy i.e. the maxilloturbinate and the lateral wall regions.
Quantitiative measurements were performed using a Quantimed 500 image analysis system (200 x magnification) following immunochemical staining (primary antibody = mouse anti-BrdU; secondary antibody = goat anti-mouse; streptavidin/Fast Red labeling; Mayer's haematoxylin counterstain). BrdU-positive cells are characterized by a red reaction product covering the nuclei. Cell types were identified on the basis of their shape and size and the number of positively labelled epithelial cell nuclei linked to the basement membrane counted for a minimum basement membrane length of 1 mm.
Statistics:
Body weights were analysed using Dunnett’s test. Clinical pathology and organ weight parameters were compared using non-parametric one-way analysis (Kruskal-Wallis) followed by the Wilcoxon test if medians were equal. S-phase response data were analysed using the Wilcoxon test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
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:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
Environmental conditions:
Mean daily relative humidity inside the chambers was in a range 38.0 - 63.0%; mean daily temperature was in a range 21.1 - 24.0 degrees C.

General:
All animals survived to scheduled necropsy. There were no treatment-related clinical observations.

Body weight:
Mean body weight and mean body weight gain for animals from the low and intermediate exposure groups did not differ significantly from that of the controls. Mean body weight of high dose males was decreased significantly (approx. - 9 %) relative to controls on study days 7 and 14 with a corresponding significant reduction in body weight gain (decreased by approx. -71% on study day and by approx. -45% on study day 14). Female body weights/weight gains were unremarkable. The Study Director considered these findings in males to reflect a transitory response to the irritative properties of TBHP vapor.

Histopathology:
Treatment-related changes were present in the nasal cavity from 5 males and 3 females from the 18 ppm (66.7 mg/m3) treatment groups and comprised (i) minimal to mild increased thickness of the transitional epithelium and (ii) slight hyperplasia/metaplasia (e.g. reflecting a change towards squamous epithelium). The tip of the maxillary turbinate and the lateral wall of the nasal cavity were mainly affected while other levels of the nasal cavity were free of lesions. No microscopic changes were present in any part of the nasal cavity from animals exposed to 6 ppm or 2 ppm TBHP vapor. Microscopic findings in other tissues examined (e.g. larynx, mediastinal lymph nodes, traecheobronchial lymph nodes, trachea, lung, liver, kidneys) were considered spontaneous and unrelated to treatment by the Study Director.

S-phase response:
Unit length labelling indices were increased significantly in transitional epithelium lining Area 1 (maxillary turbinate) and Area 2 (lateral wall) from level I of the nasal cavity in high dose males and females i.e. the S-phase response was limited to those sites where histopathological lesions were present. No increased cell proliferation was detected in levels II-IV of the nasal cavity.

Effect levels

open allclose all
Dose descriptor:
NOAEC
Effect level:
66.7 mg/m³ air (analytical)
Sex:
male/female
Basis for effect level:
other: systemic effects: no substance-related changes
Dose descriptor:
LOAEC
Effect level:
66.7 mg/m³ air (analytical)
Sex:
male/female
Basis for effect level:
other: local effects: minimal to mild hyperplasia/metaplasia of transitional epithelium in level I of the nasal cavity with significantly increased cell proliferation; no pathology in any other tissue
Dose descriptor:
NOAEC
Effect level:
22.2 mg/m³ air
Sex:
male/female
Basis for effect level:
other: local effects: no changes in nasal tissue

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Histopathology:

Minimal to mild hyperplasia/metaplasia of the transitional epithelium lining level I of the nasal cavity was present as follows:

 

Nasal cavity level I

Male animals

Female animals

Dose - ppm

0

2

6

18

0

2

6

18

Organs examined

6

6

6

6

6

6

6

6

Hyperplasia/metaplasia transitional epithelium

0

0

0

5

0

0

0

3

Grade 1 (minimal)

 

 

 

4

 

 

 

3

Grade 2 (slight, mild)

 

 

 

1

 

 

 

 

 

S-phase response:

Unit length labelling indices were increased significantly in transitional epithelium lining Area 1 and Area 2 as follows:

 

 

ULLI

Males

Females

Dose ppm

 

Area 1

Area 2

Area 1

Area 2

0

M

6.68

5.89

9.04

5.21

 

SD

2.18

2.14

3.38

2.37

 

n

6

6

6

6

2

M

7.45

7.19

8.97

5.79

 

SD

5.75

5.98

5.21

3.94

 

n

6

6

6

6

6

M

10.19*

6.56

10.81

6.77

 

SD

4.08

3.86

2.58

2.98

 

n

6

6

6

6

18

M

111.78**

83.6**

75.39*

31.63**

 

SD

56.45

52.23

56.03

27.30

 

n

6

6

6

6

*: p <= 0.05, **: p <= 0.01

Wilcoxon test, one sided

ULLI     = unit length labeling index; M = mean; SD = standard deviation; n = number of animals The increase in ULLI at 6 ppm in Area 1 of males is within historical control responses and is not considered to be a treatment related finding.

Applicant's summary and conclusion

Conclusions:
TBHP vapor is irritating and cytotoxic to rat nasal epithelium with a NOAEC (sub-acute local toxicity) of 6 ppm (22.2 mg/m3).
Executive summary:

The sub-acute inhalation toxicity of TBHP was investigated in groups of male and female Wistar rats exposed head-nose only to 0, 2, 6 or 18 ppm vapor (0, 7.4, 22.2 or 66.7 mg/m3) 6 hr/d, 5 d/wk for 4 wk. Following the last exposure, blood was sampled and clinical chemistry and haematology parameters determined using standard methods. The animals were then subject to gross necropsy, including macroscopic examination of the major internal organs and collection of organ weight data. Selected tissues were processed and examined microscopically with particular emphasis on the nasal cavity (4 levels) and larynx (3 levels). Determinations of cell proliferation (BrdU incorporation) were also performed on nasal tissue. The investigation was GLP compliant and followed OECD guideline 412.

All animals survived to scheduled necropsy with no treatment-related clinical observations. Body weight and body weight gain were decreased in high dose males on exposure days 7 and 14 (possibly reflecting a transitory response to the irritative properties of TBHP vapor) but were similar to control values thereafter.

Findings at gross necropsy were unremarkable, however histopathological assessment treatment-related alterations in the nasal cavity from high dose males and females comprising (i) minimal to mild increased thickness of the transitional epithelium and (ii) slight hyperplasia / metaplasia (e.g. reflecting a change towards squamous epithelium). The tip of the maxillary turbinate and the lateral wall of the nasal cavity were mainly affected while other levels of the nasal cavity were free of lesions. Microscopic findings in other tissues examined (e.g. larynx, mediastinal lymph nodes, traecheobronchial lymph nodes, trachea, lung, liver, kidneys) were spontaneous in origin.

Cell proliferation determinations revealed significantly increased unit length labeling indices in transitional epithelium lining Area 1 (maxillary turbinate) and Area 2 (lateral wall) from level I of the nasal cavity in high dose males and females i.e. responses present to those sites exhibiting histopathological lesions. No increased cell proliferation was detected in levels II-IV of the nasal cavity.

The results demonstrate that Inhalation exposure to TBHP vapour for 28 days resulted in hyperplasia / metaplasia of the transitional epithelia of the nasal cavity with an associated increased S-phase response (the latter present only in tissue exhibiting microscopic changes). Based on these findings, the NOAEC for local toxicity of TBHP following inhalation exposure was 6 ppm (22.2 mg/m3).