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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld, from SPF colony
- Age at study initiation: young adult rats, 8 weeks old at starting
- Weight at study initiation: 265-293g for males, 188-216g for females
- Housing: Group caging (up to 5 animals, by sex, per cage) in polycarbonate solid floor cages (type III) with stainless steel mesh lids.
- Diet (ad libitum): ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice
- Water (ad libitum): tap water
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.3-24.9
- Humidity (%): 30 - 65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
nose only
Vehicle:
clean air
Remarks on MMAD:
MMAD / GSD: The particle size of the test atmosphere was not determined since the test item was evaporated and non-condensing.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: TSE Systems GmbH, Bad Homburg, Germany. This system comprises of two, concentric anodised aluminium chambers and a computer control system incorporating pressure detectors and mass flow controllers.
- Method of holding animals in test chamber: The animals were held in polycarbonate restraint tubes located around the chamber which allow only the animals’ nares to enter the exposure port.
- Source of air: compressed air used to vaporize the test item was supplied by an oil-less compressor and passed through a respiratory quality filter train and condensate traps prior to use.
- System of generating vapor: atmosphere generation was dynamic. Fresh vapour from the generation system was constantly supplied to the inner plenum (distribution chamber) of the exposure system from where, under positive pressure, the vapour was distributed to the individual exposure ports.
- Temperature, humidity: Test atmosphere temperatures in some cases were increased over the limit of 25ºC (up to 29.0ºC) due to temporary malfunction of the cooling system. The relative humidity was lower than required range of 30-70% due to use of the filtered, dry air for the dispersion of the test item.
- Air flow rate: the flow of air through each port was at least 0.7 L/min.
- Air change rate:
- Method of particle size determination: the particle size of the test atmosphere was not determined since the test item was evaporated and non-condensing.
- Treatment of exhaust air: after passing through the animal’s breathing zone, spent vapour enters the outer cylinder from where it was exhausted through a suitable filter system.

TEST ATMOSPHERE
- Brief description of analytical method used: actual achieved test atmosphere concentrations in the animal’s breathing zone were measured using infrared (IR) analysis by Foxboro MIRAN 1a-CVF as required by the characteristics of the test item.
Additional confirmatory analytical analysis by Gas Chromatography (GC) was performed after the in-life phase where was confirmed that degradation of the Trigonox TAHP-W85, specifically tert-amyl hydroperoxide, did not occurred during the inhalation exposure.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The actual concentration of generated atmospheres was normally measured by IR analysis by Foxboro MIRAN 1a-CVF at regular intervals during an exposure. Sampling was normally performed shortly after chamber equilibration and then at regular intervals during the exposure. The actual sampling schedule employed was dependent on the required sample volume in order to obtain adequate quantities of test item. Actual chamber concentration was measured at least twice (control group) or three times (low, mid and high groups) during each exposure for each dose concentrations.

Samples were taken from an unoccupied exposure port (representing the animal’s breathing zone) by pulling a suitable, known volume (45 L direct sample) of test atmosphere and then the samples were analyzed using a calibrated Miran 1A CVF gas analyser containing a 5.64 L measuring loop.

The actual achieved test atmosphere concentration was calculated based on the valid calibration curve using the infra red absorbance values provided by the Miran and recorded by a TESTO 175-S2 Data Logger.
Duration of treatment / exposure:
28 days
Frequency of treatment:
6 hours/day, 5 days/week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0.025, 0.103 and 0.405 mg/L
Basis:
other: analytical conc. of Trigonox TAHP-W85
Remarks:
Doses / Concentrations:
0.208, 0.085 and 0.336 mg/l
Basis:
other: analytical conc. of t-amyl hydroperoxyde
No. of animals per sex per dose:
Main groups: 5 animals/sex/group, 4 groups
Recovery groups: 5 animals/sex/group, 2 groups, control and high dose
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: CiToxLAB study code: 12/354-103PE, RSS in section 7.5.2
- Post-exposure recovery period in satellite groups: 4 weeks

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: As a minimum, individual clinical observations were performed prior to exposure on the working days, and at least twice during the exposure whilst the animals were still restrained. Following exposure, clinical observations were performed at least twice (as soon as practicable after removal from restraint, and approximately one hour after completion of the exposure). After the end of the 28-day exposure period, checks were made once daily for recovery groups.
- Cage side observations checked: changes in the skin and fur, eyes and mucous membranes and also respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to exposure on the working days, and at least twice during the exposure whilst the animals were still restrained. practicable after removal from restraint, and approximately one hour after completion of the exposure). All animals were then examined once per week until termination and these observations were performed within approximately one-hour of completion of exposure.

BODY WEIGHT: Yes
- Time schedule for examinations: twice-weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on Day 29 (main) and 57 (recovery)
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes
- How many animals: 5/sexe
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on Day 29 (main) and 57 (recovery)
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes
- How many animals: 5/sexe
- Parameters checked in table 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: one day prior to the necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes but water available
- Parameters checked in table 3 were examined.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Gross necropsy was performed on each animal terminally, on Day 29 or 57, all animals were euthanized under pentobarbital anaesthesia by exsanguination.
After exsanguination, the external appearance was examined, cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed macroscopically.

ORGAN WEIGHTS: Yes (see table 4)

HISTOPATHOLOGY: Yes (see table 4)
Statistics:
The statistical analysis may be performed using SPSS PC+4.0 software. The heterogeneity of variance between groups were checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan’s Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. If the data was not normal distributed, the non-parametric 0, method of Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using Mann-Whitney U-test. The frequency of clinical observations and necropsy and histopathology findings were calculated as applicable. In addition, T-test was used for the recovery groups.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
at concentrations of 0.405 mg/L (high dose) only slight sneezing was recorded in both sexes following the end of exposure, it was not considered to be adverse
Mortality:
no mortality observed
Description (incidence):
at concentrations of 0.405 mg/L (high dose) only slight sneezing was recorded in both sexes following the end of exposure, it was not considered to be adverse
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
There was no mortality observed during the study.
No significant clinical signs were observed during the study. In the animals exposed at concentrations of 0.405 mg/L (high dose) only slight sneezing was recorded in both sexes following the end of exposure. This transient sign was observed in all animals of high dose during Weeks 1 to 3 of the exposure, however decreasing tendency was observed, i.e. during the last week of the study sneezing was noted occasionally in some animals of both sexes. This clinical sign was considered to be a possible response to irritation in upper respiratory track. However, it was not considered to be adverse as it was not associated with any morphological changes and was transient in nature.
Slight sneezing was also occasionally observed in two males at 0.103 mg/L.
In addition, wet fur and fur staining were commonly recorded during the study period in all groups. These observations were considered to be related to the restraint and exposure procedures and were considered not to be toxicologically significant.

BODY WEIGHT AND WEIGHT GAIN
Bodyweight and bodyweight gain of both males and females in all experimental groups was comparable with control means.
Occasionally, variations occurred witch attained statistical significance (e.g. during exposure period, higher gain value of males at low dose and females at mid dose, lower values of females at mid and high doses; during recovery period higher or lower values in males of high group). These variations were considered to be of no toxicological significance.

FOOD CONSUMPTION
There were no variations observed in food consumption that were considered indicative of an adverse reaction to the test item.

HAEMATOLOGY
No effect of the test item on the haematology parameters was noted.
Haematology parameters were generally within the normal range. Occasional individual values fell slightly outside the normal range, but were not considered biologically significant.
In males slightly higher platelet counts (PLT, K/¿L) (p<0.05) were observed in the high dose group at the end of the 28-day treatment period, however this was not observed after 28 days recovery period. The same parameter was slightly higher/lower in females in mid and high dose groups at the end of treatment period, but this tendency was not observed after 28 days recovery period. These findings were not considered toxicologically significant.

CLINICAL CHEMISTRY
No effect of the test item on clinical chemistry parameters was observed.
There was one statistically significant difference reported between the control and the treatment groups for one mean clinical chemistry parameter at the end of the recovery period.
The high glucose concentration (mmol/L) (p<0.05, high dose group) which attained statistical significance in the males after 28-days recovery period was not evident after the treatment period.
These statistically significant differences were low in magnitude (+ 12%) and were considered to be of no toxicological significance.

URINALYSIS
The urinalysis parameters were comparable to the controls following the 28-day exposure or recovery periods.
There were some statistically significant differences between the control and the high groups in the volume reported at the end of the 28-day exposure period (p<0.05, male high dose group) or recovery period (p<0.05, males high dose group). These differences were small and were considered to be of no toxicological significance.

NECROPSY FINDINGS
There was no evidence of test item-related macroscopic changes in animals necropsied on Day 29 and 57.

ORGAN WEIGHTS
There were no toxicologically significant differences among experimental groups in the organ weights.
However, statistically significant variations were noted in the liver in males at 0.405 mg/L concentration (p<0.05 for bodyweight relative value). Compared with control the difference was approximately 10%, but was not associated with any microscopic observations or changes in biochemical parameters. In isolation, changes are not considered toxicologically significant.

Higher than control absolute and relative adrenals weights were recorded for females at 0.405 mg/L (p<0.05). When compared with control the difference was approximately 20%, but was not associated with any microscopic changes. As no similar observation was made in males the causative role of the test item is equivocal.

Slightly higher kidney weight was found in females at 0.405 mg/L. Compared to the control mean, the difference was approximately 10% and attained statistical significance for the bodyweight related value (p<0.05). As the differences were of low magnitude and were not associated with any morphological observations or changes in biochemical parameters, they were not considered to be adverse.

Following the 28-day recovery period lower epididymis weights were recorded for males and when compared with the control mean, the difference was approximately 15%, attaining statistical significance for both absolute and relative values (p<0.05). However, as no similar observation was made in the males following the end of the exposure period, and microscopic examination revealed normal structure of the organ, this change is considered unlikely to be related to the test item. It should be noted in two males (413 and 414) unilateral minimal to mild tubular atrophy was found in testes, but was considered to be background observation, routinely observed in Wistar rats.

Slightly lower kidneys weights were recorded for males from high group by approximately 12% when compared to the control mean, attaining statistical significance for bodyweight and brain related values (p<0.05). In addition, slightly higher liver weights were recorded for females at the same dose by 11% compare to the control mean, attaining statistical significance for brain related value (p<0.05) for recovery animals.

HISTOPATHOLOGY: NON-NEOPLASTIC
Main Groups: No test-item related microscopic findings were observed.
Various microscopic changes were recorded, such as mild focal tubular degeneration/atrophy of the testes (1/5 Low dose males) and mild reduced sperm content with minimal ductal atrophy in the epididymis (1/5 Low dose males), moderate interstitial inflammation (1/5 High dose males) of the kidneys, minimal multifocal chronic-active pyelitis and mild multifocal chronic inflammation of the bladder with lymphoid follicle formation (1/5 Control females) and minimal pelvic dilatation (1/5 Low dose males) of the kidneys, minimal or mild congestion/haemorrhages in the thymus (1/5 Control males, 2/5 Control females, 2/5 Low dose females and 2/5 Mid dose females), minimal luminal dilatation and glandular proliferation in the uterine horns (in correlation with necropsy, 1/5 Control females and 2/5 High dose females). Based on the low incidence and/or severity as well as distribution in the control and dosed animals, these observations were regarded as incidental, not toxicologically significant or associated with test-item administration.

Recovery Groups: No test item-related microscopic changes were noted in the recovery animals.
In the testes, minimal to mild focal/multifocal tubular degeneration/atrophy were present in 2/5 males from the High dose group and in one High dose male, minimal, multifocal congestion/haemorrhages occurred in the thymus as incidental.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEC
Effect level:
> 0.405 other: mg/L air (analytical) as TRIGONOX TAHP-W85
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEC
Effect level:
> 0.337 other: mg/L air (analytical) as t-amyl hydroperoxide
Based on:
act. ingr.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Table 5: Mean achieved actual and nominal aerosol concentrations of TRIGONOX TAHP-W85

Group

Dose

Target Concentration

(mg/L)

Mean Achieved

Concentration
(mg/L)

Standard Deviation of Achieved Concentration (mg/L)

Nominal

Concentration
(mg/L)

1

Control

0

0.0

0.0

0.0

2

Low

0.025

0.025

0.003

0.162

3

Mid

0.100

0.103

0.007

0.223

4

High

0.400

0.405

0.009

0.425

Applicant's summary and conclusion

Conclusions:
Exposure to Trigonox TAHP-W85 in the form of vapour to Wistar rats during a 28 day period at concentration levels of 0.025, 0.103 and 0.405 mg/L for six hours per day was not associated with adverse effects. Slight sneezing was observed following the exposure at 0.405 mg/L vapour concentration in both sexes and was considered related to irritant properties of the test item.
There were no observed changes seen in the exposed animals from each treated groups in bodyweight, food consumption and clinical pathology data when compared to the control group. No test item-related microscopic findings were noted in any examined organs including the respiratory tract, including associated lymph nodes or nasal cavity at dose levels of 0.025, 0.103 and 0.405 mg/L.
The NOAEC for Trigonox TAHP-W85 in this study was therefore considered to be higher than 0.405 mg/L corresponding to a concentration of 0.337 mg/L as pure tert-amyl hydroperoxide.
Executive summary:

A 28-Day Inhalation Toxicity Study (Nose-Only) in the Rat with a 4-Week Recovery Period was performed in order to obtain information on the local and systemic toxicity of Trigonox TAHP-W85, containing 83.2%tert-amyl hydroperoxide, when administered by nose-only inhalation exposure to vapors for 6 hours per day in Wistar rats. The animals were exposed for 28 days (5 days per week) using a nose-only exposure system. The recovery animals from the control and high groups were observed for an additional 4-week period after the last exposure to give data on the reversibility of any effects detected.This study has been performed in accordance with the OECD test guideline no. 412 and the Principles of Good Laboratory Practice. The animals in groups 2 to 4 inhaled achieved concentrations of 0.025, 0.103 and 0.405 mg/L of Trigonox TAHP-W85 (corresponding to 0.208, 0.085 and 0.336 mg/l of t-amyl hydroperoxide), respectively for six-hours/day (5 days per week). A control group received the clean air only. Reversibility of any treatment-related changes was evaluated following a 28-day recovery period. The mortality observations were performed twice daily. General clinical observations were recorded on 5 occasions per day on each week day of treatment were recorded daily. Detailed clinical observations were performed once a week. Bodyweights were recorded twice a week and food consumption was measured at weekly interval during the 28-day treatment period and 28-day recovery period of the study. Gross macroscopic examination was performed at necropsy one day after the last treatment. Selected organs were weighed and full histopathology investigation was performed on control and high dose animals in the study.The test atmosphere concentration was monitored based on the MIRAN analysis. The results of the test atmosphere characterization were considered suitable for the study purposes. Additional confirmatory analytical analysis by Gas Chromatography was performed where was confirmed that degradation of the Trigonox TAHP-W85, specifically of tert-amyl hydroperoxide, did not occurred during the inhalation exposure.

There was no mortality during the study. No significant clinical signs were observed during the study. In the animals exposed at the concentrations of 0.405 mg/L (group 4) only slight sneezing was occasionally recorded during the course of the study. There were no differences observed in bodyweight gain and food consumption that were considered indicative of an adverse reaction to the test item. There were no changes in haematology, clinical chemistry and urine parameters that were considered as treatment-related. There were no macroscopic abnormalities, changes in organ weights and microscopic findings that were considered as treatment-related.

The No-Observed Adverse Effect Concentration (NOAEC) for Trigonox TAHP-W85 in this study was therefore considered to be higher than of 0.405 mg/L, corresponding to a concentration of 0.337 mg/L as pure tert-amyl hydroperoxide.