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Administrative data

Description of key information

Two repeated dose toxicity studies were available in the rat, an oral OECD 422 study and a 90-day inhalation study (OECD 413). For the 90-day study, the inhalation route was chosen because of the relatively high vapour pressure and as such likely exposure via inhalation.  

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9 months (approx)
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 99.2 % pure and was analytically demonstrated to be stable in the test vehicle (corn oil).
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
study plan "amendments": "Male animals aged 8 weeks minimum instead of 10 weeks. Lymph nodes for histopath specified as “one lymph node covering the route of administration and another one distant from the route of admin. to cover systemic effect."
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: HanRcc:WIST (SPF)
Sex:
male/female
Details on test animals and environmental conditions:
Species Rat: HanRcc:WIST (SPF)
Rationale: Specified by the international guidelines as the recommended test system
Source: RCC Ltd
Laboratory Animal Services
Wolferstrasse 4
4414 Fullinsdorf / Switzerland

Number of animals: 40 males, 10 per group; 40 females, 10 per group
Age at delivery: 8 weeks (male animals); 10 weeks (female animals)
Body weights: Males: 243 - 291 grams (at first treatment)
Females: 181 - 219 grams (at first treatment)
Acclimatization: Seven days under test conditions with an evaluation of the health status

Identification
Parental animals: Individual animal number tattooed on the pinnae and individual cage card.
F1 pups: Individual intra-litter pup number tattooed with Indian ink on day 1 post partum.

This study was performed under standard laboratory conditions: the animal room was air-conditioned with 10 - 15 air changes per hour; the environment was monitored continuously with recordings of temperature (range 22+/- 3°C) and relative humidity (range 30 - 70%), 12 hours artificial fluorescent light / 12 hours dark with background music played at a centrally defined low volume for at least 8 hours during the light period.

Accommodation: Animals were housed in Makrolon cages (type-3) with wire mesh tops and standard granulated softwood bedding (Lignocel, Schill AG, 4132 Muttenz/Switzerland). During the pre-pairing period, males and females were housed individually. Cages of males were interspersed among those holding females to promote the development of regular estrus cycles. During the pairing period, rats were housed one male / one female in Makrolon pairing cages. After mating, the males and the females were housed individually again. During lactation period (until day 4 of lactation), dams were housed together with their litters. Throughout the study, each cage was identified by a colored label according to the group and recording the study schedule number, animal number(s) and details of treatment.

Diet: Pelleted standard Kliba 3433 rat/mouse maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst/ Switzerland) was available ad libitum (Batch No. 67/06). Results of analyses for contaminants are presented in the final report.

Water Tap water from Fullinsdorf in bottles was available ad libitum. Results of the bacteriological, chemical and contaminant analyses scheduled to be conducted at least once yearly by RCC (contaminant analyses only) and by the Official Chemist of the Kanton Basel-Landschaft (bacteriological and chemical analyses) are part of the final report.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
Vehicle
Identity – Corn oil
CAS-No. 8001-30-7
EG No. 2322812
Supplier – Carl Roth GmbH & Co., 76185 Karlsruhe / Germany
Batch number – 38679499
Expiry date – 14-NOV-2016
Storage conditions -- Room temperature (20°C +/- 5°C)

Dose formulations were prepared in terms of material as supplied by the Sponsor.
Frequency of dose formulation -- Weekly, based on the results of RCC Study No. A79874
Storage of dose formulations -- Room temperature (20°C +/-5°C)
The test item was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle were added (w/v). Using an appropriate homogenizer a homogenous mixture was prepared. Having obtained a homogeneous mixture, the vehicle was added until the required final volume was achieved. Separate formulations were prepared for each concentration. During the daily administration period, homogeneity of the test item in the vehicle was maintained using a magnetic stirrer.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of Dose Formulations
Samples for determination of concentration, homogeneity and stability (4 hours and 7 days) of the dose formulations were taken during the first week of the administration period. Additionally, samples for determination of concentration and homogeneity were taken during the last week of
the administration period

On each occasion three samples of approximately 2 g were taken from the top, middle and bottom of each formulation and transferred into flat bottomed flasks. For determination of stability, samples were taken before dosing from the middle and stored at room temperature for 4 hours and for 7 days in the dark. The samples were stored at approximately 20°C in the dark pending analysis. Samples were shipped to Itingen / Switzerland. Analysis was performed using a method provided by the Sponsor and adapted by the test facility.

After analysis, the analytical results were communicated to the Study Director. Upon receipt and evaluation of these results the Study Director decided about discarding the samples. The report of the Analytical Phase is presented as part of the final report.
Duration of treatment / exposure:
Administration--After acclimatization (7 days), animals of both sexes received the test item for 14 days prior to pairing and during the pairing period. Daily dosing of the females was continued throughout pregnancy and up to day 4 of lactation. Dosing of males was continued until the first dams had reached day 4 post partum (42 days of administration).

The test item was administered orally, by gavage, once daily. All animals received a dose volume of 4 mL/kg body weight with a daily adjustment of the individual volume to the actual body weight. Control animals were dosed with the vehicle alone.

Mating—After the animals had received the test item for 14 days, the pairing period was initiated while dosing was continued. During the pairing period females were housed with males (one male : one female) in special automatic mating cages, i.e. with synchronized timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the different females. Females were removed and housed individually if: a) a copulation plug was observed, and / or b) the daily vaginal smear was sperm-positive. This day was designated day 0 post coitum.
Frequency of treatment:
Administration--After acclimatization (7 days), animals of both sexes received the test item for 14 days prior to pairing and during the pairing period. Daily dosing of the females was continued throughout pregnancy and up to day 4 of lactation. Dosing of males was continued until the first dams had reached day 4 post partum (42 days of administration).

The test item was administered orally, by gavage, once daily. All animals received a dose volume of 4 mL/kg body weight with a daily adjustment of the individual volume to the actual body weight. Control animals were dosed with the vehicle alone.

Mating—After the animals had received the test item for 14 days, the pairing period was initiated while dosing was continued. During the pairing period females were housed with males (one male : one female) in special automatic mating cages, i.e. with synchronized timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the different females. Females were removed and housed individually if: a) a copulation plug was observed, and / or b) the daily vaginal smear was sperm-positive. This day was designated day 0 post coitum.
Remarks:
Doses / Concentrations:
0; 100; 300; 1000 mg/kg bw
Basis:
other: administered gavage dose
No. of animals per sex per dose:
10 males; 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
Dose levels were selected in agreement with the Sponsor, based on the results of a preliminarydose range-finding study, where 1000 mg/kg/day were used as highest dose level.
Positive control:
None
Observations and examinations performed and frequency:
STUDY SCHEDULE
MALES FEMALES
Acclimatization 7 days (minimum) 7 days (minimum)
Treatment beginning Day 1 of pre-pairing Day 1 of pre-pairing
Pre-pairing 14 days 14 days
Pairing until mating (maximum 14 days) until mating (maximum 14 days)
Gestation N/A about 21 days
Parturition N/A expected : on day 21 or 22 post coitum
Lactation N/A until day 4 post partum
Treatment ending One day prior to the actual day of necropsy on day 4 post partum
(at least 28 days of treatment)
Blood collection On the day of necropsy On the day before necropsy or on the day of necropsy
Termination After the first dams had reached pups on day 4 post partum
day 4 post partum dams on day 5 post partum


++++++++++++++++++++++++++++++++++++++

Mortality rate -- All animals were checked at least twice daily for any mortalities. All rats found dead were subjected to a detailed macroscopic examination to establish, if possible, the cause of death.

Signs and/or symptoms -- All animals were observed at least twice daily for signs of reaction to treatment and/or symptoms of ill health. Additionally, the females were observed for signs of difficult or prolonged parturition.

Detailed clinical observations -- Once prior to the first test item administration and weekly thereafter, detailed clinical observations were performed outside the home cage. Animals were observed for thefollowing: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behaviour were also recorded.

Functional observation battery -- At one time during the study (males: shortly before scheduled sacrifice; females: on day 3 or 4 post partum) relevant parameters were performed for five P generation males and five P generation females randomly selected from each group. This FOB assessment was conducted following the daily dose administration. Animals were observed for the following:
a) Cage side observations: unusual body movements (e.g. tremors, convulsions), abnormal behaviour (e.g. circling, stereotypy) and posture as well asresistance to removal.
b) Hand-held observations: palpebral closure, pinna reflex, lacrimation, pupil size, pupil reactivity, salivation, muscle tone, extensor thrust response, righting reflex, and reactivity to handling.
c) Open field observations: level of ambulatory activity including rearing (one minute evaluation), responsiveness to sharp noise, paw pinch, gait evaluation, quantity of urine and fecal pellets voided.
d) Categorical observations (can be made any time during the FOB): hair coat, behaviour, respiration, muscle movements, eyes, hearing ability (Preyer’s reflex), urine or faeces, soiling, general abnormalities, posture.
e) Measurements / Counts: hind limb / fore limb grip strength, landing foot splay, rectal temperature.

A description of all test parameters is listed in the final report.
Any abnormal findings were recorded and, where appropriate, graded in severity.
Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with Activity Monitor AMS-0151 (FMI, Germany). Activity of the animals (basing on beam count) was recorded for 6- minute intervals over a period of 30 minutes. These data and the total activity over 30 minutes were reported.

Body weight--The animals were weighed daily during the entire study.

Food consumption
Males: Food consumption was recorded weekly during the prepairing and post-pairing period.
Females: Food consumption was recorded for the following periods: days 1-8 and 8-14 of the pre-pairing period; days 0-7, 7- 14 and 14-21 post coitum and days 1-4 post partum.
Food consumption was not recorded during the pairing period (mixed values of males and females).

Lactation and litter data -- Day 0 of lactation was the day on which a female had delivered all her pups. The litters were examined for litter size, live birth, stillbirth and any gross anomalies. The sex ratio of the pups was recorded. The dams were caged together with their litters until day 4 of lactation. Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum. The dams and pups were observed daily for survival and behavioural abnormalities in nesting and nursing.

Sacrifice and pathology:
PATHOLOGY
Males were sacrificed after the first dams had reached postmortem examination day 4 post partum (42 days of administration). Females were sacrificed on day 5 post partum. Pups were sacrificed on day 4 post partum. Males and females were killed by exsanguination following an intraperitoneal injection of sodium pentobarbital. Pups were killed by an intraperitoneal injection of sodium pentobarbital (EuthaR 77). The animals were examined macroscopically for any structural abnormalities or pathological changes, with special attention paid to the organs of the reproductive system. The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites. Dead pups (except if excessively cannibalized) and pups killed at day 4 of lactation were examined macroscopically. If birth did not occur on the expected date (day 21 post coitum), the female was treated until day 24 post coitum, sacrificed on day 25 post coitum and examined as described.

ORGAN WEIGHTS
The testes* and epididymides* of all parental males were weighed. In addition for five adult males and females, randomly selected from each group, the following organs were trimmed from any adherent tissue, as appropriate, and their wet weight taken:• liver • spleeen • adrenals* • brain • thymus • heart • kidneys* [ * weighed as pairs]


TISSUE PRESERVATION
Of all parental males the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution: • prostate • testes (in Bouin's fixative) • seminal vesicles with coagulation gland • epididymides (in Bouin's fixative)

Of all parental females the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution: • ovaries

In addition, of the five males and females per group selected for organ weights, the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution: • gross lesions • heart • brain • thymus • spinal cord • thyroid • small and large intestines (incl. Peyer's patches) • trachea and lungs (preserved by inflation with fixative and then immersion) • stomach • uterus (with vagina) • liver • urinary bladder • kidneys • lymph nodes (mandibular, mesenteric) • adrenals • peripheral nerve • spleen • bone marrow


HISTOPATHOLOGY
Full histopathology was carried out on the preserved organs and tissues of the animals in the vehicle control and high dose group (with special emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure). Examinations were extended to the animals of the other dosage groups, if treatment-related changes were seen in the highest dose group. All gross lesions were examined. Histological examination of ovaries was carried out on any female that did not give birth. Microscopic examination of the reproductive organs of all infertile males was made, ifnecessary.
Other examinations:
CLINICAL LABORATORY INVESTIGATIONS
Blood samples were obtained on the day before or on the day of scheduled necropsy from all P generation males after they had been fasted overnight. Blood samples of P generation females were obtained on day 5 post partum after the females had been fasted overnight. Blood samples were collected sublingually with the animal under light isoflurane anaesthesia. Blood samples were collected early in the working day to reduce biological variation caused by circadian rhythms.

HAEMATOLOGY
The following haematology parameters were determined:
Erythrocyte count Reticulocyte count
Haemoglobin Reticulocyte maturity index
Haematocrit Total leukocyte count
Mean corpuscular volume Differential leukocyte count
Red cell volume distribution width Platelet count
Mean corpuscular haemoglobin
Mean corpuscular haemoglobin concentration Thromboplastin time
Haemoglobin concentration distribution width Activated partial thromboplastin time

CLINICAL BIOCHEMISTRY
The following parameters were determined:
Glucose Sodium
Urea Potassium
Creatinine Chloride
Bilirubin, total Calcium
Cholesterol, total Phosphorus inorganic
Aspartate aminotransferase Protein, total
Alanine aminotransferase Albumin
Bile acids Globulin
Alkaline phosphatase Albumin/Globulin ratio
Gamma-glutamyl-transferase
Statistics:
Means and standard deviations of various data were calculated. If the variables could be assumed to follow a normal distribution, the Dunnett t-test, based on a pooled variance estimate, was used for inter-group comparisons (i.e. single treatment groups against the control group). The Steel test (rank test) was applied when the data could not be assumed to follow a normal distribution. Fisher's Exact test for 2x2 tables was applied if the variables could be dichotomized without loss of information.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food efficiency:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
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:
no effects observed
Details on results:
PARENTAL ANIMALS
MORTALITY
All male and female animals survived until scheduled necropsy.

GENERAL CAGESIDE OBSERVATIONS (DAILY)
In group 4, all male and female animals were noted with moving their head through the bedding material starting on days 11 and 12, respectively, until the last administration. About one to 5 male and 2 female animals were noted with ruffled fur starting on day 12 of the study. It continued until day 19 of the after pairing period for one male (no. 31). In two female animals (nos. 79 and 80), ruffled fur disappeared on days 6 and 7, respectively, of the gestation period. No treatment related clinical signs were noted for male and female animals in groups 2 and 3. Male no. 14 in group 2 was noted with a black tip of tail starting on day 8 until day 13, with body weight loss on day 8. On day 14, the tip of tail fell off. No survival and behavioural abnormalities were noted in the female animals and pups during nesting and nursing.

DETAILED CLINICAL OBSERVATIONS (WEEKLY)
Four male animals (nos. 31, 33, 36, and 39) and two female animals (nos. 79 and 80) in group 4 were noted with ruffled fur on day 26 of study, respectively (after pairing period / gestation period). One male animal (no. 31) was again noted with ruffled fur on day 40 of study (after pairing period). No other findings were noted during the detailed clinical observations in any other animal in any group.

FOOD CONSUMPTION - MALES
Pre-pairing and After Pairing Period
In group 4, the mean food consumption was statistically significantly decreased during the prepairing period. During after pairing period mean food consumption was not influenced. Mean food consumption in groups 2 and 3 was considered to be not influenced during pre- and after pairing period.

BODY WEIGHTS - MALES
Pre-pairing, Pairing and After Pairing Period
In group 4, mean body weight started to be statistically significantly decreased on day 11 of the pre-pairing period and continued to be statistically significantly decreased until the end of study in the after pairing period. Mean body weight gain was statistically significantly decreased between day 6 and day 14 of the pre-pairing period and on days 5, 6, and 11 of the after pairing period. In groups 2 and 3, mean body weight was not influenced by treatment with the test item during the whole study. Mean body weight gain was statistically significantly decreased in group 3 between day 3 and 9, andin group 2 on day 3, respectively, during the after pairing period.

FOOD CONSUMPTION - FEMALES
Pre-pairing, Gestation and Lactation Period
Mean food consumption in groups 2, 3, and 4 was not influenced during pre-pairing, and gestation period. During lactation period, mean food consumption was statistically not significantly increased in groups 3 and 4.

BODY WEIGHTS - FEMALES
Pre-pairing, Pairing, Gestation and Lactation Period
In groups 2, 3, and 4, mean body weight and mean body weight gain was considered to be not influenced by treatment with the test item during the whole study.

FUNCTIONAL OBSERVATIONAL BATTERY
None of the parameters under investigation was considered to be affected by exposure to the test item during the functional observational battery. One male animal was noted with reduced activity in group 4. Three males in group 1, one male in group 3 and two males in group 4 were noted with a decreased rearing (n<3). A few or small puddles of urine were noted for 3 males each in groups 1 and 2. Spontaneous vocalization, when removed was noted in two females in group 1 and one female in group 2. One female animal was noted with reduced activity in group 1. One female in group 1 and one female in group 2 were noted with a decreased rearing (n<3). A few or small puddles of urine were noted for 2 females in groups 1 and one female in group 2.

Grip Strength, Landing Foot Splay and Body Temperature
In groups 2, 3, and 4, mean values for grip strength (fore- and hind paws), landing foot splay, and body temperature (rectal) were considered not influenced by treatment with the test item.

Locomotor Activity In groups 2, 3, and 4, locomotor activity, as assessed in terms of low beam counts in an activity monitor, was considered to be notinfluenced by treatment with the test item.

REPRODUCTION DATA
FERTILITY AND MATING PERFORMANCE
All mated females except one (no. 79 in group 4) were pregnant. In groups 2, 3, and 4, mating performance was considered to be not influenced by treatment with the test item. In groups 1, 2, 3, and 4, the median pre-coital time was 3, 3, 2, and 3 days, and the mean precoital time was 2.4, 2.7, 2.3, and 3.0 days, respectively. The fertility index was 100.0, 100.0, 100.0, and 90.0% in groups 1, 2, 3, and 4, respectively.
DURATION OF GESTATION
In groups 2, 3, and 4, the gestation length was considered to be not influenced by treatment with the test item. In groups 1, 2, 3, and 4, the mean duration of gestation was 21.6, 21.5, 21.7, and 21.8 days, respectively.
CORPORA LUTEA COUNT
In groups 2, 3, and 4, the mean number of corpora lutea was considered to be not influenced by treatment with the test item. In groups 1, 2, 3, and 4, the mean number of corpora lutea was 13.8, 13.9, 14.6, and 14.7, respectively.
IMPLANTATION RATE AND POST-IMPLANTATION LOSS
In groups 2, 3, and 4, the implantation rate and the post implantation loss were considered to be not influenced by treatment with the test item. In groups 1, 2, 3, and 4, the mean implantation rate was 12.2, 13.0, 14.1, and 13.7, respectively. In groups 1, 2, 3, and 4, the total post implantation loss was 12, 16, 12, and 10, in 8, 9, 5, and 6 litters, respectively. These data correspond to 9.8%, 12.3%, 8.5%, and 8.1% of implantations.
LITTER SIZE AT FIRST LITTER CHECK
In groups 2, 3, and 4, the number of living pups at first litter check, were considered to be not influenced by treatment with the test item. In groups 1, 2, 3, and 4, the mean number of living pups was 11.0, 11.4, 12.9, and 12.6, respectively. One, 1, and 2 dead pups were noted in 1, 1, and 2 litters of groups 2, 3, and 4, respectively.
POSTNATAL LOSS DAYS 0 - 4 POST PARTUM
In groups 2, 3, and 4, the postnatal loss was considered to be not influenced by treatment with the test item. In groups 1, 2, 3, and 4, the postnatal loss was 2, 1, 1, and 3 dead pups noted in 2, 1, 1, and 3 litters, respectively. These data correspond to 1.8%, 0.9%, 0.8%, and 2.7% of dead pups during the postnatal period.

LITTER DATA - F1 PUPS
ABNORMAL FINDINGS AT FIRST LITTER CHECK UNTIL DAY 4 POST PARTUM
At first litter check, no test item-related findings were noted. Female pup no. 14 in litter 46 (group 1) was noted with a missing tail and an anal atresia. It was killed for ethical reasons on day 1 post partum. Female pup no. 14 in litter 77 (group 4) was noted with a missing tail. It was necropsied as scheduled.


SEX RATIOS
Sex ratios were considered to be not influenced by treatment with the test item. Sex ratios (% male/% female) were 49 / 51, 48 / 52, 43 / 57, and 48 / 52 in groups 1, 2, 3, and 4, respectively, at first litter check.
PUP WEIGHTS TO DAY 4 POST PARTUM
The mean body weight of pups up to day 4 post partum was considered to be not influenced by treatment with the test item. Compared with the other groups’ values, slightly higher pup body weights were noted for male and female pups in group 4 that were considered to reflect the normal biological variability.

NECROPSY FINDINGS
At scheduled necropsy, no test item-related findings were noted.

CLINICAL LABORATORY INVESTIGATIONS
HEMATOLOGY
None of the parameters under investigation for hematology was considered to be affected by exposure to the test item. In males, the mean hemoglobin concentration distribution width and the mean relative reticulocyte count were noted with statistically significantly increased values in group 4. Mean prothombin time was statistically significantly increased in groups 3 and 4. The mean activated partial thromboplastin time was statistically significantly decreased in group 4. These findings were considered incidental as these statistically significant values were within the range of reference values. In females, the mean relative reticulocyte count was noted with increased values in group 4. This finding was considered incidental as this parameter exceeded the reference values in all groups. The parameters mean absolute reticulocyte count and mean total leukocytes also exceeded the referencevalues in all groups. Therefore, these findings were also considered incidental.
CLINICAL BIOCHEMISTRY
None of the parameters under investigation for clinical biochemistry was considered to be affected by exposure to the test item. A few parameters, like creatinine, cholesterol, sodium, and chloride in males, were noted with statistically significantly values in groups 3 and 4. Yet, these were considered incidental as the statistically significant values were within the range of reference values. In females in group 4, statistically significant values were noted at the parameters of glucose and potassium. The value for potassium exceeded the reference values for females from 13 to 18 weeks of age. As the parameter of phosphorus in all groups also exceeded the reference values, these incidences were considered incidental.

TERMINAL FINDINGS
NECROPSY
All male and female animals in groups 2, 3, and 4 were noted without any treatment-related finding at necropsy.
ORGAN WEIGHTS
Males
In male animals of groups 3 and 4, respectively, mean organ weights of liver and kidneys were slightly increased. For organ / body weight ratio these organs weights were statistically significantly increased in groups 3 and 4, respectively. These findings may be due to an increased metabolism and excretion of the test item and was therefore considered test item related. All other mean organ weights were considered to be not influenced by the treatment with the test item. For organ / body weight ratio the weight of the testes was statistically significantly increased in group 4. As the mean body weight in group 4 was statistically significantly decreased at the end of study, this relative mean organ weight increase of the testes was considered incidental.

Females
In female animals of group 4, the mean liver weight was statistically significantly increased. The mean kidney weights were slightly increased in groups 3 and 4. For organ / body weight ratio, the relative liver weight was statistically significantly increased in group 4, and the relative kidney weights were statistically significantly increased in groups 3 and 4. These findings may be due to an increased metabolism and excretion of the test item and was therefore considered test item related. All other mean organ weights were considered to be not influenced by the treatment with the test item. For organ / body weight ratio the heart was statistically significantly increased in group 4. This relative mean organ weight increase of the heart was considered incidental.

HISTOPATHOLOGY
The histopathological evaluation of the reproductive organs did not reveal any relevant changes in high-dose animals. Especially the emphasis on stages of spermatogenesis and histopathology of interstitial testicular cell structure did not reveal any differences between control (group 1) and high-dose (group 4) males.

The oral application of Di-tert-butyl peroxide at the dose levels of 300 and 1000 mg/kg/day (groups 3 and 4) induced the following findings likely related to an effect of the test item in organs other than reproductive:

Liver: Minimal centrilobular hepatocellular hypertrophy was present in 3/5 males and 3/5 females of group 3 and 2/5 males and 2/5 females of group 4. Minimal to slight diffuse hepatocellular hypertrophy occurred in 1/5 males of group 3 and 3/5 males and 3/5 females of group 4. In one male, the hypertrophy was associated with focal single cell necrosis.

Thyroid gland: Minimally to moderately increased incidence and minimally increased severity of diffuse follicular cell hypertrophy was noted in 4/5 males and 5/5 females and was likely consequent to an enhanced liver cell metabolism due to the hepatocellular hypertrophy.

Kidneys: Moderate diffuse tubular degeneration/regeneration was found in 5/5 males, in all males associated with minimal to slight multifocal single cell necrosis, and in 3/5 males associated with minimal to moderate hyaline casts. In addition, slightly increased severity of hyaline droplets occurred in proximal convoluted tubules of group 4 males, representing alpha-2μ-globulin in the cytoplasm (phagolysosomes) of proximal convoluted tubules of sexually mature males.

Forestomach: Minimally increased incidence and severity of diffuse hyperkeratosis was diagnosed in group 4 males and in group 3 and 4 females.


These microscopic findings occurring in treated group 4 animals or being increased in group 4 rats, were considered to be likely related to an effect of the test item. All other microscopic findings noted in various organs and in all groups examined were considered to be incidental in nature since their morphology, severity, and incidence were indistinguishable from controls.
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
LOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified

SUMMARY OF PERFORMANCE                      F0 ANIMALS BREEDING FOR F1 LITTERS

Group

1

2

3

4

(mg/kg/day)

0

100

300

1000

Female numbers

41-50

 

51-60

61-70

71-80

Number of females paired

10

10

10

10

Number of females mated

10

10

10

10

Number of non pregnant females(A)

0

0

0

1

Number of females, which lost their

litters

0

0

0

0

Number of females which reared

their pups until day 4 post partum

10

10

10

9

(A) = Female No. 79 was not pregnant

 

 

 

Conclusions:
Treatment at 1000 mg/kg was associated with body weight effects in male animals. 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 was associated with increased liver and kidney weights. Histopathological effects were noted 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), 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), and fore stomach (minimally increased incidence and severity of diffuse hyperkeratosis 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 these data, it can be concluded that the No Observed Effect Level (NOEL) was at 100 mg/kg body weight/day.
Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of Di-tert- butyl peroxide on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. In addition, information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition was provided.

Di-tert-butyl peroxide was administered once daily orally (by gavage) at dosages of 0, 100, 300, and 1000 mg/kg/day, to male rats for 42 days in total and to female rats throughout the prepairing, 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. 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 was associated with increased liver and kidney weights. Histopathological effects were noted 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), 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), and fore stomach (minimally increased incidence and severity of diffuse hyperkeratosis 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 these data, it can be concluded that the No Observed Effect Level (NOEL) was at 100 mg/kg body weight/day.

The microscopic findings occurring in the high dose group animals or being increased in the high dose animals, were considered to be likely related to an effect of the test item. All other microscopic findings noted in various organs and in all groups examined were considered to be incidental in nature since their morphology, severity, and incidence were indistinguishable from controls. Therefore, 300 mg/kg/day will be used as the NOAEL for DNEL calculation.

Endpoint:
chronic toxicity: oral
Data waiving:
other justification
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Reliable without restrictions.

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:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
A micronucleus assay was included
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals 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:
RF study: the animals 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.
Main study: the animals of groups 1-4 (control, low, mid and high concentration) were exposed on 5 days per week, 6 hours per day, over a 13-week study period (65 exposure days in total), for the last time on the day before scheduled sacrifice. The animals of the positive control group received a single intraperitoneal injection with Mitomycin C on the day prior to scheduled sacrifice.
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:
RF study: 5
Main study: 10
Control animals:
yes, concurrent vehicle
Details on study design:
Concentrations for the main study were based on the results of the RF study.

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:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
A micronucleus assay was included
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals 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:
RF study: the animals 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.
Main study: the animals of groups 1-4 (control, low, mid and high concentration) were exposed on 5 days per week, 6 hours per day, over a 13-week study period (65 exposure days in total), for the last time on the day before scheduled sacrifice. The animals of the positive control group received a single intraperitoneal injection with Mitomycin C on the day prior to scheduled sacrifice.
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:
RF study: 5
Main study: 10
Control animals:
yes, concurrent vehicle
Details on study design:
Concentrations for the main study were based on the results of the RF study.

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

Additional information

Di-tert-butyl peroxide was tested for systemic toxicity and reproductive effects in an OECD 422 study. No effects on reproductive performance or developing animals was found. Systemic effects were limited to changes in organ weight and histopathology in the mid- and high-dose groups. The NOEL in this study was 100 mg/kg/day.

The microscopic findings occurring in the high dose group animals or being increased in the high dose animals, were considered to be likely related to an effect of the test item. All other microscopic findings noted in various organs and in all groups examined were considered to be incidental in nature since their morphology, severity, and incidence were indistinguishable from controls. Therefore, 300 mg/kg/day will be used as the NOAEL for DNEL calculation.

A 90-day inhalation study was conducted in the rat at target concentrations of 100, 300 and 1000 mg/m3. Actual exposure levels were 101, 299, and 993 mg/m3 and resulted in a few modest changes at the highest concentration tested (increases in relative liver and kidney weight (statistically significant in males only) and altered plasma levels of cholesterol and creatinine). No local effects were seen in the respiratory tract, and no treatment-related changes were observed at the lower concentrations.Considering the modest magnitude of the organ weight changes and the absence of corroborative histopathological alterations or clinical chemical indicators of organ damage, the slightly increased relative liver and kidney weights in male rats of the high-concentration group were considered not to represent adverse effects of the test material. This exposure level (993 mg/m3, actual concentration) was a No-Observed-Adverse-Effect Concentration (NOAEC).


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Apparently well conducted GLP study.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Apparently well conducted GLP study.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
Apparently well conducted GLP study.

Justification for classification or non-classification

No adverse effects were reported in a 90 -day repeat dose study in rats. The effects reported in an OECD 422 study were minor, considered adaptive in nature and/or species specific.