2,4-dimethylpentan-3-one

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Effects on fertility were examined in a whole-body inhalation GLP-compliant study performed according to OECD Test guideline 421 using male and female Wistar rats (BASF 2018). The NOAEL for fertility and reproductive performance was the highest tested concentration of 1000 ppm (nominal) / 4715 mg/m3 (measured) for the F0 parental rats. The NOAEL for general systemic toxicity was 100 ppm (nominal) / 417 mg/m3 (measured) based on adverse clinical findings and adverse effects on food consumption, body weight development as well as liver effects (high-dose only).

Link to relevant study records

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.

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

4 715 mg/m³

Study duration:

subacute

Species:

rat

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

To evaluate the developmental toxicity profile of the test substance 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 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 (~1414 mg/m³) and 1000 ppm (~4715 mg/m³). A concurrent control group was exposed to conditioned air.

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. 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). It was assessed as possibly treatment-related without any adverse consequences on parturition. The gestation index was 100.0% in all test groups.

In detail, the following was observed:

1000ppm

F0 PARENTAL ANIMALS

·        Clinical findings (e.g. salivation, lacrimation, reddish discolored fur, reduced attention, insufficient maternal care and plough nose-first into bedding) in both sexes during different periods of the study

·        Reduction in food consumption in both sexes (up to around -24% in males and -30% in females below concurrent control)

·        Body weight loss and decrease in body weight (change) in both sexes (up to around -8% in males and -10% in females below concurrent control)

·        Increased absolute (+33%) and relative (+44%) liver weights in male animals

·        Increased absolute (+21%) and relative (+27%) liver weights in female animals

·        Minimal to moderate centrilobular hepatocellular hypertrophy in 10/10 male and female animals

·        Alpha-2µ-nephropathy in 10/10 male animals with correlating increased absolute and relative kidney weights

·        Lower mean duration of gestation (22.0 / 22.1 / 22.0 / 21.4* [p ≤ 0.05] days in test groups 0-3, respectively) (assessed as possibly treatment-related without any adverse consequences on parturition since the apparently shorter duration of pregnancy had no consequence for the peri-/postnatal survival and postnatal development of the offspring).

F1 PUPS

·        Reduced nutritional condition in males and females during lactation

·        Delayed body weight development in males and females starting with the inhalation exposure on PND 4 (secondary effect due to direct exposure)

·        Increased percentage of male pups having nipple / areolae and mean number of nipples in male pups

·        No developmental effects in female pups

300 ppm

F0 PARENTAL ANIMALS

·        Clinical findings (e.g. salivation and reddish discolored fur) in both sexes during different periods of the study

·        Reduction in food consumption in both sexes (up to around -7% in males and -13% in females below concurrent control)

·        Decrease in body weight (change) of males

·        Alpha-2µ-nephropathy in 10/10 male animals with correlating increased absolute and relative kidney weights

F1 PUPS

·        Delayed body weight development in males and females starting with the inhalation exposure on PND 4 (secondary effect due to direct exposure)

·        No developmental effects in male and female pups

100 ppm

F0 PARENTAL ANIMALS

·        Alpha-2µ-nephropathy in 10/10 male animals with correlating increased absolute and relative kidney weights

F1 PUPS

·        No relevant findings

Effects on developmental toxicity

Description of key information

Effects on development were examined GLP-compliant according to OECD Testguideline 421 using male and female Wistar rats. The F1 pups showed clinical signs (a reduced nutritional condition during lactation) in the high-dose group and a delay in body weight development in the mid- and high-dose group from PND 4 onwards (start of exposure). In addition, high dose group male pups had an increased percentage of pups with nipples /areolae and an overall increased number of nipples.

The NOAEL for developmental toxicity in the offspring was 1000 ppm in females and 300 ppm in males due to retained areolae / nipples.

Clinical signs and effects on body weight gain are consistent with the effects observed for the parental animals and were therefore assessed as secondary (due to the direct exposure of the pups) and were not considered to represent specific developmental toxicity.

Link to relevant study records

Reference

Endpoint:

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

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29 Jul 2016

Deviations:

no

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:

TEST MATERIAL:
- 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

Remarks:

Crl:WI(Han)

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 rats (F0) were uniquely identified by ear tattoo. Pups: All pups were identified by skin tattoo on PND 1 and with picric acid markings on PND 10 or 11.
- Randomization: Animals were distributed according to 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 6 pm 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.

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 exposure to generate the respective concentrations.

Analytical methods of determination:
The samples were analyzed by calibrated online microGC.

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

Duration of treatment / exposure:

6 hours per day (exception: no exposure on the day of Functional Observation Battery/Motor activity)

Frequency of treatment:

On 7 consecutive days per week for the desired period of time (exception: no exposure on the days of FOB and MA)

Duration of test:

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.

Maternal 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))

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes

Examinations included:
- Number of implantations: Yes
- Number of early resorptions: Yes

Fetal examinations:

- 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.

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

Indices:

Reproductive toxicity indices: Male and female mating indices and male, female fertility indices, gestation index (for details see "any other information on materials and methods")

Offspring viability indeces: Viability index, survival index, anogenital index, percentage live males, percentage live females (for details see "any other information on materials and methods")

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 upt o 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.

Dermal irritation (if dermal study):

not examined

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 staistically 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.
Consitently 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-realted and adverse.

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:

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

Description (incidence and severity):

The significantly increased absolute and relative liver weights in male and female animals of test group 3 and the increased absolute and relative kidney weights in male animals of test groups 1 to 3 were regarded as treatment-related. The slightly increased absolute and relative kidney weights in female animals of test group 3 are assumed to be treatment-related, the absolute weights were above the range of historical control values. The marginally but significantly increased relative kidney weights in test group 2 females were assumed to be not related to the treatment, since only the relative weight parameters were affected, no female animal showed any correlating histopathological changes and the weight parameters were within the range of historical control values. The significantly increased relative weights of adrenal glands in test groups 2 and 3 males were regarded as treatment-related. Since the significantly increased relative weights of seminal vesicles in test group 3 males were above the range of historical control data, it cannot be excluded that the weight increase is treatment-related.
The significantly decreased weights of epididymides in test groups 1 and 2 were regareded as incidental, since there was no dose-response relationship and only the absolute weight parameters were significantly changed.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

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

Other effects:

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.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

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.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Dose descriptor:

NOAEL

Effect level:

99.1 ppm (analytical)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

see below
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Mean body weights of the male and female F1 pups of test group 3 (1000 ppm) were satistically significantly reduced from PND 7 (24% below control, both sexes combined) onwards until scheduled sacrifice on PND 13 (38% below control, both sexes combined). Furthermore, mean body weights of the mid-dose male and female F1 pups (300 ppm) were statistically significantly reduced on PND 13 (9% below control, both sexes combined). The mean pup body weight changes of the high- and mid-dose F1 pups were statistically significantly below the concurrent control values on PND 4-13 (test group 3: up to -54%; test group 2: -14%) and if calculated for the entire lactation period (PND 1-13, -45% in test group 3 and -12% in test group 2, both sexes combined, respectively).

Up to PND 4, body weight development was not affected in any of the test groups showing comparable mean body weights after birth. With start of inhalation exposure from PND 4 onwards, a delayed body weight development of test groups 2 and 3 pups was observed and assessed as secondary due to the combination of the direct exposure of the pups and the irritant effect of the test substance. It was not assessed as developmental effect.

One male runt was seen in test group 3. This single finding was not assessed as treatment-related.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

not examined

Visceral malformations:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Pup clinical observations:
A large number of pups in test group 3 showed a reduced nutritional condition during lactation period, i.e. 37 (out of 49) male pups and 35 (out of 57) female pups. This is most likely a secondary effect caused by the combination of insufficient maternal care of all high-dose females during the lactation and the exposure of the pups. It is assessed to be test substance-related.

The mean number of nipples was statistically significantly increased in test group 3 (1.10/1.30/1.58/2.66* (p ≤ 0.05) in test groups 0-3) and outside the historical control range (HCD, nipple number, range of 0.0-2.5). In addition, the percentage of high-dose male pups having nipple/areolae was also higher in group 3 (81% versus control: 49%), without statistical significance and barely within the historical control trange (HCD: range of 0.0-81.7%). However, a relation to treatment cannot be excluded. Therefore, it was assessed as treatment-related and adverse.

In test group 3 (1000 ppm) some findings occurred which were assessed as substance-related:
- a discolored liver in five male and five female pups,
- a discolored liver lobe in two male and two female pups,
- a discolored intestinal content in ten male and eight female pups.
Furthermore, some spontaneous findings were noted in a few F1 pups at gross necropsy, such as discolored testis, discolored thymus, dilated ureter, distended urinary bladder, dilated renal pelvis and post mortem autolysis. These findings occurred without a relation to dosing and/or some of them can be found in the historical control data at comparable incidences. None of the latter findings were considered to be related to the treatment.

Dose descriptor:

NOAEL

Effect level:

305.8 ppm (analytical)

Based on:

test mat.

Sex:

male

Basis for effect level:

fetal/pup body weight changes

other: retained areolae/nipples

Remarks on result:

other: Pups were directly exposed to the vapour of the substance. Effects observed may be the result of direct toxicity; as also parental animals showed effects on food consumption and body weight at this dose level.

Dose descriptor:

LOAEL

Effect level:

1 008.9 ppm (analytical)

Based on:

test mat.

Sex:

female

Basis for effect level:

fetal/pup body weight changes

other: reduced nutritional condition during lactation related to insufficient maternal care

Abnormalities:

not examined

Developmental effects observed:

yes

Lowest effective dose / conc.:

1 009 ppm (analytical)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Table 1: Absolute organ weights of parental animals

	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 1.1: Relative organ weights of parental animals

	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 2: Incidences and gradings of liver histopathological findings of parental animals

	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 3: Incidences and gradings of kidney histopathological findings of parental animals

	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

Table 4: Incidences and grading of adrenal glands of histopathological findings of parental animals

	Male animals
Test group (ppm)	0 (0)	1 (100)	2 (300)	3 (1000)
No. of animals	10	10	10	10
Vacuolation increased	2	1	1	6
- Grade 1	2	1		5
- Grade 2			1	1

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, the NOAEL for males for human health is 100 ppm.

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.

The NOAEL for developmental toxicity in the offspring was 1000 ppm in females and 300 ppm in males due to retained areolae/nipples. The F1 pups showed clinical signs (a reduced nutritional condition during lactation) in the high-dose group and a delay in body weight development in the mid- and high-dose group from PND 4 onwards (start of exposure). They were assessed as secondary (due to the direct exposure of the pups) and were not considered to be development effects.

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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

1 414 mg/m³

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

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, the NOAEL for males for human health is 100 ppm.

The NOAEL for developmental toxicity in the offspring was 1000 ppm in females and 300 ppm in males due to retained areolae/nipples. The F1 pups showed clinical signs (a reduced nutritional condition during lactation) in the high-dose group and a delay in body weight development in the mid- and high-dose group from PND 4 onwards (start of exposure). They were assessed as secondary (due to the direct exposure of the pups) and were not considered to be development effects.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available Reproductive / Developmental Toxicity Screening Test is reliable and - at the screening level - suitable for classification purposes under Regulation 1272/2008. No adverse effects on fertility were observed. Developmental effects (lower body weight gain and nipple retention) were considered to be the consequence of general toxicity and the direct exposure of the pups and to the reduced maternal care (dams showed a body weight loss of 13%). As a result the substance is not considered to be classified as a reproductive toxicant under Regulation (EC) No. 1272/2008, as amended for the seventh time in Regulation (EC) No. 2015/1221.