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Toxicological information

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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Cross-reference
Reason / purpose for cross-reference:
reference to other study
Remarks:
NOAEL systemic toxicity inh.
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 2017 - Mar 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 Jul 2016
Principles of method if other than guideline:
The conduct of inhalation exposures was performed following the general provisions of OECD Testguideline 413 (Subchronic Inhalation Toxicity: 90-day Study).
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
- Name of test substance: Diisopropyl Ketone; 2,4-Dimethyl-3-Pentanone
- Batch No. 151005
- Purity: 99.2 area-%
- Homogeneity: Given
- Storage stability: Expiry date: 06 Dec 2018
- Physical state/Appearance: Liquid/yellowish, clear
- Storage conditions: Room temperature

OTHER:
- the test substance was used unchanged
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Crl:WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Females nulliparous and non-pregnant: Yes
- Age: 10-11 weeks (males), 9 weeks (females)
- Housing: From delivery until randomization: Polysulfonate cages type 2000P (H-Temp), floor area about 2065 cm2 (610 x 435 x 215 mm); supplied by TECHNIPLAST, Hohenpeißenberg, Germany. From randomization onwards: Polycarbonate cages type III (floor area about 800 cm2)
- No. of animals per cage: Polysulfonate cages: up to 5 animals per sex and cage. Polycarbonate cages: 1 animal (except During mating: 1 male/1 female per cage and during rearing up to PND 13: 1 dam with her litter)
- Enrichment: Wooden gnawing blocks (Typ Lignocel block large), J. Rettenmaier & Söhne GmbH + Co KG, Rosenberg, Germany. In addition in Polysulfonate Gages: play tunnel large (Art. 14153), PLEXX B.V., Elst, Netherlands
- Nesting material: Cellulose wadding toward the end of gestation (pregnant females)
- Bedding: Dust-free wooden bedding
- Diet: Ground Kliba maintenance diet mouse/rat "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland; ad libitum
- Water: Drinking water ad libitum
- Acclimation period: at least 5 days
- Identification: Pre-treatment period for cycle determination and randomization: tail-markings. After randomization, parental animals (F0) were uniqueliy identified by ear tattoo. Pups: All pups were identified by skin tattoo on PND1 and with picric acid markings on PND 10 or 11.
- Randomization: Animals were distributed according to their body weight among the individual test groups. The list of randomization instructions was compiled with a computer.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20- 24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12 (6 am to 6pm light phase)

IN-LIFE DATES:
- 03 May 2017 - 04 Jul 2017 (parental males)
- 03 May 2017 - 25 Jul 2017 (parental females)
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Piston metering pumps KP 2000 (DESAGA; SARSTED AG & Co, Nürnbrecht, Germany), Atomization vaporizer (glass) with thermostat (BASF SE, Ludwigshafen, Germany), Thermostat (JULABO Labortechnik GmbH, Seelbach, Germany)
- Method of holding animals in test chamber: Whole-body exposure system. The animals were kept single in wire cages located in a glass-steel inhalation chamber, volume of ca. 1.1 m³.
- Source and rate of air: Compressed air was produced by an oil-free compressor. The compressed air was conducted to the laboratories via pipes from a 1500 or 5000 l storage tank, and in the laboratory, the compressed air was taken as required. Compressed air is filtered air pressurized to about 6 bar.
- Method of conditioning air: The central air conditioning system provided cold air of about 15°C. This cold air passed through an activated charcoal filter, was adjusted to room temperature of 20 to 24°C and passed through a second particle filter (H13 (HEPA) Camfil Farr, Germany). The so generated
conditioned air was used to generate inhalation atmospheres.
- Air change rate: 15 per hour
- Air flow rate: Test group 1 (100 ppm) 5-15 ml/h, test group 2 (300 ppm) 25-35 ml/h, and test group 3 (1000 ppm) 90-130 ml/h
- Temperature, humidity, pressure in air chamber: 21.2 - 23.8°C, 39.5 - 64.2%, a negative pressure was maintained
- Treatment of exhaust air: The exhaust air was filtered and conducted into the exhaust air of the building
- Substance preparation flow: 5 - 15 ml/h (Test group 100 ppm), 25 - 35 ml/h (test group 300 ppm), and 90 - 130 ml/h (test group 1000 ppm)
- Supply air 1 (total air flow of conditioned air): 14.5 - 18.5 m³/h
- Supply air 2 (compressed air): 0.8 - 1.2 m³/h
- Supply air 3 (partial flow through generatot, conditioned): 4.0 - 8.0 m³/h


TEST ATMOSPHERE:
- Brief description of analytical method used: The concentration of the test substance in the atmospheres were determined by gas chromatography method (GC) using online MicroGC. To calibrate the microGC, appropriate amounts of test substance were weighed into gas sampling tubes with defined volume. The atmospheres were sampled by microGC and a calibration line was formed by the pair of substance concentration and peak area.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: 1:1
- Length of cohabitation: mated overnight, from about 16.00 h until 06.00 - 09.00 h of the following morning for a maximum of two weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
ANALYSIS OF THE INHALATION ATMOSPHERE:
Nominal concentration:
The nominal concentration was calculated from the study means of the test pump rates and the supply air flows used during expsoure to generate the respective concentrations.

Analytical methods of determination:
The samples were analyzed by calibrated online microGC.
Duration of treatment / exposure:
6 hours per day
Frequency of treatment:
On 7 consecutive days per week for the desired period of time
Details on study schedule:
Exposure period for male animals: 35 days, females: 56 days
Dose / conc.:
99.1 ppm (analytical)
Remarks:
~471 mg/m3
Dose / conc.:
305.8 ppm (analytical)
Remarks:
~1414 mg/m3
Dose / conc.:
1 008.9 ppm (analytical)
Remarks:
~4715 mg/m3
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
OTHER:
- Reason for species selection: The test guideline requires the rat to be used as the animal species. This rat strain was selected since extensive historical control data are available for Wistar rats.
Positive control:
no positive control included in the study
Parental animals: Observations and examinations:
MORTALITY:
- Check for moribund and dead animals was made twice daily from Mondays to Fridays and once daily on Saturdays, Sundays and public holidays.

CLINICAL SIGNS:
- Was recorded once during the pre-exposure period and on post-exposure observation days and at least three times (before, during, and after exposure) on exposure days. During exposure only a group-wise examination was possible.
- Abnormalities and changes were documented daily for each animal.
- The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis.
- On weekdays (except Saturdays, Sundays and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings.

FOOD CONSUMPTION:
Generally, food consumption was determined once a week for male and female parental animals.
- Food consumption was not determined after the 2nd premating week and during the mating period
- Food consumption of F0 females with evidence of sperm was determined for GD 0-7, 7-14 and 14-20.
- Food consumption of F0 females which gave birth to a litter was determined for PND 1-4, 4-7, 7-10 and 10-13.
Food consumption was not determined in females without positive evidence of sperm during mating and gestation periods and in females without litter during lactation period.

BODY WEIGHTS:
In general, the body weight of the maternal animals was determined once a week at the same time of the day (in the morning) until sacrifice. Body weight change was calculated from this results.
The following exceptions are notable for the female animals:
- During the mating period, the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
- Females with litter were weighed on the day after parturition (PND1) and on PND 4, 7, 10 and 13.
Females without litter or waiting for necropsy, were weighed weekly.

ESTROUS CYCLE DETERMINATION
- Estrous cylce length was evaluated by daily analysis of vaginal smear for all F0 female parental rats for a minimum of two weeks prior to mating. At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 female with scheduled sacrifice.

FUNCTIONAL OBSERVATION BATTERY (FOB)
- A FOB was carried out in the first five surviving parentla males and females with litter (in order of delivery) per group.
- Pasisve observations
- Removal from the home cage
- Open field observations
- Sensorimotor tests and reflex tests

Home cage observation:
The animals were observed for a short period (about 10-30 seconds) in their closed home cages; Attention was paid to:
- Posture
- Tremors
- Convulsions
- Abnormal movements
- Impairment of gait

Open field observation:
Animals were transferred to a standard arena and observed for at least two minutes. Following parameters were examined:
- Behavior when removed from cage
- Fur
- Skin
- Salivation
- Nasal discharge
- Lacrimation
- Eyes/ pupil size
- Posture
- Palpebral closure
- Respiration
- Tremors
- Convulsions
- Abnormal movements/ stereotypes
- Impairment of gait
- Activity/ arousal level
- Feces (number of fecal pellets/appearance/consistency) within two minutets
- Urine (amount/color) within two minutes
- Number of rearings within two minutes

Sensorimotor tests/Reflexes:
The animals were removed from the open field and subjected to the sensorimotor or reflex tests:
- Approach response
- Touch response
- Vision ('Visual placing response")
- Pupillary reflex
- Pinna reflex
- Audition ("Startle response")
- Coordination of movements ("Righting response")
- Behavior during "handling"
- Vocalization
- Pain perception ("Tail pinch")
- Grip strength of fore-limbs
- Grip strength of hind-limbs
- Landing foot-splay test

MOTOR ACTIVITY MEASUREMENT (MA):
MA was measured on the same day and in the same animals as FOB was performed. The measurement was conducted in the dark with 18 infrared beams per cage. The number of beam interrupts were counted over 12 intervals, each lasting five minutes. Measurements ended 60 minutes after the first beam was interrupted. During the measurements the animals received no food and no water.

CLINICAL PATHOLOGY:
Blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The results of clinical pathology examinations were expressed in International System (SI) units.

THYROID HORMONES
Blood samples were taken from all surplus pups per litter at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anaesthesia. Additionally, blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retro-bulbar venous plexus under isoflurane anaesthesia. The adults were fastened before the blood sampling. Blood samples from the adults males, the dams at PND 14 and the PND 13 pups were assessed for serum levels for thyroid hormones (T4 and TSH).

PATHOLOGY:
Necropsy:
All parental animals were sacrificed under pentobarbitone anesthesia. The left and right brachial vessels were opened by deep cuts through the pectoral muscles along both sides of the rib cage. Caution was exercised to avoid destruction of the axillary lymph nodes. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.

Weight parameters:
The following weights were determined in all animals sacrificed on schedule:
- Anesthetized animals
- Adrenal glands
- Epididymides
- Kidneys
- Liver
- Ovaries
- Seminal vesicles with coagulating glands
- Testes
- Thyroid glands
- Uterus (with cervix)

Organ / Tissue fixation:
The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or in modified Davidson's solution:
- All gross lesions
- Cervix
- Coagulating glands
- Epididymides (modified Davidson's solution)
- Kidneys
- Larynx/Pharynx
- Liver
- Lungs
- Lymph nodes (tracheobronchial and mediastinal)
- Nose (nasal cavity)
- Ovaries (modified Davidson's solution)
- Oviducts
- Prostate glands
- Seminal vesicles
- Testes (modified Davidson's solution)
- Trachea
- Thyroid glands
- Vagina
- Uterus (uteri of all apparently non-pregnant animals or empty uterus horns were stained according to Salewski E (1964))
Oestrous cyclicity (parental animals):
For all females estrous cycle normality was evaluated before the beginning of the administration period.
Estrous cycle length was evaluated by daily analysis of vaginal smear for all F0 females for aminimum of 2 weeks prior to mating. Determination was continued throughout the pairing period until the female exhibited evidence of copulation. At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 female with scheduled sacrifice.
Sperm parameters (parental animals):
Parameters examined in all male parental generations:
testis weight, epididymis weight
Litter observations:
- Pup number and litter size at delivery:
The status (sex, live-born or stillborn) and number of all pups delivered from the parents were determined as soon as possible after birth. At the same time, the pups were also examined for gross-morphological changes.

- Pup viability/mortality:
In general, a check was made for any dead or moribund pups twice daily (once in the morning and once in the afternoon). Pups which died accidentially or had to be sacrificed due to maternal death were not included in these calculations.

- Clinical signs:
All live pups were examined daily for clinical symptoms (including gross-morphological findings), once on non-exposure days and twice (before and after exposure) on exposure days. If pups showed particular findings, these were documented for each pup.

- Body weights:
The pups were weighed on the day after birth (PND 1) and on PND 4 (before standardization), 7 and 13. "Runts" were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups.

- Anogenital distance (AGD):
AGD was determined in all live male and female pups on PND 1

- Nipple/areola anlagen:
All surviving male pups were examined for the presence of nipple/areola anlagen on PND 13. The number of nipple/areola anlagen was counted.

- Pup necropsy observations:
External examinations: Yes, all pups were examined externally and eviscerated
Soft tissue examinations: Yes, organs were assessed macroscopically
Biochemistry: Blood was sampled from one male and one female pup per litter for determination of thyroid hormone concentrations.

Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All male animals were sacrificed on study day 35 (34 exposures) after the beginning of the administration, and examined.
- Maternal animals: All females were allowed to litter and rear their pups until day 13 after parturition.

GROSS NECROPSY
- the exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs (see parental animals: observations and examinations)
Postmortem examinations (offspring):
On PND 4, as a result of standardization, the surplus pups were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. After sacrifice, the pups were examined externally and eviscerated, and the organs were assessed macroscopically.
On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anestesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and were transferred to the Pathology Laboratory for possible further processing. The remaining pups were sacrificed under isoflurane with CO2. After sacrifice, the pups were examined externally and eviscerated, and the organs were assessed macroscopically.
Statistics:
STATISTICS OF THE CLINICAL EXAMINATIONS:

DUNNETT test (two-sided) * for p ≤ 0.05; ** for p ≤ 0.01:
Food consumption (parental animals), body weight and body weight change (parenal animals and pups; for the pup weights, the litter means were used), gestation days, anogenital distance, anogenital index

FISHER'S EXACT test (one-sided) * for p ≤ 0.05; ** for p ≤ 0.01:
Male and female mating indices, male and female fertility indices, females mated, females delivering, gestation index (females with liveborn pups), females with stillborn pups, females with all stillborn pups

WILCOXON test (one-sided+) with BONFERRONI-HOLM * for p ≤ 0.05; ** for p ≤ 0.01:
Mating days until day 0 pc, % post-implantation loss, pups stillborn, % perinatal loss, nipple development

WILCOXON test (one-sided-) with BONFERRONI-HOLM * for p ≤ 0.05; ** for p ≤ 0.01:
Implantation sites, pups delivered, pups liveborn, live pups day x, viability Index, survival index

WILCOXON test (two-sided) * for p ≤ 0.05; ** for p ≤ 0.01:
% live male day x, % live female day x

KRUSKAL-WALLIS (two-sided) and WILCOXON test (two-sided) * for p ≤ 0.05; ** for p ≤ 0.01:
Number of cycles and cycle length, rearing, grip strength of fore-limbs and hind-limbs. landing foot-splay test, motor activity

STATISTICS OF CLINICAL PATHOLOGY:

KRUSKAL-WALLIS and WILCOXON-Test (two-sided) * for p ≤ 0.05; ** for p ≤ 0.01:
Blood parameters

STATISTICS OF PATHOLOGY:

KRUSKAL-WALLIS test (two-sided) and WILCOXON test (two-sided) * for p ≤ 0.05; ** for p ≤ 0.01:
Weight parameters
Reproductive indices:
Male and female mating indices, male and female fertility indices, gestation index, live birth index, postimplantation loss
Offspring viability indices:
Viability index, survival index, anogenital index, sex ratio
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
During pre-mating and mating periods, all parental male and female animals (10 out of 10) of the test groups 2 and 3 (300 and 1000 ppm) showed salivation during/after exposure. In these periods, all high-dose male and female animals had lacrimation during exposure (pre-mating only) and the attention of males and females was reduced. Furthermore, a discolored fur (reddish) occurred in three and four high-dose females during pre-mating and mating, respectively, and in seven high-dose males during mating. Salivation lasted up to scheduled sacrifice (during post-mating period) in all male animals of test group 3. Furthermore, a discolored fur (reddish) was recorded in 5 out of 10 high-dose males.
During gestation period, all female animals of test group 3 showed salivation during/after exposure and their attention was reduced. Salivation was also recorded in 5 mid-dose females (300 ppm). A discolored fur (reddish) occurred in six females of test group 3 and two females of test group 2. One high-dose female plough nose-first into bedding.
During lactation period, all high-dose females with litter (9 out of 9) showed an insufficient maternal care and plough nose-first into bedding. The above mentioned clinical findings in the mid- and high-dose male and female animals were assessed as treatment-related and adverse.
One sperm-positive high-dose female (No. 133 -1000 ppm) did not deliver F1 pups.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
During pre-mating period, the high-dose F0 male animals lost weight at all time points, thus, the mean body weight change was statistically significantly reduced during the whole premating period. Their mean body weight was constantly decreased (up to -8%) compared to control during pre-mating, mating and post-mating. The mean body weights of the F0 parental high-dose females (1000 ppm) were statistically significantly decreased compared to the concurrent control values on GD 7 and 14 (up to -6%) and during PND 1-13 (up to -10%). In the high-dose F0 females mean body weight change was statstically significantly reduced during pre-mating days 0-7 and PND 4-7 showing a body weight loss and, if calculated for the entire lactation period (PND 1-13: -29% in comparison to the concurrent control). In test group 2 (300 ppm), F0 parental males gained less weight (8 g) compared to control (18 g) during pre-mating, attaining statistical significance if calculated for days 1-13. This was likely caused by the reduction in food consumption during this time (see above) and, therefore, assessed as treatment-related and adverse. The statistically significantly reduced mean body weight change of the mid-dose F0 females during GD 0-7 (21% below control) was considered as spontaneous in nature and not treatment-related as no dose-response relationship occurred.
Overall, the body weight decrease of mid- and high-dose males and high-dose females was assessed to be treatment-related and adverse.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption of the F0 parental males of the test group 3 (1000 ppm) was statistically significantly reduced during pre-mating period (up to around 24% below concurrent control). If calculated for the whole pre-mating period (days 0-13), these males consumed approximately 20% less food than the controls. The food consumption of the mid-dose male animals (300 ppm) was lower than the concurrent control group (up to -7%) during the pre-mating period. Although it showed no statistical significance, this decrease was assessed as treatment-related, adverse finding.
Consistently to the high-dose males, food consumption of the high-dose F0 females (1000 ppm) was statistically significantly decreased during pre-mating (up to 24%; for days 0-13: approximately 18% below control), gestation days 0-7 (-8%) and lactation days 4-13 (up to -30%; for PND 1-13: approximately 22% below control). In test group 2 (300 ppm), food consumption of the F0 females was statistically significantly decreased during lactation days 4-7 (-13% in comparison to the control) and onwards (up to -6.5% without statistical significance). During the remaining study periods (pre-mating and gestation), the food consumption of the mid-dose females was comparable to the concurrent control group. Although the mid-dose females were only affected in one study period (lactation), the reduction was assessed as treatment-related and adverse.
Overall, the reduction in food consumption of the high- and mid-dose males and females was assessed as treatment-related and adverse.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Motor activity measurements (single values) were statistically significantly above the concurrent control value in males of test group 1, 2 and 3 (100, 300 and 1000 ppm) during interval 4 (108.6/90.6/91.6 vs. 12.2 in control). Females of all test groups were not affected. Since there was no dose-response relationship, it was assessed as incidental and not treatment-related.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings were observed in the livers and kidneys of male animals of test groups 1 to 3, in the livers of female animals of test groups 2 and 3, and in the adrenal glands of male animals of test group 3.

Liver
The livers showed a minimal to moderate centrilobular hepatocellular hypertrophy. Additionally, 4 out of 10 female animals of test group 3 had a minimal periportal vacuolation, characterized by multiple, small, sharply demarcated, clear cytoplasmic vacuoles. Based on the morphology, this was interpreted to be fat vacuoles. Incidences and gradings are shown in table 2 in "any other information on results and tables". The centrilobular hypertrophy in male and female animals was regarded as treatment-related and correlated to significantly increased liver weights in test group 3 animals.

Kidneys
The kidneys of male animals of test groups 0 to 3 showed eosinophilic droplets within proximal convoluted tubules. The droplets proved to be positive in the CAB-stain, which was used for the assessement of severity (grading) of this finding. The immunohistochemical staining pattern was similar to that described by Cesta et al. (2013). Therefore, the presence of alpha2u-globulin (nephropathy) is likely. However, the incidence and severity were markedly increased in test groups 1 to 3. Additionally, animals of test group 1 to 3 showed intratubular granular casts, which were composed of proteinaceous material admixed with cellular debris and an increased incidence and severity of chronic progressive nephropathy characterized by variable degrees of basophilic tubules, interstitial lypmphoid infilitrates, tubular dilation and interstitial fibrosis. Both, the granular casts and the chronic progressive nephropathy are characteristic for an alpha2u-nephropathy. Incidences and gradings are shwon in table 3 in "any other information on results and tables". The increased incidence and severity of alpha2u-nephropathy correlated to significantly increased kidney weights and was regarded as treatment-related.

Adrenal glands:
The adrenal glands of males in test group 3 showed an increased cytoplasmic vacuolation of the inner cortical cells. The clear vacuoles were sharply demarcated and of variable size. They were interpreted to be fat vacuoles. Incidences and grading are shown in table 4 in "any other information on results and tables". The increased incidence of cytoplasmic vacuoles correlated to significantly increased adrenal gland weights and was regarded as treatment-related.
Histopathological findings: neoplastic:
no effects observed
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
Estrous cycle data:
The mean estrous cycle duration was statistically significantly increased in test group 3 (1000 ppm): 3.9/3.9/3.9/5.0* (p ≤ 0.05) days in test groups 0-3, respectively. The prolonged value in test group 3 was mainly caused by one single high-dose female (animal no. 131: mean cycle length: 11.00 days). All other females of test group 3 had cycles between 3.7 and 5 days. This is comparable to the other test groups and the control. The standard deviation of test group 3 showed a high value of 2.2 compared to the other test groups. The prolonged value of the high-dose females is most likely caused by the outlier and, therefore, assessed as not treatment-related.
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
The mean duration of gestation was statistically significantly lower in test group 3 (22.0/22.1/22.0/21.4* (p ≤ 0.05) in test groups 0-3, respectively). Although there was an apparently shorter duration of pregnancy in the high-dose females, this had no consequence for the peri-/post-natal survival and post-natal development of the offspring. It was assessed as possibly treatment-related without any adverse consequences on parturition. The gestation index was 100.0% in all test groups.
For males, a NOAEL could not be established in this study since a alpha2u-nephropathy with correlating increased absolute and relative kidney weights was observed down to the lowest concentration of 100 ppm. As this finding is not relevant for humans, it should not be taken into account for risk assessment and thus, the NOAEL for males for human health is 100 ppm.
Dose descriptor:
NOAEL
Effect level:
99.1 ppm (analytical)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
body weight and weight gain
Dose descriptor:
NOAEL
Effect level:
>= 1 009 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: No adverse effects on fertility
Critical effects observed:
yes
Lowest effective dose / conc.:
1 009 ppm
System:
other: general systemic toxicity
Organ:
kidney
liver
Treatment related:
yes
Dose response relationship:
yes
Critical effects observed:
yes
Lowest effective dose / conc.:
306 ppm
System:
other: salivation, redish discoloured fur, reduced food consumption and males only: decreased body weight gain
Organ:
other: possibly related to narcotic effects (STOT SE Cat 3)
Treatment related:
yes
Dose response relationship:
yes
Remarks on result:
not measured/tested
Reproductive effects observed:
no

Table 1: Liver histopathology findings

 

Male animals

Female animals

Test group

(ppm)

0

(0)

1

(100)

2

(300)

3

(1000)

0

(0)

1

(100)

2

(300)

3

(1000)

No. of animals

10

10

10

10

10

10

10

10

Hypertrophy, centrilobular

0

2

7

10

0

0

10

10

- Grade 1

 

2

6

4

 

 

10

1

- Grade 2

 

 

1

5

 

 

 

8

- Grade 3

 

 

 

1

 

 

 

1

Vacuolation, periportal

 

 

 

 

0

0

0

4

- Grade 1

 

 

 

 

 

 

 

4

Table 2: Absolute organ weights

 

Male animals

Female animals

Test group

(ppm)

1

(100)

2

(300)

3

(1000)

1

(100)

2

(300)

3

(1000)

Terminal body weight

99%

97%

91%

99%

99%

96%

Epididymides

91%*

92%*

97%

 

 

 

Kidneys

112%**

110%**

118%**

101%

105%

109%*

Liver

103%

106%

133%**

99%

105%

121%**

* = p <= 0.05; ** = p <= 0.01

Table 3: Relative organ weights

 

Male animals

Female animals

Test group

(ppm)

1

(100)

2

(300)

3

(1000)

1

(100)

2

(300)

3

(1000)

Adrenal glands

111%

120%*

127%**

 

 

 

Seminal vesicles

100%

102%

131%**

 

 

 

Kidneys

114%**

113%**

129%**

102%

106%*

114%**

Liver

104%

109%

144%**

100%

106%

127%**

* = p <= 0.05; ** = p <= 0.01

Table 4: Kidneys histopathological findings

 

Male animals

Test group

(ppm)

0

(0)

1

(100)

2

(300)

3

(1000)

No. of animals

10

10

10

10

Eosinophilic droplets, grading based on CAB-stain

7

10

10

10

- Grade 1

6

 

 

 

- Grade 2

1

4

2

3

- Grade 3

 

6

8

7

Granular casts, tubular

0

5

5

10

- Grade 1

 

1

1

4

- Grade 2

 

3

2

3

- Grade 3

 

1

2

2

- Grade 4

 

 

 

1

Nephropathy, chronic

6

10

10

10

- Grade 1

6

4

6

1

- Grade 2

 

6

4

3

- Grade 3

 

 

 

6

Conclusions:
In this present Reproductive/Developmental Toxicity Screening Test, groups of 10 male and 10 female Wistar rats (F0 animals) per test group were exposed in a whole-body inhalation system to dynamic atmosphere of the test substance up to 1000 ppm for 6 hours per day on each day.
The no observed adverse effect level (NOAEL) for general systemic toxicity was 100 ppm in females based on adverse clinical findings and adverse effects on food consumption, body weight and development as well as liver toxicity (high-dose only). For the males, a NOAEL could not be established in this study since a alpha2u-nephropathy with correlating increased abolsute and relative kidney weights was observed down to the lowest concentration of 100 ppm. As this findig is not relevant for humans, it should not be taken into account for risk assessment and thus, 100 ppm is used as starting point for DNEL derivation.

The NOAEL for fertility and reproductive performance was 1000 ppm for the F0 parental rats. Although there was an apperently shorter duration of pregnancy in the high-dose females, this had no consequence for the peri-/post-natal survival and post-natal development of the offspring.
Executive summary:

To evaluate the toxicity profile of Diisopropyl Ketone after inhalation exposure, groups of 10 male and 10 female Wistar rats (F0 animals) per test group were exposed in a whole-body inhalation system to dynamic atmosphere of the test substance for 6 hours per day on each day. The duration of treatment covered a 2-week pre-mating and 2-week mating period in both sexes, eight days post-mating in males, the entire gestation period as well as up to 20 days lactation period and one day post-weaning in females.

The target concentrations were 100 ppm (471 mg/m³), 300 ppm (1,414 mg/m³), and 1,000 ppm (4,715 mg/m³). A concurrent control group was exposed to conditioned air. For adaptatio to the exposure conditions the animals were placed into exposure cages before start of the exposure period (pre-expsoure period) on study days -6, -4 and -1. This pre-expsoure is performed in the animal room.

OBSERVATIONS

After two weeks of pre-mating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm was detected in the vaginal smear. F0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of postimplantation loss for all F0 females.

Clinical observation was performed at least three times on exposure days and once a days during the other days.

Food consumption of the F0 parental animals was determined once weekly during pre-mating. In dams, food consumption was determined for gestation days (GD) 0 -7, 7 -14, 14 -20, and lactation days (PND) 1 -4, 4 -7, 7 -10, and 10 -13.

Body weights of F0 parental animals were determined on pre-mating days 0, 7, 10 and 13 in males and females. Afterwards the body weights were determined once a week throughout the

study in males. During gestation and lactation period, F0 females were weighed on GD 0, 7, 14 and 20, on the day after parturition (PND 1) and on PND 4, 7, 10 and 13. Estrous cycle data were evaluated for F0 generation females over a two-week period prior to mating until evidence of mating occurred. Moreover, the estrous stage of each female was determined on the day of scheduled sacrifice. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1, 4, 7 and 13. Their viability was recorded.

Anogenital distance (defined as the distance from the anus [center of the anal opening] to the base of the genital tubercle) measurements were conducted in a blind randomized fashion,

using a measuring ocular on all live male and female pups on PND 1. At necropsy on PND 4 and PND 13, the pups were sacrificed with CO2, under isoflurane anesthesia, and examined macroscopically for external and visceral findings. On PND 13 thyroid glands and parathyroid glands were fixed for possible further processing. All surviving male pups were examined for the presence or absence of nipple/areola anlagen on PND 13. The number of nipple/areola anlagen were counted. Blood samples were taken from all surplus pups at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia for hormone measurement.

Blood samples from all dams at PND 14 and all meales at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia for hormone measurement. At the end of the exposure period a functional observational battery (FOB) was performed and motor activity was measured in five parental males and females per group.

All F0 parental animals were sacrificed under pentobarbitone anesthesia by exsanguination from the abdominal aorta and vena cava and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report date:
1990

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
May, 1981
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
September, 1984
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2,4-dimethylpentan-3-one
EC Number:
209-294-7
EC Name:
2,4-dimethylpentan-3-one
Cas Number:
565-80-0
Molecular formula:
C7H14O
IUPAC Name:
2,4-dimethylpentan-3-one
Test material form:
liquid
Details on test material:
- Name of test material (as cited in report): 2,4-Dimethylpentanon-3 / Diisopropyl keton
- Physical state: liquid
Specific details on test material used for the study:
- Name of test substance( as cited in report): Diisopropyl ketone / 2.4-Dimethyl-3-pentanone; DIPK
- Purity: The purity of the test substance was determined to be 100 ± 0.00% (mean ± SD) prior to and 99.8 ± 0.006% after the study when analyzed by gas chromatography.

Test animals

Species:
rat
Strain:
other: CD (SD) BR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Kingston, NY
- Age at study initiation: Males 46 days, Females 51 days
- Weight at study initiation: Males 231 ± 6 g, Females 194 ± 9 g (mean ± SD)
- Housing: Singly housed in multicompartmented stainless steel mesh cages
- Diet: Certified Rodent Diet (Agway Prolab RMH 3000, pellets), ad libitum during non-exposure periods
- Water: ad libitum during non-exposure periods
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.1 - 23.3 (reported as 70-74 °F)
- Humidity (%): 50 - 56
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Details on inhalation exposure:
The inhalation exposures were conducted in 420 L stainless steel and glass inhalation chambers at target concentrations of 1200, 380, 120, and 0 ppm. Exposures were conducted five days per week, 6 hour per day, for a total of 22 exposures over a four-week period.
The chambers were maintained at pressures of -0.035 to -0.05 inch water (gauge) relative to room air and at 12 air changes per hour. For each exposure level, male and female rats were singly housed and exposed simultaneously.
A vapor was produced by metering the test substance from a reservoir into a glass bead-packed column where it evaporated. The column was supplied with metered, dried, oil-free compressed air. The resultant vapor was directed into the turret of the chamber where it was mixed with filtered, conditioned air.
Chamber vapor concentrations were analytically determined by infrared spectroscopy at least 8 times during each exposure for each exposure level.
The samples were taken from a fixed reference position in the inhalation chamber. Chamber temperature and relative humidity were recorded twice per hour. Chamber air flow was set at 84 liters per minute. The nominal chamber concentration was calculated from the mass of chemical consumed and total air flow.
The concentration of background non-gaseous material was measured for the high-exposure and control chamber at least once each week during the exposure to ensure that the exposures were to a vapor and not an aerosol.

The vapor distribution of the test substance was not measured for positional variations within the inhalation chambers for this study. However, vapor distribution was determined during an LC50 study (see section Acute toxicity: inhalation) of the test substance about two months earlier using the same lot of the test material. For the samples taken from the nine cage positions in the LC50 study, the concentration (mean ± coefficient of variation) was 1300 ppm ± 7%, and from the fixed reference position was 1264 ppm ± 1%. The results indicated that positional effects were minimal and the fixed reference position selected provided a representative sample for chamber vapor concentration. The same fixed reference position was selected for the present study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber vapor concentrations were analytically determined by infrared spectroscopy at least 8 times during each exposure for each exposure level.
Duration of treatment / exposure:
Six hours per day
Frequency of treatment:
Five days per week for a total of 22 exposures over a 4-week period
Doses / concentrationsopen allclose all
Dose / conc.:
128 ppm (analytical)
Remarks:
Target concentration 120 ppm
Dose / conc.:
386 ppm (analytical)
Remarks:
Target concentration 380 ppm
Dose / conc.:
1 221 ppm (analytical)
Remarks:
Target concentration 1200 ppm
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
BODY WEIGHT:
Body weights were measured on days 0, 4, 7, 14, 21, and 28

CLINICAL OBSERVATIONS:
Rats visible through chamber windows were observed during exposure for changes in activity and compared subjectively to study controls. Immediately before and after each exposure, each rat was removed from its cage and examined by a trained technician. On the morning of necropsy, cage-side observations were conducted. Observations included, but were not limited to, examination of behavior patterns, motor activity, respiratory patters, hair, skin, eyes, feces, and urine. On weekends, rats were observed for mortality.

HEMATOLGY AND CLINICAL CHEMISTRY:
At the time of necropsy, blood was collected from the posterior vena cava while the rats were under C02 anesthesia. All hematology and clinical chemistry assays were conducted by the Animal Clinical Analysis Group, HAEL. Hematology tests included: hemoglobin concentration, hematocrit, red blood cell count, white blood cell count, differential white blood cell count, platelet count, red blood cell indices, prothrombin time, and examination of blood smears for cellular morphology. Clinical chemistry tests included: aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, urea nitrogen, glucose, creatinine, sorbitol dehydrogenase, total bilirubin, total protein, albumin, and albumin/globulin ratio.
Sacrifice and pathology:
PATHOLOGY:
Rats were fasted overnight, anesthetized with C02, and exsanguinated by severing the posterior vena cava after collecting blood for hematology and clinical chemistry examination. The lungs, liver, kidneys, adrenal glands, testes, spleen, and thymus were weighed. Paired organs were weighed together. Organ/body weight ratios were calculated. The following tissues were fixed in 10% buffered formalin: nasal passages, trachea, lungs, heart, esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, pancreas, liver, salivary glands, kidneys, urinary bladder, pituitary gland, adrenal glands, thyroid glands, parathyroid glands, thymus, spleen, mesenteric lymph nodes, bone marrow (femoral), brain, testes, epididymides, male accessory sex glands, ovaries, vagina, uterus, and Fallopian tubes. All tissues were examined microscopically from the control and high-exposure groups, target organs and gross lesions were examined from the middle- and low-exposure group.
Statistics:
Mean values were calculated for chamber concentration, chamber temperature, chamber relative humidity, body weight, organ weights, hematology, and clinical chemistry. Body weight, organ weight, hematology, and clinical chemistry data were evaluated using the following computer generated statistical tests to indicate statistical significance: Bartlett’s test (p ≤ 0.01), one-way analysis of variance (ANOVA) (p ≤ 0.05), and Duncan’s multiple range test (p≤0.05).
When the variance of means were not considered equal by the Bartlett’s test (p≤0.01), the data were evaluated using Wilk-Shapiro test for normality (p≤0.05) and the F-test for variance ratio (p≤ 0.05), after which the means of the test groups were compared individually to the control using a two-tailed t-test (p≤0.05). When the data were not considered normally distributed by the Wilk-Shapiro test (p≤0.05), the data were evaluated using Ansari-Bradley test: adjusting median (p≤0.05), after which the data were compared individually to the control using the Mann-Whitney test (p≤0.05)
W

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
During exposure, those animals near the chamber windows were observed for activity levels. Exposed animals appeared to be less active (lethargic) than control animals. Whether this was because of a depressant effect of the substance or because of the animals were avoiding the vapors due to the odor or other sensory input could not be determined. Following the second week of exposure, the animals in the low-concentration group no longer exhibited reduced activity levels during the exposure periods. Activity levels appeared normal in all groups shortly after exposures ceased, except for the animals in the high-exposure group on Days 1 and 2 when they remained less active during the post-exposure observation periods.

Other clinical signs that were seen during the post-exposure periods of the chamber included sialorrhea, discoloration of the facial haircoat, porphyrin tears, and excessive tearing. Sialorrhea was frequently observed post-exposure in one female rat from the high exposure group and in two female rats from the middle-exposure group. It was occasionally observed post-exposure in two female rats from the high-exposure group. Brown discoloration of the facial haircoat was observed periodically in most female rats from the high-exposure group. Single incidences of porphyrin tears and excessive tearing in high-exposure female rats, and a single incidence of porphyrin tears and two incidences of red discoloration of the facial haircoat in low-exposure male rats were also observed. Alopecia was not considered related to the exposure since it was not observed in rats from the high-exposure group and is frequently seen in untreated rats of this strain.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Mean body weights for the high-exposure male group were consistently slightly lower than that those of the control group. At study termination this difference was 5%. The body weight differences were not statistically significant when compared to the control group.
Mean body weights for all other groups were comparable to the control group throughout the study.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The mean prothrombin time was slightly lower (p ≤ 0.05) for all male rats exposed to the test substance when compared to the control group. Prothrombin time was comparable among the exposed groups with no evidence of a substance-concentration-related effect. The mean prothrombin time for female rats from all exposure groups comparable to the control group. The mean hematocrit value for female rats from the high-exposure group was slightly but significantly (p≤ 0.05) lower when compared to the control group. Mean hematocrit values for female rats from the middle- and low-exposure group, and for all male exposure groups were comparable to the control group. The mean white blood cell count for the female low-exposure group was significantly (p ≤ 0.05) higher than the control group, but was not increased relative to expected values. Mean white blood cell counts for all other exposure groups were comparable to the control groups. All other hematologic parameters were comparable to those of the control groups.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Mean glucose levels for the male rats from the high- and middle-exposure groups were significantly lower (p≤0.05) than those of the control group. However, compared to historical data on glucose values from male control animals, the glucose levels of all male rats in the middle-exposure group and all but one male rat in the high-exposure group were within the range of the historical control (70-201 mg/dL). The mean glucose level for the low-exposure male rats was also somewhat lower when compared to those of the study control group, but the difference was not statistically significant. The mean glucose levels for the female rats from all exposure groups were comparable to the control group. The mean sorbitol dehydrogenase level was significantly (p≤0.05) lower for the high-exposure female group and significantly higher for the middle-exposure female group when compared to the control group. In the absence of a substance concentration-dependent change the sorbitol dehydrogenase values were considered spurious. Mean sorbitol dehydrogenase levels for female rats from the low-exposure group and for male rats from all exposure groups were comparable to their respective control group. Mean total protein and albumin levels were slightly but significantly (p≤0.05) higher for the female rats from the high-exposure group when compared to the control group. Mean total protein and albumin levels for the female rats from the middle- and low-exposure groups and for male rats from all exposure groups were comparable to their respective control group. The mean urea nitrogen level for female rata from the middle-exposure group was significantly higher (p≤0.05) when compared to the control group. Since the mean urea nitrogen level for female rata from the high-exposure group was comparable to the control group, the finding was not considered exposure-related. Mean urea nitrogen levels for female rata from the low-exposure group and for male rats from all exposure groups were comparable to their respective control group. All other clinical chemistry parameters for rats from all exposure groups were comparable to those of the control groups.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The mean terminal body weight for male rats from the high-exposure group was slightly, although not significantly, lower than the control group weight. Mean terminal body weights for all other male and female exposure groups were comparable to the control group.

Mean absolute and relative liver weights were significantly (P≤0.05) increased for the high-exposure male and female groups. Although mean absolute (females) and relative (males and females) liver weights were significantly (P≤0.05) higher for the middle-exposure groups when compared to control groups, the differences were very slight. No statistically significant difference was noted between the mean absolute liver weight of the middle-exposure group males and the controls. Mean absolute and relative liver weights for the low-exposure male and female groups were comparable to controls.
The mean relative kidney weight, but not the mean absolute kidney weight, for the male high-exposure group was significantly higher (P≤0.05) than the control group. This difference was primarily due to the kidney weight of one rat. There were no statistically significant differences when this rat's weight data was eliminated from the analysis. The mean absolute and relative kidney weights for the middle- and low-exposure male groups and for all female exposure groups were comparable to their respective control group. Mean absolute and relative adrenal gland weights for female rats from the high-exposure group were higher, but not significantly (P≤0.05) different, from those of the control group. Mean absolute and relative adrenal gland weights for female rats from the middle- and low-exposure groups and for male rats from all exposure groups were comparable to their respective control group. The mean absolute spleen weight, but not the mean relative spleen weight, for the low-exposure male group, was higher (P≤0.05) than the control group spleen weight. This finding was considered spurious since mean absolute and relative spleen weights for the middle- and high-exposure male groups were comparable to the control group. All other organ weight measurements for rats from all exposure groups were comparable to the control groups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Pallor of the external surface of the kidneys was observed for three male rats from the high-, three male rats from the middle-, and four male rats from the low-exposure groups. Pallor of kidneys was not observed for female rats from any exposure group.
A small number of other potential gross changes were noted during the necropsy examination. These included enlarged kidney (males) enlarged adrenal gland (females), and edema of the mucosa of the glandular stomach. On follow-up examinations of these observations including organ weight and histopathology, none of these potential changes could be verified except that the kidney weight of one rat (male high-exposure group) was higher than that of all other rats in this study.
No other exposure-related lesions were detected for rats of either sex from any exposure group on necropsy examination.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Accumulation of hyalin droplets was observed intracellularly in the proximal convoluted tubules of the kidneys of all male rats, but the number and severity of droplets formed was greatest in the exposed rats. The severity of droplet formation appeared to increase with increasing concentrations. The droplets were morphologically similar to alpha-2µ-globulin droplets which have been described by others in the kidneys of male rats. Cytoplasmic vacuolation and basophilic cytoplasmic changes in the proximal convoluted tubules were seen in one or two male rats from the high- and middle-exposure groups, but not the low-exposure group. None of these findings were observed in female rats from any exposure level. Hypertrophy of hepatocytes was observed in four of five male rats and all five female rats from the high-exposure group, but not in rats from either of the other exposure groups.
Increased cytoplasmic vacuolization and hypertrophy of the cells in the zona fasciculata of the adrenal glands were observed in four female rats from the high-exposure group, while increased cytoplasmic vacuolization was present in two female rats from the middle-exposure group. The vacuoles were predominantly small or fine, but sometimes the cells were coarsely vacuolated. In a few cells, extensive accumulation of vacuoles resulted in the occurrence of large ballooned cells. In the control rats, the vacuoles were fine and involved a smaller number of cells. Adrenal glands from control rats typically are more heavily vacuolated than the controls in this study. The apparent increase in severity of this lesion in treated rats may have been due more to an unusually low severity of vacuolization in the concurrent control rats, rather than from a true increased incidence in the exposed rats. Therefore, this lesion may not represent a true treatment-related effect. No other exposure-related changes were observed for rats of either sex from any exposure group during the histopathology examinations.
Histopathological findings: neoplastic:
no effects observed

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEC
Effect level:
386 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
Dose descriptor:
NOEC
Effect level:
< 128 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Absolute and relative liver weights for males

Test Group 0/ M
0 ppm
Test Group 1/ M
128 ppm
Test Group 2/ M
386 ppm
Test Group 3/ M
1221 ppm
liver weight (g) 11.61 12.01 13.6 15.66
relative liver weight 3.27 3.35 3.84 4.8
rel liver increase (%) 100 102 117 147

Absolute and relative liver weights for females

Test Group 0/ F
0 ppm
Test Group 1/ F
128 ppm
Test Group 2/ F
386 ppm
Test Group 3/ F
1221 ppm
liver weight (g) 7.2 7.65 8.18 11.45
relative liver weight 3.27 3.42 3.72 5.14
rel liver increase (%) 100 105 114 157

Applicant's summary and conclusion