Di-tert-pentyl peroxide

Endpoint summary

Description of key information

For this endpoint, 3 well-conducted studies are available: one on di-tert-amyl-peroxide, and two on di-tert-butyl peroxide. Data on di-tert-butyl peroxide are presented hereafter as a read across is proposed between these 2 substances.

- Repeated dose toxicity by oral route with di-tert-amyl peroxide in rats, 28-day exposure / OECD 407 (Bentz, 2012): NOAEL is 300 mg/kg bw/day based on kidney toxicity in male rats at 1000 mg/kg bw/day (association of renal a2µ globulin hyaline droplets with tubular basophilia, suggesting previous chronic cell damage and increased cell turnover).  The liver effects are considered as adaptive.

- Repeated dose toxicity by oral route on di-tert-butyl peroxide in rats, 28-day to 42-day exposure / OECD 422 (Possnecker, 2008): based on effects on forestomach, the local NOAEL can be established at 100 mg/kg bw/day in female rats, although this effect is not relevant in humans. The effects observed in male kidney rats can be considered adverse at 1000 mg/kg although no IHC analysis has been performed to characterize a2µ globulin. The other observed effects in liver can be rather considered as adaptive.

- Repeated dose toxicity study by inhalation on di-tert-butyl peroxide in rats, 90-day exposure /OECD 413 (Jonker, 2013): The inhalation route was chosen because of the relatively high vapor pressure and as such likely exposure via inhalation.  Few modest changes at the highest concentration tested (increases in liver and kidney weight and altered plasma levels of cholesterol and creatinine) were observed and not considered adverse. Therefore the highest concentration level (0.993 g/m3 actual concentration) was a NOAEC.

Renal a2µ globulin hyaline droplets are considered to be specific to male rats and therefore not relevant for human. Forestomach effects are not relevant in human as the non-glandular forestomach tissue is not present in human.

 

Di-tert-amyl peroxide repeated dose toxicity:

The toxicity of di-tert-amyl peroxide following daily oral administration (gavage) to rats for 4 weeks was evaluated in an OECD TG 407 study (Bentz, 2012). The substance was dissolved in corn oil and administrated to groups of five male and five female Sprague-Dawley rats at 0, 100, 300 or 1000 mg/kg/day at a dose volume of 5mL/kg bw.

There were no unscheduled deaths during the study. The only relevant clinical sign was ptyalism noted from day 11 on all animals treated at 1000 mg/kg/day and was considered to be non-adverse. There were no toxicologically relevant effects on mean body weight, mean body weight change or mean food consumption. There were no test item-related effects at Functional Observation Battery, including motor activity, or at macroscopic post-mortem observation.

Some minor hematological findings and blood biochemistry findings were noted and considered as test-item related, but not adverse. At urinalysis, all males had a few cells in all fields of urine microscopically observed (only one in controls) and a higher mean urine volume. These urinary findings were considered to be non-adverse.

At pathology, there were minimally to slightly statistically higher mean liver and mean kidney weights in males, and mean liver weight in females when compared with controls (up to +45% for the liver and up to +20% for the kidney). Microscopic examination revealed centrilobular hypertrophy in the liver of males and females, and tubular hyaline droplets (which were shown to be a2µ globulin at immunohistochemistry) and basophilia in the kidney of males. These kidney findings were considered to be adverse in the current study for rats at 1000 mg/kg. Nevertheless, renal hyaline droplets are considered not to be relevant for human.

At 100 and 300 mg/kg/day, there were minimally to slightly higher mean liver and kidney weights in males at both dose-levels and mean liver weight in females at 300 mg/kg/day when compared with controls, which were statistically significant only in males at 300 mg/kg/day (up to +25% for the liver and up to +16% for the kidney). At microscopic examination, there was centrilobular hypertrophy in the liver at 300 mg/kg/day in 1/5 males and 2/5 females. In the kidney of males, tubular hyaline droplets were seen with dose-related severity from 100 mg/kg/day. In the absence of associated tubular basophilia, these renal findings were considered not to be adverse at 100 and 300 mg/kg/day.

Therefore the NOAEL was 300 mg/kg/day in males as at 1000 mg/kg/day renal a2µ globulin hyaline droplets were associated with tubular basophilia, suggesting previous chronic cell damage and increased cell turnover. There were no other effects that could be considered as adverse at any doses. The liver effects are considered as adaptive. Renal a2µ globulin hyaline droplets are considered to be specific to male rats and therefore not relevant for human.

 

Di-tert-butyl peroxide repeated dose toxicity:

The repeated dose toxicity of di-tert-butyl peroxide after oral exposure was assessed in an OECD TG 422 study (Possnecker, 2008).

Di-tert-butyl peroxide was administered once daily orally (by gavage) at dosages of 0, 100, 300, and 1000 mg/kg/day at a dose volume of 5mL/kg bw, to male rats for 42 days in total and to female rats throughout the pre-pairing, the pairing, the gestation and the lactation periods until day 4 post-partum (last dosing).

Treatment at 1000 mg/kg was associated with body weight effects in male animals (-13 % compared to control group). Food consumption was decreased in male animals during the study. A sign of discomfort was noted in all animals at 1000 mg/kg/day in the way that the animals moved their heads through the bedding material after the daily administration of test item. Some male and female animals were noted with ruffled fur.

Treatment at 1000 mg/kg/day and 300 mg/kg/day were associated with increased liver and kidney weights associated with histopathological effects in liver (minimal centrilobular and diffuse hepatocellular hypertrophy with association of a consequent increase in diffuse follicular cell hypertrophy in thyroid glands in males and females at 1000 mg/kg/day) and in kidneys (moderate diffuse tubular degeneration/regeneration with slight multifocal single cell necrosis and hyaline casts as well as hyaline droplets in males at 1000 mg/kg/day). Minimally increased incidence and severity of diffuse hyperkeratosis was also noted in forestomach in males at 1000 mg/kg/day and females at 1000 mg/kg/day and 300 mg/kg/day.

Behavioral effects were considered to be not influenced by the treatment with the test item. No effects were noted on reproduction data, for the parameters during the clinical laboratory investigations, or for macroscopic findings during necropsy.

Based on effects on forestomach, the local NOAEL can be established at 100 mg/kg bw/day in females although this effect is not relevant in humans. The effects observed in male kidney rats are considered adverse at 1000 mg/kg although also not relevant in human. The other observed effects in liver are rather considered as adaptive.

Comparison between di-tert-amyl peroxide and di-tert-butyl peroxide (same solvent, same range of administrated doses) show very close systemic effects: male kidney adverse effects at 1000 mg/kg, liver adaptive effects at lowest dose levels. Effects on forestomach were observed only with di-tert-butyl peroxide, nevertheless it should be stressed that these effects were of very minor toxicological importance in males and that the duration exposure for females was 42 days instead of 28 days for di-tert-amyl peroxide. In addition the doses are equivalent in grams but not in moles, the administrated doses in moles being 16% higher for di-tert-butyl peroxide. The slight difference in doses and in duration exposure could explain that di-tert-butyl peroxide exhibited slightly more effects both on kidney (tubular single cell necrosis) and on forestomach (hyperkeratosis). The difference in administrated volumes (5 ml/kg bw/day for di-tert-amyl peroxide vs 4 mL/kg bw/day for di-tert-butyl peroxide) could also partially explain that di-tert-butyl peroxide induced more effects in the stomach rats, since di-tert-butyl peroxide was more concentrated in stomach than di-tert-amyl peroxide.

 

Di-tert-butyl peroxide was also tested for systemic toxicity following repeated inhalation exposure in an OECD TG 413 (Jonker, 2013). A 90-day inhalation study was conducted in the rat at target concentrations of 100, 300 and 1000 mg/m3. Endpoints to assess toxicity included clinical and ophthalmoscopic observations, growth, food consumption, hematology, clinical chemistry and organ weights. In addition, the animals were examined grossly at necropsy, and a large number of organs and tissues were examined microscopically. The target concentrations were accurately achieved as demonstrated by the results of total carbon analysis of the test atmospheres. The overall mean actual concentrations (± standard deviation of the daily mean concentration) were 101 (± 3), 299 (± 3) and 993 (± 10) mg/m3 for the low-, mid- and high concentration level respectively. All animals survived until scheduled sacrifice. Clinical and ophthalmoscopic observations, growth and food consumption results, hematology values, most clinical chemistry values, most organ weights, and necropsy and histopathology findings showed no treatment-related changes.

Clinical chemistry values showed slight but statistically significant changes in the plasma levels of cholesterol (increased) and creatinine (decreased) at the high concentration in male and female rats, respectively. These findings were considered to be of no toxicological significance.

The relative weights of the liver and kidneys were slightly (about 10%) but statistically significantly increased in male rats of the high-concentration group. In female rats of this group relative liver weight was increased to about the same extent but the difference from controls was not statistically significant. Though these organ weight changes were related to treatment, they were considered not to represent adverse effects of the test material because of the modest magnitude of the increases and the absence of corroborative histopathological alterations or clinical chemical indicators of organ damage.

In conclusion, under the conditions of this study exposure to di-tert-butyl peroxide CAS# 110-05-4 resulted in a few modest changes at the highest concentration tested (increases in liver and kidney weight and altered plasma levels of cholesterol and creatinine). No treatment-related changes were observed at the lower concentrations. Since the changes at the high-concentration were considered not to constitute adverse effects, this exposure level (993 mg/m3 actual concentration) was a No-Observed- Adverse-Effect Concentration (NOAEC).

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 March 2012 - 09 October 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: breeder: Charles River Laboratories France, l’Arbresle, France.
- Age at study initiation: approximately 6 weeks old on the first day of treatment
- Mean body weight at study initiation: the males had a mean body weight of 215 g (range: 198 g to 232 g) and the females had a mean body weight of 169 g (range: 155 g to 179 g).
- Fasting period before study: no
- Housing: the animals were housed by five, in polycarbonate cages with stainless steel lids (Tecniplast 2000P, 2065 cm2) containing autoclaved sawdust
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: 8 days before the beginning of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: 23 March 2012 to 20 April 2012

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

The test item was administered as a solution in the vehicle.
It was mixed with the required quantity of vehicle. No correction factor was applied.
The frequency of dose formulation preparation was based on available stability data. The dose formulations were stored at room temperature, protected from light, and delivered to the study room in brown flasks.


VEHICLE
- Justification for use and choice of vehicle: test item soluble in corn oil
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Type of method: GC-FID.
Test item concentrations: remained within an acceptable range of variation compared to nominal values.
Homogeneity: homogeneous
Stability: stable after 4 days at ambient temperature and protected from light

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
100, 300 and 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

5 animals per sex per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose-levels were selected in agreement with the Sponsor based on the results of a 7 day toxicity study (CIT/Study No. 38492 TSR). In this preliminary study, the test item was given at 100, 300 or 1000 mg/kg/day to Sprague-Dawley rats. No mortalities or clinical signs were observed in the study. There were no obvious effects of the test item on body weight and there was a minimal trend to a dose-related reduction in mean food consumption in males. There were no test item-related macroscopic findings and the only relevant effect seen at necropsy was higher liver weights in test item-treated males, with statistical significance at 1000 mg/kg/day.

The same dose-levels as in the preliminary study have thus been chosen for the present study.


- Rationale for animal assignment: computerized stratification procedure

Positive control:

no (not required).

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
- Time schedule mortality: once a day during the acclimation period and at least twice a day during the treatment period.
- Clinical signs: once a day.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: before the beginning of the treatment period and then once a week until the end of the study.

BODY WEIGHT:
- Time schedule: once before group allocation, then on the first day of treatment and at least once a week until the end of the study.

FOOD CONSUMPTION:
- Time schedule: once a week until the end of the study.

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule: each animal was evaluated once in week 4.

HAEMATOLOGY, CLINICAL CHEMISTRY, URINALYSIS:
- Time schedule: at the end of the treatment period.

Sacrifice and pathology:

ORGAN WEIGHTS: see table below

GROSS PATHOLOGY:
Complete macroscopic post-mortem examination of all study animals.

HISTOPATHOLOGY:
- on all tissues listed in the table below for the control and high-dose animals (groups 1 and 4) sacrificed at the end of the treatment period,
- liver (males and females) and kidneys (males only) from the low- and intermediate-dose animals (groups 2 and 3) sacrificed at the end of the treatment period,
- immunostained kidneys from the control and high-dose males (groups 1 and 4) sacrificed at the end of the treatment period,
- on all macroscopic lesions from all low- and intermediate-dose animals (groups 2 and 3) sacrificed on completion of the treatment period.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

(for clinical signs but no mortality)

Mortality:

mortality observed, treatment-related

Description (incidence):

(for clinical signs but no mortality)

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:

effects observed, treatment-related

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:

MORTALITY:
There were no unscheduled deaths during the study.

CLINICAL SIGNS:
All animals treated at 1000 mg/kg/day had ptyalism from day 11. This was considered to be related to treatment with the test item but non adverse. Incidental findings included reflux at dosing, scabs, soft feces, thinning of hair and chromodacryorrhea and were generally observed in isolated animals.

BODY WEIGHT (GAIN):
There were no toxicologically relevant effects on mean body weight and mean body weight change.

FOOD CONSUMPTION:
There were no toxicologically relevant effects on mean food consumption.

NEUROBEHAVIOURAL EXAMINATION:
There were no test item-related effects at Functional Observation Battery, including motor activity.

HAEMATOLOGY:
At 1000 mg/kg/day, there were in males statistically significantly higher mean white blood cell count, due to higher basophil but mainly lymphocyte counts, when compared to controls. There were also statistically significantly lower mean hemoglobin concentration and packed cell volume. Females of that group had a statistically significantly shortened prothrombin time than in the other groups.
These findings were considered to be test item-related but non adverse in the absence of any pathological correlates or in view of the amplitude of difference from controls.

At 100 and 300 mg/kg/day, there were no test item-related effects.

CLINICAL CHEMISTRY:
At 1000 mg/kg/day, there were statistically significant low mean glucose and high mean cholesterol levels in both sexes, associated in females with a statistically significantly higher mean triglyceride concentration, when compared with controls. The trend to a high mean cholesterol level appeared in a dose-related manner from 100 mg/kg/day, especially in males. These variations were considered to be test item-related but non adverse in the absence of any correlating adverse effect at pathology or in view of the amplitude of difference from controls.

At 1000 mg/kg/day, there were also in females statistically significant variations in mean chloride and calcium levels when compared with controls. In the absence of any other correlating findings in this sex, these variations were considered not to be toxicologically relevant.

The statistically significantly low alkaline phosphatase activity recorded at 1000 mg/kg/day in both sexes (350 and 198 IU/L in males and females, respectively, vs. 491 and 314 IU/L in controls, p<0.01) was considered not to be toxicologically relevant.

URINALYSIS:
At 1000 mg/kg/day, all males had a few cells in all fields of urine microscopically observed (one in controls) and a higher mean urine volume (also seen in two females) when compared with controls. These finding were considered to be test item-related but non adverse. The cells observed in urine could be related to the effects seen in male kidneys at microscopy.

At 100 and 300 mg/kg/day, there were no test item-related effects.
The higher urine specific gravity noted in males at 100 mg/kg/day (1044, vs. 1030 in controls, p<0.05) was considered to be unrelated to the treatment with the test item as not observed in the highest dose-levels.

ORGAN WEIGHTS:
When compared with controls, there were minimal to slight increases in mean absolute and relative liver weights in males at all dose-levels and in females at 300 and 1000 mg/kg/day. These increases were statistically significant in males at 300 and 1000 mg/kg/day and in females at 1000 mg/kg/day.

The mean absolute and relative kidney weights were increased in males given the test item at all dose-levels, reaching statistical significance for the relative weight at 300 and 1000 mg/kg/day.

These variations in liver and kidney weights were considered to be related to the test item administration in view of the correlated microscopic changes.

Other statistically significant changes for heart, prostate with seminal vesicles, spleen and uterus were considered not to be toxicologically significant as there were no microscopic correlates.

GROSS PATHOLOGY:
The few macroscopic findings noted at the end of the treatment period were of those commonly recorded in the Sprague-Dawley rat and none were considered to be related to the test item administration.

HISTOPATHOLOGY: NON-NEOPLASTIC:
The test item administration induced centrilobular hypertrophy in the liver from 300 mg/kg/day in males and females, and tubular hyaline droplets in the kidney of males at all dose-levels.

. Liver
Minimal to slight centrilobular hypertrophy was seen in 3/5 males and 5/5 females at 1000 mg/kg/day. This finding was also seen with minimal severity in 1/5 males and 2/5 females at 300 mg/kg/day. There were no associated degenerative changes at any of the dose-levels.

. Kidney
Moderate hyaline droplets were noted in the proximal tubular epithelium of the renal cortex from 5/5 males at 1000 mg/kg/day, and were associated with evidence of regeneration characterized by multifocal minimal to slight tubular basophilia. Hyaline droplets were observed with lesser severity at 100 and 300 mg/kg/day. Hyaline droplets were variably sized, round, dense eosinophilic and intracytoplasmic. Moderate to marked staining with an anti-a2µ globulin antibody was seen in droplets located in the cytoplasm of tubular cells and in the lumen of tubules from all high-dose males. Minimal to slight staining with this antibody was also seen in all control males, but it was located in small droplets in the cytoplasm of tubular cells only and was not seen within the lumen.
Minimal tubular basophilia was seen in 4/5 control males but was less extensive than in rats given the test item at 1000 mg/kg/day. At 100 and 300 mg/kg/day, incidence and severity of tubular basophilia were similar to that seen in controls.
Hyaline droplets were not observed in any of the females.

Other microscopic findings noted in treated animals were considered incidental changes, as they also occurred in controls, were of low incidence, had no dose-relationship in incidence or severity, and/or are common background findings in the Sprague-Dawley rat.

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Critical effects observed:

not specified

Conclusions:

Under the experimental conditions of this study, the NOAEL is 300 mg/kg bw/d in males based on the association of renal alpha2µ-globulin hyaline droplets with tubular basophilia, suggesting previous chronic cell damage and increased cell turnover.
The NOAEL in females is 1000 mg/kg.
There were no other effects that could be considered as adverse at any doses. Renal alpha2µ globulin hyaline droplets are considered to be specific to male rats and therefore not relevant for human.

Executive summary:

The toxicity of di-tert-amyl peroxide following daily oral administration (gavage) to rats for 4 weeks was evaluated in an OECD 407 study.

The substance was dissolved in corn oil and administrated to groups of five male and five female Sprague-Dawley rats at 0, 100, 300 or 1000 mg/kg/day.

 

The animals were checked at least twice daily during the dosing period for mortality and morbidity and once daily for clinical signs. In addition, detailed clinical examinations were performed at least once weekly. Body weight was recorded once before the beginning of the treatment period, and then at least once a week during the study as food consumption. Towards the end of the dosing period, a Functional Observation Battery including motor activity measurement, and hematology, blood biochemistry and urinalysis were performed on all animals. Blood was also taken for a possible further thyroid hormone investigation, but no analysis was performed.

On completion of the treatment period, the animals were euthanized and submitted to a full macroscopicpost-mortemexamination. Designated organs were weighed and selected tissues were preserved. A microscopic examination was performed on selected tissues (including liver and kidneys) from control- and high-dose animals sacrificed at the end of the treatment period, on liver (males and females) and kidneys (males only) from the low- and mid-dose animals sacrificed at the end of the treatment period and on all macroscopic lesions.

 

There were no unscheduled deaths. The only relevant clinical sign was ptyalism noted from day 11 in all animals treated at 1000 mg/kg/day and was considered to be non adverse. There were no toxicologically relevant effects on mean body weight, mean body weight change or mean food consumption. There were no test item-related effects at Functional Observation Battery, including motor activity, or at macroscopicpost-mortemobservation.

 

At 1000 mg/kg/day in males, some hematological findings were considered to be test item-related but non adverse (higher mean white blood cell count, mainly due to higher mean lymphocyte count, lower mean hemoglobin concentration and mean packed cell volume).

At blood biochemistry, some variations were noted and considered to be test item-related but non adverse (lower mean glucose level in males and females respectively, higher mean cholesterol level in females, with a higher mean triglyceride concentration).

At urinalysis, all males had a few cells in all fields of urine microscopically observed (only one in controls) and a higher mean urine volume. These urinary findings were considered to be non adverse. The cells observed in urine could be related to the effects seen in male kidneys at microscopy.

 

At pathology, there were minimally to slightly statistically higher mean liver and mean kidney weights in males, and mean liver weight in females when compared with controls (up to +45% for the liver and up to +20% for the kidney). Microscopic examination revealed centrilobular hypertrophy in the liver of males and females, and tubular hyaline droplets (which were shown to bea2µ globulin at immunohistochemistry) and basophilia in the kidney of males. These kidney findings were considered to be adverse in the current study for rats at 1000 mg/kg. Nevertheless, renal hyaline droplets are considered not to be relevant for human.

 

At 100 and 300 mg/kg/day, there were no test item-related effects on hematology, blood biochemistry or urinalysis parameters, except a trend to a high mean cholesterol level from 100 mg/kg/day, especially in males. At pathology, there were minimally to slightly higher mean liver and kidney weights in males at both dose-levels and mean liver weight in females at 300 mg/kg/day when compared with controls, which were statistically significant only in males at 300 mg/kg/day (up to +25% for the liver and up to +16% for the kidney). At microscopic examination, there was centrilobular hypertrophy in the liver at 300 mg/kg/day in 1/5 males and 2/5 females. In the kidney of males, tubular hyaline droplets were seen with dose-related severity from 100 mg/kg/day. In the absence of associated tubular basophilia, these renal findings were considered not to be adverse at 100 and 300 mg/kg/day.

 

Under the experimental conditions of this study, the dose-level of 1000 mg/kg/day was considered to be an adverse Effect Level in rats as at 1000 mg/kg/day renal a2µ globulin hyaline droplets were associated with tubular basophilia, suggesting previous chronic cell damage and increased cell turnover. There were no other effects that could be considered as adverse at any doses. Renal a2µ globulin hyaline droplets are considered to be specific to male rats and therefore not relevant for human.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP guideline study on di-tert-amyl peroxide and on di-tert-buty peroxide.

System:

urinary

Organ:

kidney

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 2012 - March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This is a well documented study conducted according to modern standards of protocol, quality assurance, and good laboratory practices. The test material was >99 % pure.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

yes

Remarks:

A micronucleus assay was included

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Young adult, male and female Wistar Hannover outbred rats (RccHan®:WIST) were obtained from a colony maintained under specific pathogenfree
(SPF) conditions at Harlan Laboratories, The Netherlands. On the day of randomization (shortly before the first exposure day), the age of the rats was about 7-8 weeks, and the initial body weight variation did not exceed ± 20% of the mean weight for each sex. Mean body weights at the start of treatment in the range-finding study were 295 and 179 grams for male and female animals, respectively. Mean body weights at the start of treatment in the main study were 264 and 176 grams for male and female animals, respectively.

Upon arrival, the rats were taken to a quarantine room and checked for overt signs of ill health and anomalies. During the quarantine period, serological investigation of the microbiological status was conducted in blood samples taken from three (range-finding study) or five (main study) randomly selected animals. Two days after arrival, the results of serological tests were passed on by telephone and indicated an acceptable microbiological status. Subsequently, the animals were released for experimental use. The duration of the acclimatization period to the conditions in the experimental room prior the first exposure was 16 days (range-finding study) or 10 (males) / 11 (females) days (main study).

From their arrival, the rats were housed under conventional conditions in one room separated by sex. No other test system was housed in the same room during the study. Lighting was artificial (fluorescent tubes) with a sequence of 12 hours light and 12 hours dark. The room was ventilated with about 10 air changes per hour. The temperature and relative humidity in the room were 22 ± 2°C and 45-65%, respectively, with the following exceptions. During the main study, the temperature was just below 20°C (minimum 19.8°C) for about half an hour on 22 January 2013.
The relative humidity exceeded 65% during occasional brief periods following cleaning activities. In addition, during the main study, the relative humidity was lower than 45% on three occasions: on 8 December 2012 for about half an hour due to technical malfunction (minimum 25.9%); on 10 January 2013 for about an hour due to maintenance (minimum 34.3%); and on 13 February 2013 for about five hours due to technical malfunction (minimum 20.2%).

The animals were housed in groups of five, separated by sex, in Makrolon® cages (type IV) with a bedding of wood shavings (Lignocel, Rettenmaier & Söhne GmbH & Co, Rosenberg, Germany) and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment (Lillico, Betchworth, England). Sentinel animals were housed similarly (individually in the range-finding study, in groups of four of the same sex in the main study). During exposure, the animals were kept individually in the exposure units. Immediately after each exposure, the animals were returned to their home cages. After treatment with the mutagen Mitomycin C, the five animals of the positive control group were kept in smaller Makrolon® cages with filter tops (one or two animals per cage; bedding: wood shavings; enrichment:
strips of paper) until sacrifice the next day.

Feed and drinking water were provided ad libitum from the arrival of the animals until the end of the study, except during inhalation exposure and, in the main study, during the fasting period before scheduled sacrifice. The animals received a proprietary cereal-based rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3) from a commercial supplier (SDS Special Diets Services, Whitham, England). Each batch of RM3 diet is analysed by the supplier for nutrients and contaminants. The feed was provided as a powder in stainless steel cans, covered by a perforated stainless steel plate to
prevent spillage. The feed in the feeders was replaced with fresh portions once weekly and filled as needed.
Each cage was supplied with domestic mains tap-water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC). The water was given in polypropylene bottles, which were cleaned weekly and filled as needed. Results of the routine physical, chemical and microbial examination of the drinking water as conducted by the supplier are made available to the test facility. In addition, the supplier periodically (twice per year) analyses water samples taken on the premises of the test facility for a limited number of variables.

Route of administration:

inhalation: vapour

Type of inhalation exposure:

nose/head only

Vehicle:

clean air

Remarks on MMAD:

MMAD / GSD: Not applicable, vapour test atmosphere

Details on inhalation exposure:

The animals were exposed to the test atmosphere in a nose-only inhalation chamber (Institute’s design) consisting of a cylindrical PVC column with a volume of about 75 litres, surrounded by a transparent hood. The test atmosphere was introduced at the bottom of the central column and was exhausted at the top. Each column included three rodent tube levels of 20 ports each. In both the rang-finding study and the main study the animals were placed at the top level. Empty ports were used for measurement of temperature and relative humidity (high concentration groups only). The animals were secured in plastic animal holders (Battelle), positioned radially through the outer hood around the central column. Only the nose of the rats protruded into the interior of the column. The remaining ports were closed. Male and female rats were placed in alternating order. Animals were rotated weekly with respect to the position in the column. From 25 February 2013, larger sized animal holders were used for the male rats of the main study because these animals had reached a size for which the standard holders were too small.

The animal's body does not exactly fit in the animal holder which always results in some leakage from the high to the low pressure side. By securing a positive pressure in the central column and a slightly negative pressure in the outer hood, which encloses the entire animal holder, air leaks from nose to thorax rather than from thorax to nose. This way, dilution of test atmosphere at the animals’ noses was avoided.

The units were illuminated externally by normal laboratory fluorescent tube lighting. The total airflow through the unit was at least 1 litre/min per animal. The air entering the unit was maintained between 22 ± 3°C and the relative humidity between 30% and 70%.

The inhalation equipment was designed to expose rats to a continuous supply of fresh test atmosphere. To generate the test atmospheres, a liquid flow of test material, controlled by a peristaltic pump (Gilson France SA, Villiers le Bel, France), was evaporated in a glass evaporator. The temperature of the evaporator was controlled at 22.5°C (exceptions: 22.0°C on the first six days of the rangefinding study; occasionally 22.7 or 22.8°C in the main study) using a temperature controlled flow of circulating water. The vapour was transported in a stream of humidified compressed air, the flow of which was controlled by a mass flow controller (Bronkhorst, Hi Tec, Ruurlo, The Netherlands).
In the range-finding study, the test atmospheres for group 2 (low-concentration, target 0.1 g/m3) and 3 (mid-concentration, target 1 g/m3) were obtained by diluting the high-concentration (group 4, target 10 g/m3). For this purpose, a mass flow controlled stream of the high-concentration test atmosphere was supplemented with a mass flow controlled stream of humidified compressed air via an eductor (Fox Eductor from Fox Valve Development Corp., Dover, NJ, USA). The generated test atmospheres were directed to the bottom inlets of the exposure units.
In the main study, all three test atmospheres (target concentrations 0.1, 0.3 and 1 g/m3) were obtained by diluting a pre-mixture containing about 3 g/m3 of the test material in humidified compressed air. First, mass flow controlled streams of the pre-mixture were supplemented with a mass flow controlled stream of humidified compressed air via an eductor to obtain the low- and mid-concentration. Next, the remaining stream of the pre-mixture was diluted with a mass flow controlled stream of humidified compressed air to obtain the high-concentration. The generated test
atmospheres were directed to the bottom inlets of the exposure units.
The exposure unit for the control animals (range-finding and main study) was supplied with a measured stream of humidified compressed air only.

The animals were placed in the exposure unit after stabilization of the test atmosphere.

The nominal concentration was determined, for each exposure day, by dividing the total amount of test material used (by weight) by the total volume of air passed through the exposure unit. The generation efficiency was calculated from the actual and the nominal concentration (efficiency = actual concentration as percentage of nominal concentration). In the range-finding study, the nominal concentration was calculated only for the high-concentration because the low- and mid-concentration were obtained by diluting the high-concentration.

The chamber airflow of the test atmospheres was recorded about hourly using a Rotameter or a mass flow controller.

The temperature and the relative humidity of the test atmospheres were measured continuously and recorded every minute using a CAN transmitter with temperature and relative humidity probes (G.Lufft Mess- und Regeltechnik GmbH, 70719 Fellbach, Germany) or were (additionally) measured about hourly by means of a RH/T device (TESTO 635-1, TESTO GmbH & Co, Lenzkirch, Schwarzwald, Germany)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The actual concentration of the test material in the test atmospheres was measured by total carbon analysis (Sick Maihak EuroFID total hydrocarbon analyser; Sick Instruments Benelux, Hedel, the Netherlands). The response of the analyser was recorded on a PC every minute using a CAN transmitter (G. Lufft Mess- und Regeltechnik GmbH, 70719 Felbach, Germany). The responses were converted to concentrations by means of calibration graphs. For each exposure day, the mean concentration was calculated from the values determined every minute. Representative test atmosphere samples were taken continuously from the exposure unit at the animals’ breathing zone and were passed to the total carbon analyser (TCA) through a sample line.
Prior to the first exposure, the output of the flame ionization detector of the TCA was calibrated using PET sample bags with known volumes of clean dry air and known amounts (by weighing) of test material. For each target concentration three calibration concentrations were prepared, at least in duplicate, and analysed (approximately 80, 100 and 120% of the target concentration). The calibrations were checked weekly by measuring the concentration in a sample bag with a theoretical concentration close to the target concentration. If the measured concentration deviated more than 5% from the theoretical concentration and this was confirmed with a second sample bag, the TCA was recalibrated.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

5 days/week, 6 h/day

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000 mg/m3
Basis:
other: target concentrations

Remarks:

Doses / Concentrations:
0, 101, 299, and 993 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Concentrations for the main study were based on the results of the RF study.
The animals (5/sex) were exposed on 5 days per week, 6 hours per day, over a 2-week study period (total of 10 exposure days), for the last time on the day before scheduled sacrifice.
Shortly before initiation of exposure (range finding study: day -1; main study days -4 and -5 for males and females, respectively), the animals were allocated to the various groups by computer randomization proportionally to body weight (males and females separately). The surplus animals were kept in reserve to serve as sentinels (range-finding study: one/sex; main study: four/sex). These animals were discarded at the end of the inlife phase of the study.

Positive control:

For the in vivo MN test (see also section 7.6.2) the animals of the positive control group received a single intraperitoneal injection with Mitomycin C on the day prior to scheduled sacrifice.

Observations and examinations performed and frequency:

Clinical signs
On exposure days, each animal was observed daily in the morning, prior to exposure, by cage-side observations and, if necessary, handled to detect signs of toxicity. All animals were thoroughly checked again after exposure. During exposure in the main study, a group-wise observation was made about halfway the 6-hour exposure period. During exposure in the range-finding study, observations were made more frequently on account of abnormalities noted in the groups exposed to the test material (tail trembling and restlessness). During weekends and on public holidays only one check per day was carried out. All abnormalities, signs of ill health, and reactions to treatment were recorded.

Ophthalmoscopic examination
In the main study, ophthalmoscopic observations were made prior to the start of the exposure in all rats (on day -6) and in the last week of the exposure period in all rats of the control group and the high-concentration group (on day 92). Eye examination was carried out using an ophthalmoscope after induction of mydriasis by a solution of atropine sulphate.

Body weight
Range-finding study
The body weight of each animal was recorded once before the start of exposure (these pre-test weights served as a basis for animal allocation, once prior to exposure on the first exposure day (day 0) and twice weekly thereafter, for the last time on the day of scheduled sacrifice.
Main study
The body weight of each animal was recorded once before the start of exposure. These pre-test weights served as a basis for animal allocation. Animals were weighed prior to exposure on the first exposure day (day 0), twice weekly for the first four weeks of exposure (Mondays and Fridays), then once weekly from the fifth week onward. At the end of the in-life phase, animals were weighed the day before overnight fasting prior to scheduled sacrifice. They were weighed again on the day of sacrifice; this was the terminal body weight used for calculation of organ weights relative to body weight.

Food consumption
Food consumption was measured per cage by weighing the feeders. In the rangefinding study, food consumption was measured over two 7-day periods, starting on day 0. In the main study, consumption was measured from day 0, over successive 7-day periods. The results were expressed in g per animal per day.

Haematology
In the main study, haematology was conducted at the end of the treatment period on all animals. Blood samples were taken from the abdominal aorta of overnight fasted rats (water was freely available) whilst under pentobarbital anaesthesia at sacrifice. Potassium-EDTA was used as anticoagulant. In each sample the following determinations were carried out: red blood cells (RBC), haemoglobin (Hb), packed cell volume (PCV), reticulocytes, total white blood cells (WBC), differential white blood cells, prothrombin time (PT), thrombocytes.
The following parameters were calculated: mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC).

Clinical chemistry
In the main study, clinical chemistry was conducted at the end of the treatment period on all animals, after overnight fasting, at the same time blood samples for haematology were collected. Blood was collected from the abdominal aorta in heparinized plastic tubes. Plasma was prepared by centrifugation. The following measurements were made in the plasma: alkaline phosphatase activity (ALP), cholesterol (total), aspartate aminotransferase activity (ASAT), phospholipids, alanine aminotransferase activity (ALAT), triglycerides, gamma glutamyl transferase activity (GGT), creatinine, bilirubin (total), urea, total protein, inorganic phosphate (PO4), albumin, calcium (Ca), ratio albumin to globulin (calculated), chloride (Cl), glucose, potassium (K), sodium (Na).

Sacrifice and pathology:

Pathology
Range-finding study
Macroscopic examination
At the end of the exposure period, the animals were sacrificed in such a sequence that the average time of killing was approximately the same for each group. The animals were sacrificed by exsanguination from the abdominal aorta under pentobarbital anaesthesia and then examined grossly for pathological changes.

Organ weights
At scheduled sacrifice, the following organs of all animals were weighed (paired organs together) as soon as possible after dissection to avoid drying and the relative organ weights (g/kg body weight) were calculated from the absolute organ weights and the terminal body weight: adrenals, heart, kidneys, liver, lung with trachea and larynx, spleen, testes (after weighing, the lung was infused with the fixative).

Tissue preservation
The organs listed above and the complete respiratory tract of all animals were preserved with a 10% solution of Formalin in a neutral aqueous phosphate buffer (final formaldehyde concentration 4 per cent).

Histopathological examination
The main lung lobe (one level), the larynx (three levels, including the base of the epiglottis) and the nasal tissues (levels 3 and 5, as described by Woutersen et al., 1994) of all male animals were examined microscopically. Tissue for microscopy was embedded in paraffin wax, sectioned at 5 µm and stained with haematoxylin and eosin.

Main study
Gross necropsy
On study day 93 (13 and 14 March 2013 for males and females, respectively), animals were fasted overnight but allowed free access to water. The next day (study day 94; 14 and 15 March 2013 for males and females, respectively), the animals were killed in such a sequence that the average time of killing was approximately the same for each group. The animals were anaesthetized by intraperitoneal injection of sodium pentobarbital then killed by exsanguination from the abdominal aorta and examined grossly for pathological changes.

Organ weights
At scheduled necropsy, the following organs were weighed (paired organs together) as soon as possible after dissection to avoid drying and the relative organ weights (g/kg body weight) were calculated from the absolute organ weights and the terminal body weight: adrenals, lung with trachea and larynx, brain, ovaries, epididymides, spleen, heart, testes, kidneys, thymus, liver, thyroid, uterus (after weighing, the lung was infused with the fixative).

Tissue preservation
Samples of the tissues and organs listed below of all animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde (10% solution of formalin). The carcass containing any remaining tissues was retained in formalin until completion of the histopathological examination and then discarded. The following were preserved, all gross lesions, ovaries, adrenals, pancreas, aorta, parathyroids, axillary lymph nodes, parotid salivary glands, brain, pharynx, caecum, pituitary, colon, prostate, duodenum, rectum, epididymides, seminal vesicles / coagulating glands, exorbital lachrymal glands, skeletal muscle (thigh), eyes (with optic nerve), skin (flank), femur with joint, spinal cord, NALT (nose associatedlymphoid tissue, spleen, sternum with bone marrow, heart, stomach, ileum, sublingual salivary glands, jejunum, submaxillary salivary glands, kidneys, testes, larynx, thymus, liver, thyroid, lung, tongue, mammary gland (female), trachea, mandibular (cervical) lymph nodes, tracheobronchial (mediastinal) lymph nodes, nasopharyngeal tissues (with teeth), ureter, nerve-peripheral (sciatic), urethra, oesophagus, urinary bladder, olfactory bulb, uterus (with cervix).
- For the micronucleus test, bone marrow cells of one of the femurs (left femur) were collected from the first five male rats of each group
- Brain: Three levels were examined microscopically (brain stem, cerebrum, cerebellum).
- Larynx: Three levels (one including the base of the epiglottis) were examined microscopically.
- Lung: Each lung lobe was examined microscopically at one level, including main bronchi.
- Nasopharyngeal tissues: Six levels (Woutersen et al., 1994) were examined microscopically (one including the nasopharyngeal duct and the draining lymphatic tissue [NALT]).
- Spinal cord: Retained in vertebral column, at least three levels were examined microscopically (cervical, mid-thoracic and lumbar).
- Stomach: Non-glandular (‘forestomach’) and glandular (fundus, pylorus) parts were examined microscopically.
- Trachea: Three transverse sections and three longitudinal sections (at least one through the carina of the bifurcation of the bronchi) were examined microscopically.

Histopathological examination
The tissues for microscopic examination were embedded in paraffin wax, sectioned at 5 µm and stained with haematoxylin and eosin. Histopathological examination (by light microscopy) was performed on all tissues and organs listed above of all animals of the control group and the high-concentration group. In addition, all gross lesions were examined in the low- and mid-concentration groups.

Other examinations:

Micronucleus test (see also section 7.6.2)
In the main study, five male animals per group (negative controls, males treated with test material and positive controls) were used for the micronucleus test (from groups 1-4 the animals with the lowest animal identification numbers were used, i.e. those of the first cage of each group).

Statistics:

Body weight data collected after initiation of treatment:
‘AnCova & Dunnett’s Test’ (abbreviation ANCDUN) with ‘Automatic’ data transformation method (abbreviation AUTO). Day 0 body weight data were used as
covariate in the analysis of the post-treatment data unless removed during data preprocessing. The ANCDUN (AUTO) is an automatic decision tree consisting of: (1) Data preprocessing tests, (2) A group test assessing whether or not group means were all equal (one-way analysis of covariance [Ancova], or one-way analysis of variance [Anova] if the covariate is removed), (3) Post-hoc analysis. If the group test showed significant (p<0.05) nonhomogeneity of group means, pairwise comparisons with the control group were conducted by Dunnett’s multiple comparison test.

Pre-treatment body weight, haematology, clinical chemistry and organ weight data: ‘Generalised Anova/Ancova Test’ (abbreviation GEN AN) with ‘Automatic’ data transformation method (abbreviation AUTO). This test is an automatic decision tree consisting of: (1) Data pre-processing tests, (2) A group test assessing whether or not group means were all equal (parametric for untransformed or log-transformed data: one-way analysis of variance [Anova]; non-parametric for rank transformed data: Kruskal-Wallis test), (3) Post-hoc analysis. If the group test showed significant (p<0.05) nonhomogeneity of group means, pairwise comparisons with the control group were conducted by Dunnett’s multiple comparison test (parametric after Anova, non-parametric after Kruskal-Wallis

Food consumption data main study: Dunnett’s multiple comparison test.
Food consumption data range-finding study: no comparative statistics (one cage per sex per group).

Incidences of histopathological changes: Fisher’s exact probability test.

Tests were performed as two-sided tests with results taken as significant where the probability of the results is <0.05 or <0.01.

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:

no effects observed

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

Clinical signs
All animals survived until scheduled sacrifice. There were no treatment-related clinical signs. The few signs observed were incidental findings unrelated to treatment. No abnormalities were seen at the group-wise observations made about halfway each 6-hour exposure period.

Ophthalmoscopic examination
Ophthalmoscopic examination did not reveal any treatment-related abnormalities.

Body weight
Mean body weights of animals exposed to the test material showed no biologically or statistically significant differences from controls.

Food consumption
Food consumption was not affected by the exposure to the test material.

Haematology
Red blood cell and coagulation values and total and differential white blood cell values showed no treatment-related changes.

Clinical chemistry
Compared to controls, animals exposed to the test material showed the following statistically significant differences:
- Higher cholesterol in high-concentration group males
- Lower creatinine in high-concentration group females

Organ weights
The organ weight results showed the following statistically significant differences between animals exposed to the test material and controls:
- Higher relative liver weight at the high-concentration in males (relative difference from control 9%). Mean relative liver weight in females of the high-
concentration group was also higher than in controls (9%) but the difference was not statistically significant.
- Higher relative kidney weight at the high-concentration in males (relative difference from control 11%).
- Higher thyroid weights in males (higher absolute weight at the midconcentration, higher relative weight at the low- and high-concentration). In the
absence of a concentration-related response, these differences in thyroid weights were judged to be chance findings.
- Lower absolute lung weight at the mid-concentration in females. This difference was considered to be a chance finding because there was no concentration-related response.

Pathology
Macroscopic examination
There were no macroscopic findings attributable to the exposure to the test material. The few gross changes observed represented background pathology in rats of this strain and age and occurred only incidentally.
Microscopic examination
Microscopic examination did not reveal treatment-related histopathological changes. The histopathological changes observed in the high-concentration group were considered unremarkable because they represented background findings and occurred in only one or two animals or at about the same incidence in the highconcentration group and the control group.

Dose descriptor:

NOAEC

Effect level:

> 993 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: At this level only a few modest changes (increases in liver and kidney weight and altered plasma levels of cholesterol and creatinine); these changes were considered not to constitute adverse effects

Critical effects observed:

not specified

Table – Clinical chemistry at the end of the exposure period (mean value±SD):

 

Males	Cholesterol (mmol/L)	Creatinine (µmol/L)
0 mg/m3	1.544±0.205	33.6±3.4
100 mg/m3	1.847±0.340	34.5±4.0
300 mg/m3	1.629±0.287	31.4±5.3
1000 mg/m3	1.926±0.312*	29.8±3.8

N=10; * p<0.05

 

Females	Cholesterol (mmol/L)	Creatinine (µmol/L)
0 mg/m3	1.248±0.397	37.7±5.3
100 mg/m3	1.096±0.216	35.6±3.5
300 mg/m3	1.395±0.280	35.8±4.0
1000 mg/m3	1.483±0.424	32.3±3.5*

N=10; * p<0.05

 

Table – Absolute and relative organ weights (mean value±SD):

 

Males	Liver (g)	Liver (g/kg)	Kidneys (g)	Kidneys (g/kg)
0 mg/m3	7.756±0.541	21.50±1.30	1.920±0.070	5.326±0.228
100 mg/m3	7.791±0.618	21.75±1.59	1.992±0.138	5.572±0.501
300 mg/m3	8.240±0.762	21.93±1.14	2.102±0.183	5.592±0.158
1000 mg/m3	8.449±1.052	23.51±1.88*	2.118±0.258	5.891±0.383**

N=10; * p<0.05; ** p<0.01

 

 

Females	Liver (g)	Liver (g/kg)	Kidneys (g)	Kidneys (g/kg)
0 mg/m3	5.064±0.492	24.19±2.15	1.336±0.105	6.378±0.411
100 mg/m3	5.189±0.363	24.97±1.52	1.335±0.069	6.429±0.358
300 mg/m3	5.037±0.470	24.60±2.54	1.284±0.049	6.268±0.276
1000 mg/m3	5.440±0.551	26.45±1.88	1.355±0.066	6.600±0.256

N=10

 

Conclusions:

Exposure to di-tert-butyl peroxide CAS# 110-05-4 resulted in a few modest changes at the highest concentration tested (increases in liver and kidney weight and altered plasma levels of cholesterol and creatinine). No treatment-related changes were observed at the lower concentrations. Since the changes at the high-concentration were considered not to constitute adverse effects, this exposure level (993 mg/m3 actual concentration) was a No-Observed-Adverse-Effect Concentration (NOAEC).

Executive summary:

The toxicity of di-tert-butyl peroxide CAS# 110-05-4 upon repeated exposure by inhalation was studied in a sub-chronic (90-day) study with Wistar Hannover rats. The study included a micronucleus test. The target concentrations for this study (100, 300 and 1000 mg/m3 as low-, mid- and high-concentration, respectively) were selected on the basis of a 14-day range-finding study in which groups of five male and five female Wistar Hannover rats were exposed to target concentrations of 100, 1000, and 10,000 mg/m3 for 6 hours/day, 5 days/week. In this 14-day study treatment-related findings at 10,000 mg/m3 consisted of signs of discomfort during exposure in rats of both sexes (trembling of the tail, restlessness), slightly retarded growth in males (mainly on exposure days), and increased relative weights of the liver in both sexes and of the adrenals and kidneys in males. Trembling of the tail and restlessness during exposure were also observed at the lower exposure levels but to a lesser extent than at the high exposure level. The only other treatment-related change at the lower exposure levels was a slight increase in relative kidney weight in males of the mid-concentration group.

The sub-chronic (main) study included four groups of 10 rats/sex. The animals were exposed nose-only, 6 hours/day, 5 days/week for 13 consecutive weeks (resulting in 65 exposure days in total) to the above target concentrations or to clean air for the control group. Endpoints to assess toxicity included clinical and ophthalmoscopic observations, growth, food consumption, haematology, clinical chemistry and organ weights. In addition, the animals were examined grossly at necropsy, and a large number of organs and tissues were examined microscopically. Five male rats of each group were used to examine possible damage to the chromosomes and/or mitotic apparatus in bone marrow cells collected at scheduled sacrifice.

The target concentrations were accurately achieved as demonstrated by the results of total carbon analysis of the test atmospheres. The overall mean actual concentrations (± standard deviation of the daily mean concentration) were 101 (± 3), 299 (± 3) and 993 (± 10) mg/m3 for the low-, mid- and highconcentration level respectively.

All animals survived until scheduled sacrifice. Clinical and ophthalmoscopic observations, growth and food consumption results, haematology values, most clinical chemistry values, most organ weights, and necropsy and histopathology findings showed no treatment-related changes.

Clinical chemistry values showed slight but statistically significant changes in the plasma levels of cholesterol (increased) and creatinine (decreased) at the high concentration in male and female rats, respectively. These findings were considered to be of no toxicological significance.

The relative weights of the liver and kidneys were slightly (about 10%) but statistically significantly increased in male rats of the high-concentration group. In female rats of this group relative liver weight was increased to about the same extent but the difference from controls was not statistically significant. Though these organ weight changes were related to treatment, they were considered not to represent adverse effects of the test material because of the modest magnitude of the increases and the absence of corroborative histopathological alterations or clinical chemical indicators of organ damage.

Microscopic examination of bone marrow smears of male rats revealed no signs of toxicity to the bone marrow and no evidence of chromosomal damage and/or damage to the mitotic apparatus of bone marrow erythrocytes. There was no reason to assume that the negative bone marrow response was due to lack of systemic exposure because treatment-related systemic effects (including increases in liver and kidney weight) occurred in male rats of the high-concentration group. Positive controls (five male rats treated with the mutagen Mitomycin C) showed the

expected bone marrow response (cytotoxicity and increased number of micronucleated polychromatic erythrocytes).

Under the conditions of this study exposure to di-tert-butyl peroxide CAS# 110-05-4 resulted in a few modest changes at the highest concentration tested (increases in liver and kidney weight and altered plasma levels of cholesterol and creatinine). No treatment-related changes were observed at the lower concentrations. Since the changes at the high-concentration were considered not to constitute adverse effects, this exposure level (993 mg/m3 actual concentration) was a No-Observed- Adverse-Effect Concentration (NOAEC).

The micronucleus test incorporated in this study revealed no chromosomal damage and/or damage to the mitotic apparatus of bone marrow erythrocytes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

993 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

Reliable without restrictions.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 2012 - March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This is a well documented study conducted according to modern standards of protocol, quality assurance, and good laboratory practices. The test material was >99 % pure.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

yes

Remarks:

A micronucleus assay was included

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Young adult, male and female Wistar Hannover outbred rats (RccHan®:WIST) were obtained from a colony maintained under specific pathogenfree
(SPF) conditions at Harlan Laboratories, The Netherlands. On the day of randomization (shortly before the first exposure day), the age of the rats was about 7-8 weeks, and the initial body weight variation did not exceed ± 20% of the mean weight for each sex. Mean body weights at the start of treatment in the range-finding study were 295 and 179 grams for male and female animals, respectively. Mean body weights at the start of treatment in the main study were 264 and 176 grams for male and female animals, respectively.

Upon arrival, the rats were taken to a quarantine room and checked for overt signs of ill health and anomalies. During the quarantine period, serological investigation of the microbiological status was conducted in blood samples taken from three (range-finding study) or five (main study) randomly selected animals. Two days after arrival, the results of serological tests were passed on by telephone and indicated an acceptable microbiological status. Subsequently, the animals were released for experimental use. The duration of the acclimatization period to the conditions in the experimental room prior the first exposure was 16 days (range-finding study) or 10 (males) / 11 (females) days (main study).

From their arrival, the rats were housed under conventional conditions in one room separated by sex. No other test system was housed in the same room during the study. Lighting was artificial (fluorescent tubes) with a sequence of 12 hours light and 12 hours dark. The room was ventilated with about 10 air changes per hour. The temperature and relative humidity in the room were 22 ± 2°C and 45-65%, respectively, with the following exceptions. During the main study, the temperature was just below 20°C (minimum 19.8°C) for about half an hour on 22 January 2013.
The relative humidity exceeded 65% during occasional brief periods following cleaning activities. In addition, during the main study, the relative humidity was lower than 45% on three occasions: on 8 December 2012 for about half an hour due to technical malfunction (minimum 25.9%); on 10 January 2013 for about an hour due to maintenance (minimum 34.3%); and on 13 February 2013 for about five hours due to technical malfunction (minimum 20.2%).

The animals were housed in groups of five, separated by sex, in Makrolon® cages (type IV) with a bedding of wood shavings (Lignocel, Rettenmaier & Söhne GmbH & Co, Rosenberg, Germany) and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment (Lillico, Betchworth, England). Sentinel animals were housed similarly (individually in the range-finding study, in groups of four of the same sex in the main study). During exposure, the animals were kept individually in the exposure units. Immediately after each exposure, the animals were returned to their home cages. After treatment with the mutagen Mitomycin C, the five animals of the positive control group were kept in smaller Makrolon® cages with filter tops (one or two animals per cage; bedding: wood shavings; enrichment:
strips of paper) until sacrifice the next day.

Feed and drinking water were provided ad libitum from the arrival of the animals until the end of the study, except during inhalation exposure and, in the main study, during the fasting period before scheduled sacrifice. The animals received a proprietary cereal-based rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3) from a commercial supplier (SDS Special Diets Services, Whitham, England). Each batch of RM3 diet is analysed by the supplier for nutrients and contaminants. The feed was provided as a powder in stainless steel cans, covered by a perforated stainless steel plate to
prevent spillage. The feed in the feeders was replaced with fresh portions once weekly and filled as needed.
Each cage was supplied with domestic mains tap-water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC). The water was given in polypropylene bottles, which were cleaned weekly and filled as needed. Results of the routine physical, chemical and microbial examination of the drinking water as conducted by the supplier are made available to the test facility. In addition, the supplier periodically (twice per year) analyses water samples taken on the premises of the test facility for a limited number of variables.

Route of administration:

inhalation: vapour

Type of inhalation exposure:

nose/head only

Vehicle:

clean air

Remarks on MMAD:

MMAD / GSD: Not applicable, vapour test atmosphere

Details on inhalation exposure:

The animals were exposed to the test atmosphere in a nose-only inhalation chamber (Institute’s design) consisting of a cylindrical PVC column with a volume of about 75 litres, surrounded by a transparent hood. The test atmosphere was introduced at the bottom of the central column and was exhausted at the top. Each column included three rodent tube levels of 20 ports each. In both the rang-finding study and the main study the animals were placed at the top level. Empty ports were used for measurement of temperature and relative humidity (high concentration groups only). The animals were secured in plastic animal holders (Battelle), positioned radially through the outer hood around the central column. Only the nose of the rats protruded into the interior of the column. The remaining ports were closed. Male and female rats were placed in alternating order. Animals were rotated weekly with respect to the position in the column. From 25 February 2013, larger sized animal holders were used for the male rats of the main study because these animals had reached a size for which the standard holders were too small.

The animal's body does not exactly fit in the animal holder which always results in some leakage from the high to the low pressure side. By securing a positive pressure in the central column and a slightly negative pressure in the outer hood, which encloses the entire animal holder, air leaks from nose to thorax rather than from thorax to nose. This way, dilution of test atmosphere at the animals’ noses was avoided.

The units were illuminated externally by normal laboratory fluorescent tube lighting. The total airflow through the unit was at least 1 litre/min per animal. The air entering the unit was maintained between 22 ± 3°C and the relative humidity between 30% and 70%.

The inhalation equipment was designed to expose rats to a continuous supply of fresh test atmosphere. To generate the test atmospheres, a liquid flow of test material, controlled by a peristaltic pump (Gilson France SA, Villiers le Bel, France), was evaporated in a glass evaporator. The temperature of the evaporator was controlled at 22.5°C (exceptions: 22.0°C on the first six days of the rangefinding study; occasionally 22.7 or 22.8°C in the main study) using a temperature controlled flow of circulating water. The vapour was transported in a stream of humidified compressed air, the flow of which was controlled by a mass flow controller (Bronkhorst, Hi Tec, Ruurlo, The Netherlands).
In the range-finding study, the test atmospheres for group 2 (low-concentration, target 0.1 g/m3) and 3 (mid-concentration, target 1 g/m3) were obtained by diluting the high-concentration (group 4, target 10 g/m3). For this purpose, a mass flow controlled stream of the high-concentration test atmosphere was supplemented with a mass flow controlled stream of humidified compressed air via an eductor (Fox Eductor from Fox Valve Development Corp., Dover, NJ, USA). The generated test atmospheres were directed to the bottom inlets of the exposure units.
In the main study, all three test atmospheres (target concentrations 0.1, 0.3 and 1 g/m3) were obtained by diluting a pre-mixture containing about 3 g/m3 of the test material in humidified compressed air. First, mass flow controlled streams of the pre-mixture were supplemented with a mass flow controlled stream of humidified compressed air via an eductor to obtain the low- and mid-concentration. Next, the remaining stream of the pre-mixture was diluted with a mass flow controlled stream of humidified compressed air to obtain the high-concentration. The generated test
atmospheres were directed to the bottom inlets of the exposure units.
The exposure unit for the control animals (range-finding and main study) was supplied with a measured stream of humidified compressed air only.

The animals were placed in the exposure unit after stabilization of the test atmosphere.

The nominal concentration was determined, for each exposure day, by dividing the total amount of test material used (by weight) by the total volume of air passed through the exposure unit. The generation efficiency was calculated from the actual and the nominal concentration (efficiency = actual concentration as percentage of nominal concentration). In the range-finding study, the nominal concentration was calculated only for the high-concentration because the low- and mid-concentration were obtained by diluting the high-concentration.

The chamber airflow of the test atmospheres was recorded about hourly using a Rotameter or a mass flow controller.

The temperature and the relative humidity of the test atmospheres were measured continuously and recorded every minute using a CAN transmitter with temperature and relative humidity probes (G.Lufft Mess- und Regeltechnik GmbH, 70719 Fellbach, Germany) or were (additionally) measured about hourly by means of a RH/T device (TESTO 635-1, TESTO GmbH & Co, Lenzkirch, Schwarzwald, Germany)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The actual concentration of the test material in the test atmospheres was measured by total carbon analysis (Sick Maihak EuroFID total hydrocarbon analyser; Sick Instruments Benelux, Hedel, the Netherlands). The response of the analyser was recorded on a PC every minute using a CAN transmitter (G. Lufft Mess- und Regeltechnik GmbH, 70719 Felbach, Germany). The responses were converted to concentrations by means of calibration graphs. For each exposure day, the mean concentration was calculated from the values determined every minute. Representative test atmosphere samples were taken continuously from the exposure unit at the animals’ breathing zone and were passed to the total carbon analyser (TCA) through a sample line.
Prior to the first exposure, the output of the flame ionization detector of the TCA was calibrated using PET sample bags with known volumes of clean dry air and known amounts (by weighing) of test material. For each target concentration three calibration concentrations were prepared, at least in duplicate, and analysed (approximately 80, 100 and 120% of the target concentration). The calibrations were checked weekly by measuring the concentration in a sample bag with a theoretical concentration close to the target concentration. If the measured concentration deviated more than 5% from the theoretical concentration and this was confirmed with a second sample bag, the TCA was recalibrated.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

5 days/week, 6 h/day

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000 mg/m3
Basis:
other: target concentrations

Remarks:

Doses / Concentrations:
0, 101, 299, and 993 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Concentrations for the main study were based on the results of the RF study.
The animals (5/sex) were exposed on 5 days per week, 6 hours per day, over a 2-week study period (total of 10 exposure days), for the last time on the day before scheduled sacrifice.
Shortly before initiation of exposure (range finding study: day -1; main study days -4 and -5 for males and females, respectively), the animals were allocated to the various groups by computer randomization proportionally to body weight (males and females separately). The surplus animals were kept in reserve to serve as sentinels (range-finding study: one/sex; main study: four/sex). These animals were discarded at the end of the inlife phase of the study.

Positive control:

For the in vivo MN test (see also section 7.6.2) the animals of the positive control group received a single intraperitoneal injection with Mitomycin C on the day prior to scheduled sacrifice.

Observations and examinations performed and frequency:

Clinical signs
On exposure days, each animal was observed daily in the morning, prior to exposure, by cage-side observations and, if necessary, handled to detect signs of toxicity. All animals were thoroughly checked again after exposure. During exposure in the main study, a group-wise observation was made about halfway the 6-hour exposure period. During exposure in the range-finding study, observations were made more frequently on account of abnormalities noted in the groups exposed to the test material (tail trembling and restlessness). During weekends and on public holidays only one check per day was carried out. All abnormalities, signs of ill health, and reactions to treatment were recorded.

Ophthalmoscopic examination
In the main study, ophthalmoscopic observations were made prior to the start of the exposure in all rats (on day -6) and in the last week of the exposure period in all rats of the control group and the high-concentration group (on day 92). Eye examination was carried out using an ophthalmoscope after induction of mydriasis by a solution of atropine sulphate.

Body weight
Range-finding study
The body weight of each animal was recorded once before the start of exposure (these pre-test weights served as a basis for animal allocation, once prior to exposure on the first exposure day (day 0) and twice weekly thereafter, for the last time on the day of scheduled sacrifice.
Main study
The body weight of each animal was recorded once before the start of exposure. These pre-test weights served as a basis for animal allocation. Animals were weighed prior to exposure on the first exposure day (day 0), twice weekly for the first four weeks of exposure (Mondays and Fridays), then once weekly from the fifth week onward. At the end of the in-life phase, animals were weighed the day before overnight fasting prior to scheduled sacrifice. They were weighed again on the day of sacrifice; this was the terminal body weight used for calculation of organ weights relative to body weight.

Food consumption
Food consumption was measured per cage by weighing the feeders. In the rangefinding study, food consumption was measured over two 7-day periods, starting on day 0. In the main study, consumption was measured from day 0, over successive 7-day periods. The results were expressed in g per animal per day.

Haematology
In the main study, haematology was conducted at the end of the treatment period on all animals. Blood samples were taken from the abdominal aorta of overnight fasted rats (water was freely available) whilst under pentobarbital anaesthesia at sacrifice. Potassium-EDTA was used as anticoagulant. In each sample the following determinations were carried out: red blood cells (RBC), haemoglobin (Hb), packed cell volume (PCV), reticulocytes, total white blood cells (WBC), differential white blood cells, prothrombin time (PT), thrombocytes.
The following parameters were calculated: mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC).

Clinical chemistry
In the main study, clinical chemistry was conducted at the end of the treatment period on all animals, after overnight fasting, at the same time blood samples for haematology were collected. Blood was collected from the abdominal aorta in heparinized plastic tubes. Plasma was prepared by centrifugation. The following measurements were made in the plasma: alkaline phosphatase activity (ALP), cholesterol (total), aspartate aminotransferase activity (ASAT), phospholipids, alanine aminotransferase activity (ALAT), triglycerides, gamma glutamyl transferase activity (GGT), creatinine, bilirubin (total), urea, total protein, inorganic phosphate (PO4), albumin, calcium (Ca), ratio albumin to globulin (calculated), chloride (Cl), glucose, potassium (K), sodium (Na).

Sacrifice and pathology:

Pathology
Range-finding study
Macroscopic examination
At the end of the exposure period, the animals were sacrificed in such a sequence that the average time of killing was approximately the same for each group. The animals were sacrificed by exsanguination from the abdominal aorta under pentobarbital anaesthesia and then examined grossly for pathological changes.

Organ weights
At scheduled sacrifice, the following organs of all animals were weighed (paired organs together) as soon as possible after dissection to avoid drying and the relative organ weights (g/kg body weight) were calculated from the absolute organ weights and the terminal body weight: adrenals, heart, kidneys, liver, lung with trachea and larynx, spleen, testes (after weighing, the lung was infused with the fixative).

Tissue preservation
The organs listed above and the complete respiratory tract of all animals were preserved with a 10% solution of Formalin in a neutral aqueous phosphate buffer (final formaldehyde concentration 4 per cent).

Histopathological examination
The main lung lobe (one level), the larynx (three levels, including the base of the epiglottis) and the nasal tissues (levels 3 and 5, as described by Woutersen et al., 1994) of all male animals were examined microscopically. Tissue for microscopy was embedded in paraffin wax, sectioned at 5 µm and stained with haematoxylin and eosin.

Main study
Gross necropsy
On study day 93 (13 and 14 March 2013 for males and females, respectively), animals were fasted overnight but allowed free access to water. The next day (study day 94; 14 and 15 March 2013 for males and females, respectively), the animals were killed in such a sequence that the average time of killing was approximately the same for each group. The animals were anaesthetized by intraperitoneal injection of sodium pentobarbital then killed by exsanguination from the abdominal aorta and examined grossly for pathological changes.

Organ weights
At scheduled necropsy, the following organs were weighed (paired organs together) as soon as possible after dissection to avoid drying and the relative organ weights (g/kg body weight) were calculated from the absolute organ weights and the terminal body weight: adrenals, lung with trachea and larynx, brain, ovaries, epididymides, spleen, heart, testes, kidneys, thymus, liver, thyroid, uterus (after weighing, the lung was infused with the fixative).

Tissue preservation
Samples of the tissues and organs listed below of all animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde (10% solution of formalin). The carcass containing any remaining tissues was retained in formalin until completion of the histopathological examination and then discarded. The following were preserved, all gross lesions, ovaries, adrenals, pancreas, aorta, parathyroids, axillary lymph nodes, parotid salivary glands, brain, pharynx, caecum, pituitary, colon, prostate, duodenum, rectum, epididymides, seminal vesicles / coagulating glands, exorbital lachrymal glands, skeletal muscle (thigh), eyes (with optic nerve), skin (flank), femur with joint, spinal cord, NALT (nose associatedlymphoid tissue, spleen, sternum with bone marrow, heart, stomach, ileum, sublingual salivary glands, jejunum, submaxillary salivary glands, kidneys, testes, larynx, thymus, liver, thyroid, lung, tongue, mammary gland (female), trachea, mandibular (cervical) lymph nodes, tracheobronchial (mediastinal) lymph nodes, nasopharyngeal tissues (with teeth), ureter, nerve-peripheral (sciatic), urethra, oesophagus, urinary bladder, olfactory bulb, uterus (with cervix).
- For the micronucleus test, bone marrow cells of one of the femurs (left femur) were collected from the first five male rats of each group
- Brain: Three levels were examined microscopically (brain stem, cerebrum, cerebellum).
- Larynx: Three levels (one including the base of the epiglottis) were examined microscopically.
- Lung: Each lung lobe was examined microscopically at one level, including main bronchi.
- Nasopharyngeal tissues: Six levels (Woutersen et al., 1994) were examined microscopically (one including the nasopharyngeal duct and the draining lymphatic tissue [NALT]).
- Spinal cord: Retained in vertebral column, at least three levels were examined microscopically (cervical, mid-thoracic and lumbar).
- Stomach: Non-glandular (‘forestomach’) and glandular (fundus, pylorus) parts were examined microscopically.
- Trachea: Three transverse sections and three longitudinal sections (at least one through the carina of the bifurcation of the bronchi) were examined microscopically.

Histopathological examination
The tissues for microscopic examination were embedded in paraffin wax, sectioned at 5 µm and stained with haematoxylin and eosin. Histopathological examination (by light microscopy) was performed on all tissues and organs listed above of all animals of the control group and the high-concentration group. In addition, all gross lesions were examined in the low- and mid-concentration groups.

Other examinations:

Micronucleus test (see also section 7.6.2)
In the main study, five male animals per group (negative controls, males treated with test material and positive controls) were used for the micronucleus test (from groups 1-4 the animals with the lowest animal identification numbers were used, i.e. those of the first cage of each group).

Statistics:

Body weight data collected after initiation of treatment:
‘AnCova & Dunnett’s Test’ (abbreviation ANCDUN) with ‘Automatic’ data transformation method (abbreviation AUTO). Day 0 body weight data were used as
covariate in the analysis of the post-treatment data unless removed during data preprocessing. The ANCDUN (AUTO) is an automatic decision tree consisting of: (1) Data preprocessing tests, (2) A group test assessing whether or not group means were all equal (one-way analysis of covariance [Ancova], or one-way analysis of variance [Anova] if the covariate is removed), (3) Post-hoc analysis. If the group test showed significant (p<0.05) nonhomogeneity of group means, pairwise comparisons with the control group were conducted by Dunnett’s multiple comparison test.

Pre-treatment body weight, haematology, clinical chemistry and organ weight data: ‘Generalised Anova/Ancova Test’ (abbreviation GEN AN) with ‘Automatic’ data transformation method (abbreviation AUTO). This test is an automatic decision tree consisting of: (1) Data pre-processing tests, (2) A group test assessing whether or not group means were all equal (parametric for untransformed or log-transformed data: one-way analysis of variance [Anova]; non-parametric for rank transformed data: Kruskal-Wallis test), (3) Post-hoc analysis. If the group test showed significant (p<0.05) nonhomogeneity of group means, pairwise comparisons with the control group were conducted by Dunnett’s multiple comparison test (parametric after Anova, non-parametric after Kruskal-Wallis

Food consumption data main study: Dunnett’s multiple comparison test.
Food consumption data range-finding study: no comparative statistics (one cage per sex per group).

Incidences of histopathological changes: Fisher’s exact probability test.

Tests were performed as two-sided tests with results taken as significant where the probability of the results is <0.05 or <0.01.

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:

no effects observed

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

Clinical signs
All animals survived until scheduled sacrifice. There were no treatment-related clinical signs. The few signs observed were incidental findings unrelated to treatment. No abnormalities were seen at the group-wise observations made about halfway each 6-hour exposure period.

Ophthalmoscopic examination
Ophthalmoscopic examination did not reveal any treatment-related abnormalities.

Body weight
Mean body weights of animals exposed to the test material showed no biologically or statistically significant differences from controls.

Food consumption
Food consumption was not affected by the exposure to the test material.

Haematology
Red blood cell and coagulation values and total and differential white blood cell values showed no treatment-related changes.

Clinical chemistry
Compared to controls, animals exposed to the test material showed the following statistically significant differences:
- Higher cholesterol in high-concentration group males
- Lower creatinine in high-concentration group females

Organ weights
The organ weight results showed the following statistically significant differences between animals exposed to the test material and controls:
- Higher relative liver weight at the high-concentration in males (relative difference from control 9%). Mean relative liver weight in females of the high-
concentration group was also higher than in controls (9%) but the difference was not statistically significant.
- Higher relative kidney weight at the high-concentration in males (relative difference from control 11%).
- Higher thyroid weights in males (higher absolute weight at the midconcentration, higher relative weight at the low- and high-concentration). In the
absence of a concentration-related response, these differences in thyroid weights were judged to be chance findings.
- Lower absolute lung weight at the mid-concentration in females. This difference was considered to be a chance finding because there was no concentration-related response.

Pathology
Macroscopic examination
There were no macroscopic findings attributable to the exposure to the test material. The few gross changes observed represented background pathology in rats of this strain and age and occurred only incidentally.
Microscopic examination
Microscopic examination did not reveal treatment-related histopathological changes. The histopathological changes observed in the high-concentration group were considered unremarkable because they represented background findings and occurred in only one or two animals or at about the same incidence in the highconcentration group and the control group.

Dose descriptor:

NOAEC

Effect level:

> 993 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: At this level only a few modest changes (increases in liver and kidney weight and altered plasma levels of cholesterol and creatinine); these changes were considered not to constitute adverse effects

Critical effects observed:

not specified

Table – Clinical chemistry at the end of the exposure period (mean value±SD):

 

Males	Cholesterol (mmol/L)	Creatinine (µmol/L)
0 mg/m3	1.544±0.205	33.6±3.4
100 mg/m3	1.847±0.340	34.5±4.0
300 mg/m3	1.629±0.287	31.4±5.3
1000 mg/m3	1.926±0.312*	29.8±3.8

N=10; * p<0.05

 

Females	Cholesterol (mmol/L)	Creatinine (µmol/L)
0 mg/m3	1.248±0.397	37.7±5.3
100 mg/m3	1.096±0.216	35.6±3.5
300 mg/m3	1.395±0.280	35.8±4.0
1000 mg/m3	1.483±0.424	32.3±3.5*

N=10; * p<0.05

 

Table – Absolute and relative organ weights (mean value±SD):

 

Males	Liver (g)	Liver (g/kg)	Kidneys (g)	Kidneys (g/kg)
0 mg/m3	7.756±0.541	21.50±1.30	1.920±0.070	5.326±0.228
100 mg/m3	7.791±0.618	21.75±1.59	1.992±0.138	5.572±0.501
300 mg/m3	8.240±0.762	21.93±1.14	2.102±0.183	5.592±0.158
1000 mg/m3	8.449±1.052	23.51±1.88*	2.118±0.258	5.891±0.383**

N=10; * p<0.05; ** p<0.01

 

 

Females	Liver (g)	Liver (g/kg)	Kidneys (g)	Kidneys (g/kg)
0 mg/m3	5.064±0.492	24.19±2.15	1.336±0.105	6.378±0.411
100 mg/m3	5.189±0.363	24.97±1.52	1.335±0.069	6.429±0.358
300 mg/m3	5.037±0.470	24.60±2.54	1.284±0.049	6.268±0.276
1000 mg/m3	5.440±0.551	26.45±1.88	1.355±0.066	6.600±0.256

N=10

 

Conclusions:

Exposure to di-tert-butyl peroxide CAS# 110-05-4 resulted in a few modest changes at the highest concentration tested (increases in liver and kidney weight and altered plasma levels of cholesterol and creatinine). No treatment-related changes were observed at the lower concentrations. Since the changes at the high-concentration were considered not to constitute adverse effects, this exposure level (993 mg/m3 actual concentration) was a No-Observed-Adverse-Effect Concentration (NOAEC).

Executive summary:

The toxicity of di-tert-butyl peroxide CAS# 110-05-4 upon repeated exposure by inhalation was studied in a sub-chronic (90-day) study with Wistar Hannover rats. The study included a micronucleus test. The target concentrations for this study (100, 300 and 1000 mg/m3 as low-, mid- and high-concentration, respectively) were selected on the basis of a 14-day range-finding study in which groups of five male and five female Wistar Hannover rats were exposed to target concentrations of 100, 1000, and 10,000 mg/m3 for 6 hours/day, 5 days/week. In this 14-day study treatment-related findings at 10,000 mg/m3 consisted of signs of discomfort during exposure in rats of both sexes (trembling of the tail, restlessness), slightly retarded growth in males (mainly on exposure days), and increased relative weights of the liver in both sexes and of the adrenals and kidneys in males. Trembling of the tail and restlessness during exposure were also observed at the lower exposure levels but to a lesser extent than at the high exposure level. The only other treatment-related change at the lower exposure levels was a slight increase in relative kidney weight in males of the mid-concentration group.

The sub-chronic (main) study included four groups of 10 rats/sex. The animals were exposed nose-only, 6 hours/day, 5 days/week for 13 consecutive weeks (resulting in 65 exposure days in total) to the above target concentrations or to clean air for the control group. Endpoints to assess toxicity included clinical and ophthalmoscopic observations, growth, food consumption, haematology, clinical chemistry and organ weights. In addition, the animals were examined grossly at necropsy, and a large number of organs and tissues were examined microscopically. Five male rats of each group were used to examine possible damage to the chromosomes and/or mitotic apparatus in bone marrow cells collected at scheduled sacrifice.

The target concentrations were accurately achieved as demonstrated by the results of total carbon analysis of the test atmospheres. The overall mean actual concentrations (± standard deviation of the daily mean concentration) were 101 (± 3), 299 (± 3) and 993 (± 10) mg/m3 for the low-, mid- and highconcentration level respectively.

All animals survived until scheduled sacrifice. Clinical and ophthalmoscopic observations, growth and food consumption results, haematology values, most clinical chemistry values, most organ weights, and necropsy and histopathology findings showed no treatment-related changes.

Clinical chemistry values showed slight but statistically significant changes in the plasma levels of cholesterol (increased) and creatinine (decreased) at the high concentration in male and female rats, respectively. These findings were considered to be of no toxicological significance.

The relative weights of the liver and kidneys were slightly (about 10%) but statistically significantly increased in male rats of the high-concentration group. In female rats of this group relative liver weight was increased to about the same extent but the difference from controls was not statistically significant. Though these organ weight changes were related to treatment, they were considered not to represent adverse effects of the test material because of the modest magnitude of the increases and the absence of corroborative histopathological alterations or clinical chemical indicators of organ damage.

Microscopic examination of bone marrow smears of male rats revealed no signs of toxicity to the bone marrow and no evidence of chromosomal damage and/or damage to the mitotic apparatus of bone marrow erythrocytes. There was no reason to assume that the negative bone marrow response was due to lack of systemic exposure because treatment-related systemic effects (including increases in liver and kidney weight) occurred in male rats of the high-concentration group. Positive controls (five male rats treated with the mutagen Mitomycin C) showed the

expected bone marrow response (cytotoxicity and increased number of micronucleated polychromatic erythrocytes).

Under the conditions of this study exposure to di-tert-butyl peroxide CAS# 110-05-4 resulted in a few modest changes at the highest concentration tested (increases in liver and kidney weight and altered plasma levels of cholesterol and creatinine). No treatment-related changes were observed at the lower concentrations. Since the changes at the high-concentration were considered not to constitute adverse effects, this exposure level (993 mg/m3 actual concentration) was a No-Observed- Adverse-Effect Concentration (NOAEC).

The micronucleus test incorporated in this study revealed no chromosomal damage and/or damage to the mitotic apparatus of bone marrow erythrocytes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

993 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

Reliable without restrictions.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to EU Regulation (EC) N0. 1272/2008 (CLP), di-tert amyl peroxide is not classified for repeated dose toxicity.