Hexafluoropropene

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 Feb 2018 to 30 Aug 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

28 July 2011

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline for the Testing of Chemicals 413, Subchronic Inhalation Toxicity: 90 Day Study

Version / remarks:

7 September 2009

Deviations:

yes

Remarks:

Only used for the inhalation exposure part.

GLP compliance:

yes

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:
- Premating exposure duration for parental (P0) animals: 10 weeks (7 days/week)
- Basis for dose level selection: The initial concentrations were selected in consultation with the sponsor and were based on the results of a preliminary dose-range finding study in rats with the same test item (Triskelion study 21058) and the prenatal development study in rats (Triskelion study 21069).
- Inclusion/exclusion of extension of Cohort 1B: Extension of Cohort 1B is not triggered, because the substance does not fulfil the requirements under Annex X column 2.
- Termination time for F2: Not applicable since the F2 generation is not included in this study.
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B: These Cohorts are not included.
- Inclusion/exclusion of developmental immunotoxicity Cohort 3: This Cohort is not included.
- Route of administration: Inhalation, this administration route was chosen because the test substance is a gas and humans may be exposed to the test substance by inhalation.
- Other considerations: Not applicable, standard OECD 443 protocol is used.

Test material

Test material information

Constituent 1

Reference substance name:

Hexafluoropropene

EC Number:

204-127-4

EC Name:

Hexafluoropropene

Cas Number:

116-15-4

Molecular formula:

C3F6

IUPAC Name:

1,1,2,3,3,3-hexafluoroprop-1-ene

Test material form:

gas under pressure: liquefied gas

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han) (SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: (P) males 5 weeks, (P) females 4 weeks
- Weight at study initiation: (P) group averages for males; 166.45 to 166.88 g. For females; 110.55 to 112.65 g. Body weight at allocation was within ± 20% of the mean weight for each sex.
- Housing: Animals were housed in Makrolon cages with a bedding of wood shavings (Lignocel, Rettenmaier & Söhne GmbH & Co, Rosenberg, Germany) and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment. After allocation, just before the start of the premating period, animals were housed 4 rats/cage (separated by sex). For mating, one male and one female were housed together, except during exposure. Mated females were housed individually in Makrolon cages, which were placed in another cage rack. After delivery, the cage containing the dam with litter was transferred to another cage rack. After allocation to the cohorts at weaning at or shortly after PN day 21, the F1 animals were housed in groups of 4 or 5 per sex/cage. During exposure periods the rats were individually housed in the exposure unit and did not have access to food or water. Immediately after each exposure, the animals returned to their home cages. During the mating period, animals were separated during exposure and were immediately returned to their mating cages after exposure.
- Diet: cereal-based (closed formula) rodent diet (VRF1 (FG)) from a commercial supplier (SDS Special Diets Services, Witham, England), ad libitum. Except during exposure and unless precluded by the performance of certain laboratory investigations.
- Water. Tap water, ad libitum. Except during exposure and unless precluded by the performance of certain laboratory investigations.
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 45 - 65
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
5 Feb 2018 to 30 Aug 2018

Administration / exposure

Route of administration:

inhalation: gas

Type of inhalation exposure (if applicable):

whole body

Vehicle:

clean air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The animals were exposed to the test atmosphere in 2.2 m3 whole body exposure units (based on the design by Hazleton Systems, Inc., Aberdeen, MD, USA). These chambers were constructed of stainless steel, with glass doors on two sides which allowed observation of the animals during exposure. The test atmosphere was introduced at the top and exhausted at the bottom of the chamber. During exposure, animals were housed individually in Type II Macrolon cages. A maximum number of 48 animals was exposed per whole body chamber. Animals were rotated at least twice weekly with respect to their position in the exposure chamber.
- Method of holding animals in test chamber: Not applicable, the animals were exposed via whole body exposure.
- Source and rate of air: Clean air available as a laboratory provided source of (non-pressurized) filtered air (HEPA filter).
- Method of conditioning air: The test atmospheres were generated by mixing a mass flow controlled (Bronkhorst Hi Tec, Ruurlo, The Netherlands) stream of gaseous test material with a controlled flow of clean air.
- Temperature, humidity, pressure in air chamber: The atmosphere in the chambers was maintained at a temperature of 22 ± 3 ºC and a relative humidity between 30 and 70%. Pressure in the air chamber was not reported.
- Air flow rate: The flow of test atmosphere was controlled using a constant volume valve and was measured in the exhaust of the exposure chamber using a KIMO air velocity sensor (type CTV110-AOD150; KIMO, Emerainville, France). The flow was continuously measured and recorded on a PC every minute using a Data acquisition system (MyriaNed, van Mierlo Ingenieursbureau BV, Eindhoven, the Netherlands).
- Air change rate: at least 10 air changes per hour
- Treatment of exhaust air: Not reported

TEST ATMOSPHERE
- Brief description of analytical method used: The actual concentration of the test material in the test atmospheres was measured by total carbon analysis (Sick Maihak GMS 810 EuroFID Total Hydrocarbon Analyzer; Sick Instruments Benelux, Hedel, the Netherlands; except during the period 5-14 February 2018 when the low concentration test atmosphere was analyzed using a Ratfisch RS55T, Munich, Germany). Test atmosphere samples were taken continuously from the exposure chamber at the animals’ breathing zone and were passed to the total carbon analyzer (TCA) through a sample line. The response of the analyzers was recorded on a PC at one minute intervals using a CAN transmitter (G. Lufft Mess- und Regeltechnik GmbH, 70719 Felbach, Germany). The responses of the analyzers were converted to concentrations by means of calibration graphs.
- Samples taken from breathing zone: yes

VEHICLE
- Composition of vehicle: Clean air

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days
- Proof of pregnancy: sperm in vaginal smear referred to as Day 0 of pregnancy
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYTICAL VERIFICATION
Prior to the first use for animal exposure, homogeneous distribution of the test material in the exposure chambers was confirmed by analysis of samples taken at five different locations in each exposure chamber (deviation of individual values from the mean of all five samples should not exceed 10%; this criterion was passed for all test atmospheres).
The actual concentration of the test material in the test atmospheres was measured by total carbon analysis (Sick Maihak GMS 810 EuroFID Total Hydrocarbon Analyzer; Sick Instruments Benelux, Hedel, the Netherlands; except during the period 5-14 February 2018 when the low concentration test atmosphere was analyzed using a Ratfisch RS55T, Munich, Germany). Test atmosphere samples were taken continuously from the exposure chamber at the animals’ breathing zone and were passed to the total carbon analyzer (TCA) through a sample line. When two exposure chambers per group were used (see paragraph 4.8), samples were taken intermittently from both chambers, with an automatic switch between chambers every ten minutes using a valve system and a digital timer (Omron H5CX). The response of the analyzers was recorded on a PC at one minute intervals using a Data acquisition system (MyriaNed, van Mierlo Ingenieursbureau BV, Eindhoven, the Netherlands). The responses of the analyzers were converted to concentrations by means of calibration graphs. For each exposure day, the mean concentration was calculated from the values determined every minute. The average concentration during exposure was corrected for the duration of the animals’ stay in the exposure chamber and the number of measurements during exposure.

CALIBRATION OF EQUIPMENT
Prior to the first exposure, the output of the flame ionization detector of each TCA was calibrated using gas sample bags. To this end, sample bags were filled with accurate (mass flow controlled) (Omron H5CX) and a valve system. The mass flow controller was calibrated using a volumetric flow meter (DryCal, Bios International Corporation, Butler, NJ, USA) at the flow settings used for filling of the sample bags. Three concentrations were thus prepared (at least in duplicate) – at approximately 80%, 100% and 120% of the target concentration of each group. A zero calibration was included for each TCA, using clean dry air only. Linear relations were found between the response of the analyzers and the concentration of the test material.
The calibrations were checked weekly during the study. To this end, gas sample bags were prepared at each target concentration as described above, and were subsequently analyzed by the TCA. If the measured concentration deviated more than 5% from the calculated concentration, the calibration check was repeated. If the deviation was more than 5% at the re-check, a complete re-calibration was carried out.

Duration of treatment / exposure:

Exposure during the different study phases:
Pre-mating period: Male and female animals were exposed daily for 10 weeks (7 days/week)
Mating period: Male and female animals were exposed daily (and returned to the mating cages after exposure)
Gestation period: Female animals were exposed daily from gestation day 0 up to and including gestation day 19
Lactation period: Females were exposed daily starting from lactation day 5 up to weaning on lactation day 21 and thereafter up to sacrifice
F1 offspring: Were exposed daily starting from one day after weaning (postnatal day 22) up to sacrifice

Frequency of treatment:

The animals were exposed to the test substance by whole body inhalation exposure for 6 hours per exposure day, seven days per week.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 ppm

Remarks:

(P) Group 2: low concentration

Doses / concentrations 2

Dose / conc.:

300 ppm

Remarks:

(P) Group 3: mid concentration

Doses / concentrations 3

Dose / conc.:

600 ppm

Remarks:

(P) Group 4: high concentration (initially the high concentration was 900 ppm, after 5 days of exposure the concentration was lowered to 600 ppm because of unforeseen mortality)

Doses / concentrations 4

Dose / conc.:

50 ppm

Remarks:

(F1) Group 2: low concentration (same low concentration as parent generation)

Doses / concentrations 5

Dose / conc.:

100 ppm

Remarks:

(F1) Group 3: mid concentration (initially the mid concentration was 300 ppm, after two days of exposure the concentration was lowered to 100 ppm because of unforeseen mortality)

Doses / concentrations 6

Dose / conc.:

200 ppm

Remarks:

(F1) Group 4: high concentration (initially the high concentration was 600 ppm, after two days of exposure the concentration was lowered to 200 ppm because of unforeseen mortality)

No. of animals per sex per dose:

Parent generation: 24/sex/group
Cohort 1A: One male and/or one female per litter were selected (20/sex/group)
Cohort 1B: One male and/or one female pup per litter were selected (20/sex/group)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The initial concentrations were selected in consultation with the sponsor and were based on the results of a preliminary dose-range finding study in rats with the same test item (Triskelion study 21058) and the prenatal development study in rats (Triskelion study 21069).

OTHER:
- Dose adjustment rationale: Since several animals of the high-concentration group (900 ppm) were found dead during exposure, the target concentration of the high-concentration group was lowered to 600 ppm for ethical reasons starting on 10 February 2018 (after five days of exposure to 900 ppm). The animals that died were replaced by surplus animals.
Since mortality was observed in the F1-animals (selected pups for Cohorts 1A and 1B) after 2 exposure days in mid- and high-concentration groups (300 and 600 ppm, respectively), the target concentrations of these exposure groups were lowered to 100 ppm for the mid-concentration group and to 200 ppm for the high-concentration group. This included both the exposure of F1 animals in Cohorts 1A and 1B as well as the exposure of the P0 females in the last week up to sacrifice. Dead F1-animals were replaced by spare animals, which were exposed from 3 or 6 June 2018 onwards.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
- Time schedule: Each animal was observed daily in the morning hours before exposure by cage-side observations and, if necessary, handled to detect signs of toxicity. The animals were also observed about halfway through the 6 hour exposure period, in particular to monitor any breathing abnormalities and restlessness. In the afternoon all animals were observed for post-exposure observations again.
- Cage side observations included all abnormalities, signs of ill health or reactions to treatment.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: prior to the first exposure and once weekly thereafter.
- Signs noted included, but were not limited to, changes in skin and fur, piloerection, changes in the eyes, gait (including posture), and presence of clonic or tonic movements, stereotypies and bizarre behaviour.

BODY WEIGHT:
- Time schedule for examinations: at least once during the acclimatization period for allocation (day -4) and at initiation of treatment (day 0). Subsequently, males were weighed weekly until sacrifice. Females were weighed once per week during the premating and mating period. Mated females were weighed on days 1, 7, 14 and 21 during presumed gestation and on day 0, 4, 7 and 14 and 21 of lactation. Non-mated females were weighed once per week after the mating period. Not-pregnant animals were erroneously weighted on day 105 of the study, the day before scheduled sacrifice of the non-mated and not-pregnant animals. In addition, the adult animals were weighed on their scheduled necropsy date in order to calculate the correct organ to body weight ratios.

FOOD CONSUMPTION:
- The food consumption was measured per cage over the same periods as the body weight were measured, except during the mating period when food intake was not recorded. Additionally, food consumption was not measured for non-mated females after the mating period and notpregnant animals after day 21 of lactation. The results are expressed in g per animal per day.

URINALYSIS:
- Time schedule: For females during the last week of the premating period and for males in the week before necropsy,
- Number of animals used: 10 animals/sex/group
- Metabolism cages used: Yes, one rat per cage, collection was done overnight for 16 hours.
- Fasted: During overnight fasting the animals were deprived of food. Water was freely available.
- Parameters checked: volume, density, appearance, pH, glucose, occult blood, ketones, protein, bilirubin, urobilinogen, red blood cells, white blood cells, epithelial cells, amorphous material, crystals, casts, bacteria, sperm cells, worm eggs

HAEMATOLOGY:
- Time schedule: at necropsy
- Number of animals used: 10 animals/sex/group
- Fasted: overnight (water was freely available)
- Anesthesia used: pentobarbital
- Parameters examined: See Table 1 in ‘Any other information on materials and methods incl. tables’.

CLINICAL CHEMISTRY:
- Time schedule: at necropsy
- Number of animals used: 10 animals/sex/group
- Fasted: overnight (water was freely available)
- Anesthesia used: pentobarbital
- Parameters examined: See Table 2 in ‘Any other information on materials and methods incl. tables’.

OTHER: T4 AND TSH HORMONE DETERMINATIONS:
- Time schedule: at necropsy
- Number of animals used: 10 animals/sex/group
- Fasted: overnight (water was freely available)
- Anesthesia used: pentobarbital
- Analysis for T4 and TSH hormones were performed with commercially available ELISA kits of Cloud-Clone Corp (kit CEA463Ra for TSH and kit CEA452Ge for T4, respectively). The ELISA was performed according to a validated method based on the manufacturer’s protocol.

Oestrous cyclicity (parental animals):

Vaginal smears to evaluate the estrous cycle length and normality were made daily during the last three weeks of the premating period and during the mating period until confirmation of mating or end of 2 weeks mating period (for not mated females). Smears were made, stained and examined in all females.
An additional vaginal smear was made at the day of sacrifice. The smears made at the day of sacrifice were stained, but the results of the microscopic examination of the uterus and vagina did not give any indication for further examination of these smears.

Sperm parameters (parental animals):

Parameters examined in male parental generation:
At scheduled necropsy, epididymal sperm was derived from the left cauda epididymis of all P0 males of each group. Sperm motility and, after sonification and DNA-staining, the cauda epididymal sperm reserves (sperm count) were measured for males of all groups. In addition, a smear of the sperm solution was prepared and stained for males of all groups, but only the smears of the control and the high-dose males were examined microscopically for morphology.
Following DNA-staining, the homogenization-resistant sperm heads were enumerated using the IVOS. The daily sperm production was calculated. Sperm counts were only conducted on the control and the high-dose males.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible). A number of 10 pups/sex/group was allocated to a spare group. Excess pups were sacrificed at culling by decapitation in random order in such a sequence that the average time of killing was approximately the same for each group (in order to account for possible diurnal variation). Blood was collected per litter and stored in a freezer at ≤-18 °C.

PARAMETERS EXAMINED
The following parameters were examined in pups that were not selected for the cohorts (non-selected pups).
- Litter evaluation: The total litter size and numbers of each sex as well as the number of stillbirths, live- and dead pups and grossly malformed pups were evaluated on days 0, 4, 7, 14 and 21 of lactation. The pups were individually observed for clinical signs and weighed on days 0, 4, 7, 14 and 21 of lactation. Mean pup weight was calculated per sex and for both sexes combined.
- Anogenital distance: At day lactation day 4 the anogenital distance (AGD) was measured of each pup before culling of the litter. The AGD is reported as such and corrected for body weight and the cube root of body weight.
- Nipple retention in male pups: On postnatal day 13 and postnatal day 21 all surviving male pups were quantitatively examined (number of nipples/areolae per pup will be counted) for the presence of nipples and/or areolae.

The following parameters were examined in pups that were selected for the cohorts (1A and 1B).
- General clinical observations: Each animal was observed daily in the morning hours before exposure by cage-side observations and, if necessary, handled to detect signs of toxicity (see Table 9 in 'Any other information on materials and methods incl. tables'). The animals were also observed about halfway through the 6 hour exposure period, in particular to monitor any breathing abnormalities and restlessness. In the afternoon all animals were observed for post-exposure observations again. On non-exposure days all cages were checked again in the afternoon for dead or moribund animals to minimize loss of animals from the study. All abnormalities, signs of ill health or reactions to treatment were recorded. Any animal showing signs of severe debility or intoxication, particularly if death appears imminent, was humanely killed to prevent loss of tissues by cannibalism or autolytic degeneration.
- Detailed clinical observations: Once weekly all animals were subjected to detailed clinical observations, e.g. during weighing.
- Body weight: Body weights of all male and female animals were recorded weekly. Additionally body weight was recorded on the day of attainment of vaginal patency or balano-preputial separation
- Food consumption: The food in the feeders was topped up when necessary and refreshed weekly. Except for during the mating period, the food consumption was measured per cage over the same periods as the body weight was measured. The results are expressed in g per animal per day.
- Sexual maturation: In males, preputial separation was recorded from PND 39 onwards. For females, vaginal opening was recorded from PND 31 onwards. Daily checks were discontinued after 95% of the animals reached sexual maturation. The day the animals were scored positive for these landmarks, the body weight was recorded.
- Estrous cycle (Cohort 1A): From the onset of vaginal opening a daily vaginal smear was made for all Cohort 1A females for 7 consecutive days to determine the first estrous stage. From animals of which no first estrous cycle was determined within 7 days, vaginal smears were made for an additional 7 days to determine the first estrous stage. In addition vaginal smears to evaluate the estrous cycle length and normality were made daily for about 3 weeks before sacrifice for Cohort 1A females. Smears were stained and evaluated for all Cohort 1A females.
- Urinalysis (Cohort 1A): Before necropsy, ten Cohort 1A animals/sex were kept in stainless-steel metabolism cages (one rat per cage) and urine was collected in glass tubes overnight for 16 hours. During this time the animals were deprived of food. Water was freely available. Parameters checked included: volume, density, appearance, pH, glucose, occult blood, ketones, protein, bilirubin, urobilinogen, red blood cells, white blood cells, epithelial cells, amorphous material, crystals, casts, bacteria, sperm cells, worm eggs
- Terminal body weight (Cohort 1A and 1B): Terminal body weight was determined for all Cohort 1A and 1B animals prior to scheduled necropsy.
- Haematology (Cohort 1A): During necropsy, blood was taken from the aorta under intraperitoneal pentobarbital anaesthesia from 10 overnight fasted Cohort 1A animals/sex/group. The parameters listed in Table 1 in ‘Any other information on materials and methods incl. tables’ were examined.
- Clinical chemistry (Cohort 1A): During necropsy, blood was taken from the aorta under intraperitoneal pentobarbital anaesthesia from 10 overnight fasted Cohort 1A animals/sex/group. Clinical chemistry data were collected from the same animals used for haematology and hormone analysis. The parameters listed in Table 2 in ‘Any other information on materials and methods incl. tables’ were examined.
- T4 and TSH hormone determinations (Cohort 1A): During necropsy blood was taken from the aorta under intraperitoneal pentobarbital anaesthesia from 10 overnight fasted Cohort 1A animals/sex/group (same animals used for haematology and clinical chemistry) for determination of TSH and T4 hormone levels in serum. Analysis was performed with commercially available ELISA kits of Cloud-Clone Corp (kit CEA463Ra for TSH and kit CEA452Ge for T4, respectively). The ELISA was performed according to a validated method based on the manufacturer’s protocol.
- At scheduled necropsy, epididymal sperm was derived from the left cauda epididymis of all male Cohort 1A animals of each group. Sperm motility and, after sonification and DNA-staining, the cauda epididymal sperm reserves (sperm count) were measured for males of all groups,In addition, a smear of the sperm solution was prepared and stained for males of all groups, but only the smears of the control and high-dose males were examined microscopically for morphology
- Following DNA-staining, the homogenization-resistant sperm heads were enumerated using the IVOS. The daily sperm production was calculated. Sperm counts were only conducted on the control and the high-dose males.
- At sacrifice, splenic lymphocyte subpopulation analysis was performed in 10 cohort 1A animals/sex/group (in the high-dose group 9 males were analysed). A single cell suspension was prepared by dissociation of the spleen. After washing, erythrocyte lysing and filtering the cell suspension, the cells were counted, viability was checked and the cells were resuspended at a density of 10million live cells/ml before flow cytometric analysis (results in raw data file). For the flow cytometric analysis of the splenocytes, the cells were incubated with a specific antibody cocktail preceded by Fc block to minimize non-specific antibody binding to surface Fc receptors. The antibody cocktail consisted of anti-CD45RA, CD161, CD3, CD4 and CD8a antibodies in order to determine the frequency of CD4+ and CD8+ T lymphocytes (i.e. T-helper and T-cytotoxic cells), B lymphocytes and natural killer cells by flow cytometry (FACS CANTO II). Addition of FVS dye enabled discriminating living and dead cells. Flowcytometric analysis was performed using a FACSCanto™ II flowcytometer and analyzed using BD FACSDiva™ 8.01 software. In addition to the absolute numbers of above-mentioned cells, frequencies were calculated as follows:
- % total T cells = (number of T cells / number of live cells) x 100%
- % T-helper cells = (number of T-helper cells / number of T cells) x 100%
- % T-cytotoxic cells = (number of T-cytotoxic cells / number of T cells) x 100%
- % B cells = (number of B cells / number of live cells) x 100%
- % NK cells = (number of NK cells / number of live cells) x 100%

GROSS EXAMINATION OF DEAD PUPS:
Necropsy was performed on stillborn pups and pups dying or sacrificed in a moribund state during the study and macroscopic abnormalities were recorded.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were sacrificed after the mating period.
- Maternal animals: All surviving animals were sacrificed shortly after weaning of the F1-pups.
All females that did not mate with their assigned male and all non-pregnant females were sacrificed at least one week after the end of the gestation period (day 106 of the study).

GROSS NECROPSY
At scheduled necropsy, all surviving male and female animals were fasted overnight and sacrificed by exsanguination from the abdominal aorta whilst under intraperitoneal pentobarbital anaesthesia and then examined grossly for pathological changes. The animals were sacrificed in random order in such a sequence that the average time of killing was approximately the same for each group. A necropsy was also performed on animals that died intercurrently.
- The organs examined are listed in Table 3 in ‘Any other information on materials and methods incl. tables’.

HISTOPATHOLOGY / ORGAN WEIGHTS
Tissues for microscopic examination were embedded in paraffin wax, sectioned and stained with haematoxylin and eosin, except for sections of the testes which were stained with PAS. Microscopic examination was performed on the collected organs of all animals of the control (group 1) and high concentration group (group 4). Since treatment-related effects were observed in the high-concentration group, evaluation of the following tissues/organs was extended to the intermediate-dose groups (2 and 3) in consultation with the sponsor: heart (females only), thymus (males only) and kidneys (both sexes). Furthermore, organs showing gross lesions of animals of all groups were microscopically examined. In addition, the reproductive organs of males that failed to sire (did not mate or mated females were not pregnant) and females that were non-mated or non-pregnant of the low- and mid-dose groups were microscopically examined.
- The tissues indicated in Table 3 in ‘Any other information on materials and methods incl. tables’ were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- Grossly malformed pups were sacrificed and examined.
- The F1 pups that were not selected for the cohorts (non-selected pups) at weaning were sacrificed on PND 21 or shortly thereafter.
- The Cohort 1A and 1B pups were sacrificed on scheduled necropsy.

GROSS NECROPSY
All surviving male and female animals were sacrificed (after fasting overnight) by exsanguination from the abdominal aorta whilst under intraperitoneal pentobarbital anaesthesia at necropsy and then examined grossly for pathological changes. Necropsy was also performed on animals that died intercurrently or were sacrificed in a moribund state and assessed by gross pathology (macroscopy).
Pups that were not selected for cohorts, as spares, and not selected for blood sampling were sacrificed on PND 21 or shortly thereafter by decapitation while under CO2/O2 anaesthesia after gross macroscopic examination.

HISTOPATHOLOGY / ORGAN WEIGTHS
At scheduled necropsy, samples of the listed tissues and organs of all animals were weighed and/or preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde; except for the testes, which were preserved in Bouin’s fixative.
- F1 pups: The organs/tissues listed in Table 4 in ‘Any other information on materials and methods incl. tables’ field of maximally 10 non-selected pups/sex/group at weaning were weighed and/or were subjected to microscopic examination.
- Cohort 1A animals: The organs/tissues listed in Table 5 in ‘Any other information on materials and methods incl. tables’ field of all Cohort 1A animals were weighed and/or were subjected to microscopic examination.
- Cohort 1B animals: The organs/tissues listed in Table 6 in ‘Any other information on materials and methods incl. tables’ field of all Cohort 1B animals were weighed and/or were subjected to microscopic examination.

OTHER:
- Since treatment-related effects were observed in the high-concentration group, evaluation of the heart (females), thymus (males) and kidneys (both sexes) was extended to the intermediate-dose groups (2 and 3) in consultation with the sponsor:
- Organs showing gross lesions of animals of all groups were microscopically examined. In addition, the reproductive organs of males that failed to sire (did not mate or mated females were not pregnant) and females that were non-mated or non-pregnant of the low- and mid-dose groups were microscopically examined.

Statistics:

The statistical procedures for analysis of the various parameters of this study are described in Table 7 in "Any other information on materials and methods incl. tables'. Other statistical tests may be performed when considered appropriate.

Reproductive indices:

See Table 8 in 'Any other information on materials and methods incl. tables'.

Offspring viability indices:

See Table 8 in 'Any other information on materials and methods incl. tables'.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

A general red appearance of the skin, skin piloerection, hunched posture, muscle weakness and ear erythema were observed in males of the high-concentration group between day 3 and 4 of exposure (900 ppm target concentration). These findings were most probably related to treatment, since these findings were transient and disappeared after lowering the high concentration to 600 ppm on day 6 of the study.
Light or white coloured discoloration of the lower incisors was observed pre- and post-exposure in several males of the mid- (300 ppm) and all males of the high-concentration (600 ppm) groups. Discolouration of the lower incisors was observed in the mid-concentration group from day 43 and in the high-concentration group from day 18 onwards. On day 32 of the study, light coloured discoloration of the upper incisors was observed in all males of the high-concentration group (600 ppm).
General pre- and post-exposure signs in males observed included skin observations (sparsely haired areas and encrustations), kink tail and eyes discharge. These signs are common in this strain and based on the distribution amongst the groups not related to treatment.

During premating, a general red appearance of the skin, skin piloerection, hunched posture, muscle weakness and ear erythema were observed in females of the high-concentration group between day 3 and 4 of exposure (900 ppm target concentration). These findings were most probably related to treatment, since these findings were transient and disappeared after lowering the high concentration to 600 ppm on day 6 of the study.
During premating, gestation and lactation, light or white coloured discoloration of the lower incisors was observed pre- and post-exposure in a few females of the mid- (300 ppm) and all females of the high-concentration (600 ppm) groups. Discolouration of the lower incisors was observed in the mid-concentration group from day 31 onwards and in the high-concentration group from day 18 onwards. On day 32 of the study, light coloured discoloration of the upper incisors was observed in 22 females of the high-concentration group (600 ppm).
General pre- and post-exposure signs in females observed during premating, gestation and lactation included skin observations (sparsely haired areas, wounds, encrustations, piloerection and soiled fur), tiptoe walking, nose encrustations, vaginal blood, malocclusion of incisors, nodule in abdomen and kink tail. These signs are common in this strain and based on the distribution amongst the groups not related to treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Three females of the high-concentration group were found dead and (partly) cannibalized between day 3-5 after exposure. Pre- and post-exposure signs of these animals included skin piloerection, nose encrustation(s) and hunched posture. These animals were replaced by spare animals on the day that these animals were found dead and the target concentration of the high-concentration group was decreased from 900 ppm to 600 ppm, starting on day 6 of the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Premating period: Mean body weights were statistically significantly decreased in males of the mid-(300 ppm) and high-concentration (600 ppm) groups from day 7 of exposure onwards compared to the control group. Body weight changes of the male animals of the mid- and high concentration groups were statistically significantly lower than controls between premating days 0-7, 28-35 and 49-56. For males of the high-concentration group body weight changes were also statistically significantly decreased compared to the control group between premating days 35-42, 56-63 and 77-84. In females, mean body weights were statistically significantly decreased in the high concentration (600 ppm) group compared to the control group from day 7 of exposure onwards. Body weight changes were statistically significantly decreased in females of the mid- (300 ppm) and high-concentration (600 ppm) groups between premating days 0-7 and 28- 35 compared to the control group. Additionally, a statistically significant higher body weight change was observed in females of the high-concentration group between days 21- 28.

Gestation period: Mean body weights of the pregnant animals of the high-concentration group (600 ppm) were statistically significantly lower than controls on gestation days 1 and 21, whereas body weight changes of the pregnant animals of the high-dose group were comparable to the controls during the gestation period, except for a decreased body weight change in high concentration females (18% decreased compared to controls, although not statistically significant).

Lactation period: Mean body weights of the lactating animals of the high-concentration group (600 ppm) were statistically significantly lower than of control animals on day 0 of lactation only. Body weight change of the animals of the mid-concentration group (300 ppm) was statistically significantly higher compared to the control group between lactation days 0-4. Body weight change of the animals of the high-concentration group was statistically significantly higher compared to the control group between lactation days 14-21.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Premating period: Food consumption of male and female animals of the high-concentration group (600 ppm) was statistically significantly lower as compared to the control group during almost the entire premating period. Food consumption of the male animals of the mid concentration group (300 ppm) was statistically significantly decreased compared to the control group between premating days 0-28 and 42-56.

Gestation period: Food consumption of the pregnant animals was comparable between all groups during the entire gestation period.

Lactation period: Food consumption of the lactating animals of the high-concentration group (600 ppm) was statistically significantly decreased compared to the control animals between lactation days 7- 21.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Red blood cell and coagulation parameters: Prothrombin time was statistically significantly decreased in males of the mid- (300 ppm) and high-concentration (600 ppm) groups. Since no concentration-relationship could be established, this effect was considered as chance finding and not related to exposure. In female animals, no statistically significant effects were observed on any of the red blood cell and coagulation parameters.
White blood cell parameters: In male animals of the high-concentration group (600 ppm), the absolute number of eosinophils was statistically significantly decreased as compared to the control group. Since no concentration-relationship could be established, this effect was considered as chance finding and not related to exposure. In female animals, no statistically significant effects were observed on any of the white blood cell parameters.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant differences were observed in clinical chemistry parameters compared to the control group:
- ALAT was increased in the males of the mid-concentration group (300 ppm), which was considered as chance finding due to the lack of a concentration response.
- Creatinine was decreased in males and females of the mid-concentration group (300 ppm). Since no concentration-response was observed, this was considered as chance finding.
- Cholesterol was increased in males of the high-concentration group (600 ppm; 37.5% increase compared to control)
- Urea was decreased in males of the low-concentration group and increased in males of the high-concentration group (600 ppm; 68% increase compared to controls).
- Potassium was increased in males of the mid- (300 ppm) and high-concentration (600 ppm) groups (16% and 14% increase, respectively, compared to control).
- Total protein was decreased in females of the mid- (300 ppm) and high-concentration (600 ppm) groups (6% and 5% decrease, respectively, compared to control).

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Volume and density: The renal concentration test showed a slight, though statistically significant decrease in specific gravity in males of the mid- (300 ppm) and high-concentration (600 ppm) groups, associated with a statistically significant higher urinary volume in low-, mid- and high-concentration males (45%, 107% and 119% increase compared to controls).

Semi-quantitative observations: Semi-quantitative (dipstick) urinary measurements showed a statistically significant increase in the incidence of urinary ketones in females of the high-concentration (600 ppm) group compared to the control group, mainly due to the low value of the control group. The urinary pH was statistically significantly increased in males of the mid-concentration group (300 ppm) and decreased in males of the high-concentration group (600 ppm). Since no concentration-response was observed, this was considered as chance finding.

Microscopic observations: Microscopic examination of the urinary sediment showed statistically significant increase in epithelial cells in males of the mid- (300 ppm) and high-concentration (600 ppm) groups. In males of the high-concentration group, the presence of amorph material was statistically significantly increased and the score for crystals was statistically significantly decreased.

Behaviour (functional findings):

not examined

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):

Microscopic examination of the sampled organs and tissues revealed treatment-related histopathological changes in the heart, kidneys and thymus.
Heart: The changes in the heart were observed in females only and comprised minimal to moderate ventricular muscle degeneration. The occurrence of this finding in 10/24 females in the 600 ppm group was considered related to treatment.
Kidney: The histopathological changes in the kidneys were observed in males as well as females and comprised minimal to moderate tubular dilatation, mononuclear inflammation, proteinaceous casts and/or basophilic tubules. Because of the clearly increased incidences of these findings in the 300 ppm animals (up to 22/24) and the 600 ppm animals (up to 24/24) they were considered related to treatment. The proteinaceous casts are indicative for protein loss from the kidneys, which may have contributed to the weight loss of the animals.
Thymus: In several treated males the thymus showed cortical atrophy. This finding may be secondary to other effects of the test substance, such as an increased demand of inflammatory cells or stress related involution, rather than a specific effect targeted at the thymus. The statistically significantly increased incidence of cortical atrophy in 5/24 of 50 ppm males is probably irrelevant because of the lack of a concentration-effect relationship.
The other organs and tissues did not reveal treatment related histopathological changes. The histopathological changes observed were about equally distributed amongst the different treatment groups or occurred in one or a few animals only. They are common findings in rats of this strain and age or occurred as individual chance findings. Therefore, they were not considered to be related to treatment.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The statistically significantly decreased incidence of follicular hyperplasia in the thyroid of the high 600 ppm males was merely caused by a relatively high incidence in the control animals and not related to treatment, since this is a common background finding.

Other effects:

no effects observed

Description (incidence and severity):

HORMONE DETERMINATIONS: Exposure to the test substance via inhalation did not affect the concentrations of TSH and T4 hormones in sera of male and female animals of the P0-generation.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Evaluation of estrous cyclicity resulted in a statistically significant decrease in the number of complete cycles per animal in 21 days in females of the high-concentration group (600 ppm) compared to the control group. Since no effect was observed on fertility, this difference was considered as chance finding.

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

Epididymal sperm motility: No statistically significant differences were observed on the epidydimal sperm motility parameters between the control and the treatment groups.
Epididymal sperm count: No statistically significant differences were observed on the epidydimal sperm count parameters between the control and the treatment groups.
Epididymal sperm morphology: Except for a statistically significant increase in the number of sperm cell with an amorphous hook in males of the high-concentration group (600 ppm), no effects were observed on the epididymal sperm morphology parameters between the control and high-concentration group. This difference, as the only affected epididymal sperm morphology parameter, was considered of no toxicological relevance.
Testicular sperm count: No statistically significant differences were observed on any of the testicular sperm count parameters between the control and high-concentration group.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

See Table 1-5 in 'Any other information on results incl. tables'.
Fertility and reproductive performance were not affected by exposure in any of the groups. Except for two females of the high-concentration group (600 ppm), all females that were placed with males were mated within 7 days. Consequently, the male and female mating indices and the pre-coital time were comparable among the groups.
Two females of the control group (0 ppm), 2 females of the mid concentration group (300 ppm) and three females of the high-concentration group (600 ppm) turned out to be not pregnant. This resulted in (male and female) fertility indices of 91.7%, 100%, 91.7% and 79.2% in the control-, low-, mid-, and high-concentration groups, respectively. The number of males that became sire and the female fecundity index were comparable among the groups.
One female of the low-concentration group (50 ppm) delivered a litter with stillborn pups, while there were no dams delivering a litter with only stillborn pups. Consequently, the live birth- and stillborn indices were comparable among the groups. The mean number of live and dead pups delivered and the mean number of live pups delivered were comparable among the groups.
The sex ratio’s on post-natal day 0 and 21, mean number of implantation sites, the number of lost implantations and post-implantation loss were also comparable among the groups.
No pregnant females died during gestation and except for one female (only implants) of the high-concentration group (600 ppm), all females completed delivery and delivered liveborn pups, resulting in comparable gestation indices between the groups. The duration of gestation (gestation days) was comparable among the groups

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEC

Remarks:

Systemic toxicity

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

urinalysis

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEC

Remarks:

Reproductive toxicity

Effect level:

>= 600 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

1

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 ppm

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

2

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 ppm

System:

cardiovascular

Organ:

heart

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

F1 GENERATION: No effects observed during the lactation period.

COHORT 1A: Effects observed, treatment related: Light coloured discoloration of the lower and/or upper incisors was observed in several males and females of the high-concentration group (200 ppm). Discolouration was observed from day 25 onwards.
During the first weeks of exposure, several animals of the mid- (100 ppm) and high concentration group (200 ppm) showed piloerection and/or respiration dyspnoea, and one animal a hunched posture. Two animals of the mid-concentration group (100 ppm) showed muscle weakness on one day after start of exposure. These clinical observations during the first weeks of exposure were transient and disappeared at the start of Cohort 1A (day 0).
General pre- and post-exposure signs in males and females observed between day -11 until sacrifice included skin observations (sparsely haired areas and encrustations), kink tail and tip of tail missing. These signs are common in this strain and based on the distribution amongst the groups not related to treatment.
Light coloured lower and/or upper incisors were observed in several male and female animals of the high-concentration group from week 4 onwards. Other findings were incidental and not related to exposure

COHORT 1B: Effects observed, treatment related: Light coloured discoloration of the lower and upper incisors was observed in several males and females of the high-concentration group (200 ppm). Discolouration was observed from day 49 onwards.
During the first weeks of exposure, several animals of the high-concentration group (200 ppm) showed piloerection and one animal a hunched posture and eyes blepharospasm. A few animals of the mid-concentration group (100 ppm) showed piloerection during the first days of exposure. These clinical observations during the first weeks of exposure were transient and were disappeared at day 12 of Cohort 1B.
General pre- and post-exposure signs in males and females observed between day -11 until sacrifice included skin observations (sparsely haired areas and encrustations), kink tail and tip of tail missing. One animal of the mid-concentration group (100 ppm) showed a local swelling of the head from study day 38 to 77. These signs are common in this strain and based on the distribution amongst the groups not related to treatment.
Light coloured lower and/or upper incisors were observed in several male and female animals of the high-concentration group from week 7 onwards. Other findings were incidental and not related to exposure.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

F1 GENERATION: Mortality observed, not treatment related: The number of pups that were born alive but that died or were missing and/or cannibalized after birth on postnatal day 0 to culling on postnatal day 4 accounted 3, 3, 3 and 4 in the control, low-, mid- and high-concentration groups, respectively. After culling, 1 pup of the mid concentration group (300 ppm) was lost and no litters were lost entirely. Consequently, the viability indices of post-natal days 0-4 and 4-21 were comparable among the groups.

COHORT 1A AND 1B: Mortality observed, treatment related: Mortality was observed in the selected animals for Cohorts 1A and 1B after 2-4 exposure days in mid- and high-concentration groups (300 and 600 ppm, respectively). Pre- and post-exposure signs of these animals included dyspnoea, muscle weakness, hunched posture, lethargic, tremors, skin piloerection, eyes blepharospasm and eyes encrustations. Due to animal ethical reasons, the target concentrations of these exposure groups were lowered to 100 ppm for the mid-concentration group and to 200 ppm for the high-concentration group. All animals that died were replaced by spare animals before start of Cohort 1A and Cohort 1B.
For the high-concentration group animals in Cohort 1B, it was not possible to replace all animals that died during the first week of exposure with spare animals. This resulted in a group size of 14 males and 15 females in the high-concentration group of Cohort 1B.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

F1 GENERATION: Effects observed, treatment related: On postnatal days 0, 4, 7, 14 and 21, mean weights of the male pups of the mid- (300 ppm) and high-concentration (600 ppm) groups were statistically significantly decreased compared to the corresponding weights of male pups of the control group, except for the pups of the high concentration group on postnatal day 4. Mean weight of the female pups was decreased on postnatal day 7 in the high-concentration group and on postnatal days 14 and 21 in the mid- and high-concentration groups compared to the corresponding weight of female pups of the control group.
In addition, the combined weight of male and female pups were also statistically significantly lower in the mid- (postnatal days 14 and 21) and high-concentration groups (postnatal days 7, 14 and 21) compared to the corresponding weights of pups of the control group.

COHORT 1A: Effects observed, treatment related: Mean body weight of the male and female animals of the high-concentration group (200 ppm) was statistically significantly decreased at all timepoints compared to the control group (approximately decreased by 30% and 28% for males and females, respectively, at Day 0 with a decrease in body weight loss at most timepoints, through Day 69, where, at Day 69, body weight was approximately decreased by 12% for males and 10% for females (i.e. body weight loss was reversing)). However, a statistically significant decrease in mean body weight change was only observed in males of the high-concentration group between day 0-7 and 14-21. In males and females of the mid-concentration group (100 ppm), mean body weight was lower than the body weights of the control animals, reaching the level of statistical significance on days 0, 7, 14, 21 and 28 (approximately decreased by 18% and 17% for males and females, respectively, at Day 0 with a decrease in body weight loss at most timepoints, through Day 69, where, at Day 69, body weight was approximately decreased by 2% for males and 3% for females (i.e. body weight loss was reversing)). However, no decrease in mean body weight change was observed at any timepoint for either mid-dose male or female animals. No effects were observed on body weight and body weight changes in the low-concentration group (50 ppm).

COHORT 1B: Effects observed, treatment related: Mean body weight of the male and female animals of the high-concentration group (200 ppm) was statistically significantly decreased at all timepoints compared to the control group (approximately decreased by 35% and 30% for males and females, respectively, at Day 0 with a decrease in body weight loss at most timepoints, through Day 76, where, at Day 79, body weight was approximately decreased by 15% for males and 12% for females (i.e. body weight loss was reversing)). However, whereas a statistically significant decrease in mean body weight change was only observed in males of the high-concentration group between day 0-7 and 14-21. In males and females of the mid-concentration group (100 ppm), mean body weight was lower than the body weights of the control animals, reaching the level of statistical significance on days 0, 7, 14, 21 and 28 (approximately decreased by 19% and 17% for males and females, respectively, at Day 0, with a decrease in body weight loss at most timepoints, through Day 28, where, at Day 28, body weight was approximately decreased by 6% for both males and females (i.e. body weight loss was reversing)), whereas statistically significant increases in body weight change were sporadically-observed in males (e.g. between days 28-35, 56-63 and 70-76) and in females only between days 0-7. In females of the low-concentration group (50 ppm), body weight was also statistically significantly decreased on days 7 and 14.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

F1 GENERATION: Not examined

COHORT 1A: Effects observed, treatment related: Food consumption of male and female animals of the high-concentration group (200 ppm) was statistically significantly decreased compared to the control group between day 0 and 28 for males and day 0 and 35 for females. This observed (both early and temporary) decrease in food consumption may be directly contributing to the observed decreases in male and female body weight in the high-dose animals. Food consumption of the male and females animals of the mid-concentration group (100 ppm) was statistically significantly decreased compared to the control group between day 0-7. Again, this observed (both early and temporary) decrease in food consumption may be directly contributing to the observed decreases in male and female body weight in the mid-dose animals observed through Day 28.

COHORT 1B: Effects observed, treatment related: Food consumption of male and female animals of the high-concentration group (200 ppm) was statistically significantly decreased compared to the control group between day 0 and 35 for males and day 0 and 28 for females. This observed decrease (both early and temporary) in food consumption may be directly contributing to the observed decreases in male and female body weight in the high-dose animals. Food consumption was statistically significantly decreased for mid-concentration group (100 ppm) males between day 0-7 and for low- (50 ppm) and mid concentration group (100 ppm) females between day 0-7 and 14-21. Again, this observed decrease (both early and temporary) in food consumption may be directly contributing to the observed decreases in male and female body weight in the mid-dose and low-dose animals.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

F1 GENERATION: Not examined

COHORT 1A: Effects observed, treatment related:
- Red blood cell and coagulation parameters: In male and female animals, no statistically significant effects were observed on any of the red blood cell and coagulation parameters, except for a statistically significant decrease in the percentage of reticulocytes in males of the high-concentration group (200 ppm), which was considered as chance finding due to the lack of a concentration response.
- White blood cell parameters: In male animals of the high-concentration group (200 ppm), the absolute number of monocytes was statistically significantly decreased (36% decrease) as compared to the control group. In female animals, no statistically significant effects were observed on any of the white blood cell parameters, except for a statistically significant increase in the relative number of neutrophils in the low-concentration group (50 ppm). Since no dose-relationship could be established, this effect was considered as chance finding and not related to exposure.

COHORT 1B: Not examined

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

F1 GENERATION: Not examined

COHORT 1A: Effects observed, treatment related: A few statistically significant differences were observed in clinical chemistry parameters compared to the control group:
- Plasma glucose was decreased in males of the high-concentration group (200 ppm; 20% decrease, compared to control).
- Urea was increased in males of the high-concentration group (200 ppm; 18% increase, compared to control).
- Potassium was increased in males of the high-concentration group (200 ppm; 12% increase, compared to control).
- ASAT was decreased in females of the low-concentration group (50 ppm), which was considered as chance finding due to the lack of a concentration-response.
- Calcium was deceased in females of the low- (50 ppm) and mid-concentration (100 ppm) groups, which was considered as chance finding due to the lack of a concentration response.
- Chloride was deceased in females of the low-concentration group (50 ppm), which was considered as chance finding due to the lack of a concentration-response.

COHORT 1B: Not examined

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

F1 GENERATION: Not examined

COHORT 1A: Effects observed, treatment related:
Volume and density: The renal concentration test showed a statistically significant decrease in specific gravity in females of the mid- (100 ppm) and high-concentration (200 ppm) groups, associated with a higher urinary volume (statistically significant in high-concentration (200 ppm) females).
Semi-quantitative observations: Semi-quantitative (dipstick) urinary measurements showed a statistically significant increase in urinary pH in females of the mid-concentration group (100 ppm) compared to the control group. Since no concentration-response was observed, this was considered as chance finding.
Microscopic observations: Microscopic examination of the urinary sediment showed statistically significant increase in red blood cells in males of the mid-concentration group (100 ppm). Since no concentration-response was observed, this was considered as chance finding. In females of the high-concentration group (200 ppm), the score for crystals was statistically significantly increased.

COHORT 1B: Not examined

Sexual maturation:

effects observed, treatment-related

Description (incidence and severity):

F1 GENERATION: Not examined

COHORT 1A and 1B: Effects observed, treatment related
Males:
- Preputial separation: All male pups included in Cohort 1A and 1B scored positive for preputial separation. The timing of preputial separation in the male pups of the high-concentration group (200 ppm) was statistically significantly delayed as compared to the corresponding control group (mean day of preputial separation in the control group was 42.4 days and in the males of the high concentration). Additionally, mean body weight at the day of preputial separation was statistically significantly decreased in the high-concentration group (200 ppm). The delayed timing of preputial separation in the high-concentration group is probably related to the (statistically significant) lower weights of the pups in this group.
- Epididymal sperm motility: No statistically significant differences were observed on the epidydimal sperm motility parameters between the control and the treatment groups.
- Epididymal sperm count: No statistically significant differences were observed on the epidydimal sperm count parameters between the control and the treatment groups.
- Epididymal sperm morphology: No effects were observed on the epididymal sperm morphology parameters between the control and high-concentration group.
- Testicular sperm count: No effects were observed on the testicular sperm count parameters between the control and high-concentration group.

Females:
- Vaginal opening: All female pups included in Cohort 1A and 1B scored positive for vaginal opening. No statistically significant differences were observed on the timing of vaginal opening in the female pups between the control and the treatment groups. Mean body weight at the day of passing vaginal opening was statistically significantly decreased in the mid- (100 ppm) and high-concentration (200 ppm) groups.
- Estrus cycle: evaluation for 3 weeks before sacrifice did not show any statistically significant difference between the control and treatment groups in mean estrus cycle length and normality of the cycle.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

F1 GENERATION: Effects observed, treatment related: Pup organ weights per litter: In the high-concentration group (600 ppm), the combined absolute brain weight of male and female pups and the absolute brain weight of male pups separately were statistically significantly decreased (4% and 5%, respectively). In the mid- concentration group, the combined relative brain weight was statistically significantly increased (8% compared to controls) and in the high-concentration group, the combined relative brain weight of male and female pups separately were statistically significantly increased (12%, 11% and 13% of controls, respectively).
The combined absolute weight of the thymus of male and female pups together was statistically significantly decreased in the high-concentration group (600 ppm; 14% compared to controls). The absolute thymus weight was also decreased in high-concentration male- (16% compared to controls) and female- (12% compared to controls) pups separately, but did not reach the level of statistical significance as compared to the corresponding controls. No effect was observed on relative thymus weight.
The combined absolute weight of the spleen of male and female pups and the absolute spleen weight of female pups separately were statistically significantly decreased (24% and 26% compared to controls, respectively) in the high-concentration group (600 ppm). The absolute spleen weight of the male pups was also decreased (13% of controls), but this difference did not reach the level of statistical significance as compared to the corresponding controls. No effect was observed on relative spleen weight. The decreased absolute weights of the brain, thymus and spleen as observed in pups of the high-concentration group were considered as related to the lower terminal weight of the pups and of no direct toxicological relevance.

COHORT 1A: Effects observed, treatment related: In males and females of the high-concentration (200 ppm) group, the mean terminal body weight was statistically significantly decreased compared to the terminal body weight of the corresponding control animals (decrease of 13% and 12%, respectively).
The following statistically significant differences with the controls in absolute and/or relative organ weights were observed:
- Absolute brain weight was decreased in males of the high-concentration group (200 ppm) and in females of the mid- (100 ppm) and high-concentration (200 ppm) groups.
- Relative weight of the lungs was increased in males and females of the mid- (100 ppm) and high-concentration (200 ppm) groups (mid: 7% males, 6% females; high: 13% males; 10% females).
- Relative weight of the heart was increased in males of the high-concentration group (200 ppm; 13%) and in females of the low- (50 ppm), mid- (100 ppm) and high concentration (200 ppm) groups (6%, 7% and 15%, respectively).
- Absolute kidney weight was increased in males and females of the mid- (100 ppm) and high concentration (200 ppm) groups (mid: 13% males, 15% females; high: 38% males; 33% females). Relative kidney weight was increased in males and females of the mid- (100 ppm) and high-concentration (200 ppm) groups (mid: 17% males, 20% females; high: 58% males, 51% females).
- Relative liver weight was increased in males and females of the mid- (100 ppm) and high-concentration (200 ppm) groups (mid: 9% males, 7% females; high: 13% males, 11% females).
- Absolute weight of the testes was decreased in males of the high-concentration group (200 ppm; 6%).
- Relative weight of the epididymis was increased in males of the high-concentration group (200 ppm; 10%).
- Relative weight of the ovaries was increased in females of the high-concentration group (200 ppm; 16%)

COHORT 1B: effects observed, treatment-related: In males and females of the high-concentration (200 ppm) group, the mean terminal body
weight was statistically significantly decreased compared to the terminal body weight of the corresponding control animals (decrease of 16% and 12%, respectively).
Absolute weight of the testes was decreased in males of the high-concentration group (200 ppm; 8%), whereas relative weight of the testes was increased in males of the highconcentration group (200 ppm; 11%). This was probably related to the decreased terminal body weight.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

F1 GENERATION: Effects observed, non-treatment related: Macroscopic examination of stillborn pups and pups that died on postnatal day 0 did not reveal any effect. Obviously, macroscopy could not be performed on the pups that were missing during the lactation period.
Macroscopic observations of selected pups (10/sex/group) at necropsy on postnatal day 21 did not reveal any treatment-related effects. One pup of the mid-concentration group (300 ppm) showed a hernia in the diaphragm and a discoloured thymus, which was considered to be a fortuitous finding.

COHORT 1A: Effects observed, treatment related: At necropsy of the Cohort 1A animals treatment related macroscopic changes were observed in the kidneys of two and four animals of the mid- (100 ppm) and high-concentration (200 ppm) groups and comprised pale discoloration and/or a pitted surface and enlargement.

COHORT 1B: Effects observed, treatment related: At necropsy treatment related macroscopic changes were observed in the kidneys of severalanimals of the mid- and high-concentration groups and comprised pale discoloration, enlargement and/or a pitted surface.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

F1 GENERATION: Not examined

COHORT 1A: Effects observed, treatment related: Microscopic examination of the sampled organs and tissues revealed treatment related histopathological changes in the kidneys. The histopathological changes in the kidneys were observed in males as well as females and comprised minimal to moderate tubular dilatation, mononuclear inflammation, proteinaceous casts and/or basophilic tubules. Because of the clearly increased incidences of these findings in the 100 ppm animals (up to 16/20) and the 200 ppm animals (up to 20/20) they were considered related to treatment.
The other organs and tissues did not reveal treatment related histopathological changes. The histopathological changes observed were about equally distributed amongst the different treatment groups or occurred in one or a few animals only. They are common findings in rats of this strain and age or occurred as individual chance findings. Therefore, they were not considered to be related to treatment.

Differential ovarian follicle count: No treatment-related effects were observed on the differential ovarian follicle count (absolute numbers of small, growing and antral follicles and of corpora lutea and the relative numbers of each of these follicles).

Splenic lymphocyte subpopulation analysis: The resulting data had robust discriminative value, and could be effectively used to identify all planned lymphocyte populations. In both male and female animals, there were no treatment-related effects on any of the splenic lymphocyte subpopulation parameters, except for a statistically significant increased absolute number of B-lymphocytes in the spleen of male animals of the low- (50 ppm) and mid concentration (100 ppm) groups. Since this effect was not observed in the high-concentration group (200 ppm) and in males only, it was considered to be a chance finding and not related to treatment.

COHORT 1B: Not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

F1 GENERATION: Effects observed non-treatment related
- Anogenital distance: In male pups, the absolute anogenital distances as measured on postnatal day 4 was slightly, but statistically significantly decreased in the mid-concentration group (300 ppm) compared to the controls, while the corrected anogenital distance was comparable among all groups. Therefore, this difference was considered to be related to the lower pup weight. In female pups, both the absolute- and corrected anogenital distances were comparable among all groups.
- Nipple retention in male pups: On post-natal days 13 and 21, there were no effects on nipple retention in male pups.
- Hormone determinations: Exposure to the test substance via inhalation did not affect the concentrations of TSH and T4 hormones in sera of male and female animals of the F1-generation.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEC

Generation:

other: F1 and Cohort 1A

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEC

Remarks:

Sexual development

Generation:

other: Cohort 1A and 1B

Effect level:

>= 200 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 ppm

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Concentrations achieved

All achieved concentrations were very close to the respective target concentrations.

The time to reach 95% of the steady state concentration (T95), based on chamber volume and average total air flow was calculated to be 16.6 – 16.8 minutes.

The test material consumption as calculated from the actual concentrations and flows, was close to the consumption as determined from the weight decrease of the test material cylinder. Thus, the overall generation efficiency was close to 100% (i.e. 91.8%) which is expected for test atmosphere generation from a gaseous test material. The slight deviation from 100% was likely related to small inaccuracies or variations in flow measurements, limited precision of the scales used for weighing of the test material cylinder, and/or test material use for activities other than animal exposure (e.g. calibration of analyzers).

The oxygen concentration during exposure was in the range of 20.6 – 20.7% (v/v) and the carbon dioxide concentration was in the range of 0.071 – 0.114% (v/v), which was well within the limits of >19% oxygen and <1% carbon dioxide described in the OECD guideline.

Table 1. F0 Mating report

Sex: Both (Litter: A)		0 ppm	50 ppm	300 ppm	600 ppm
Females Placed with Males	N+ve	24	24	24	24
Females Mated	N+ve	24	24	24	22
Females Not Mated	N+ve	0	0	0	2
Female Mating Index	%	100.0	100.0	100.0	91.7
Males Placed with Females	N+ve	24	24	24	24
Males Mated	N+ve	24	24	24	22
Males Not Mated	N+ve	0	0	0	2
Males that became sire	N+ve	22	24	22	19
Male Mating Index	%	100.0	100.0	100.0	91.7
Male Fertility Index	%	91.7	100.0	91.7	79.2
Pre-coital time (days) [k]	Mean	2.4	2.7	3.2	3.3
	SD	1.1	1.0	1.5	1.2
	N	24	24	24	22
Day 1-4 [f]	N+ve	24	24	22	19
	%	100.0	100.0	91.7	86.4
Day 5-7 [f]	N+ve	0	0	2	3
	%	0.0	0.0	8.3	13.6
Day 8-14 [f]	N+ve	0	0	0	0
	%	0.0	0.0	0.0	0.0
Day 1-14 [f]	N+ve	24	24	24	22
	%	100.0	100.0	100.0	100.0

Litter: A = First litter

Female mating index: no. of females mated * 100/no. of females placed with males

Male mating index: no. of males mated * 100/no. of males placed with females

Male fertility index: no. of males that became sire *100/no. of males placed with females

[k] - Kruskal-Wallis & Wilcoxon

[f] - Chi-Squared & Fisher's Exact

 

Table 2. F0 Pregnancy report

Sex: Female (Litter: A)		0 ppm	50 ppm	300 ppm	600 ppm
Females Pregnant [f]	N+ve	22	24	22	19
Females Not Pregnant	N+ve	2	0	2	5
Pregnant, Implants, No Pups	N	.	.	.	1
Pregnant, Found Dead	N+ve	0	0	0	0
Pregnant, Killed Moribund	N+ve	0	0	0	0
Females Completing Delivery	N+ve	22	24	22	18
Females with Liveborn [f]	N+ve	22	24	22	18
Female Fecundity Index	%	91.7	100.0	91.7	86.4
Female Fertility Index	%	91.7	100.0	91.7	79.2
Gestation Index	%	100.0	100.0	100.0	94.7
Gestation Days [k]	Mean	22.4	22.5	22.3	22.3
	SD	0.5	0.5	0.5	0.5
	N	22	24	22	18
Females with Stillborn Pups [f]	N+ve	0	1	0	0
	%	0.0	4.2	0.0	0.0
Females with all Stillborn Pup [f]	N+ve	0	0	0	0
	%	0.0	0.0	0.0	0.0

[f] - Chi-Squared & Fisher's Exact

[k] - Kruskal-Wallis & Wilcoxon

Litter: A = First litter

Female fecundity index: no. of females pregnant * 100 /no. of females mated

Female fertility index: no. of females pregnant *100/no. of females placed with males

Gestation index: no. of females with a viable litter *100/no. of females pregnant

 

Table 3. F0 Delivery report

Sex: Female (Litter: A)		0 ppm	50 ppm	300 ppm	600 ppm
Pups Delivered (Total) [k]	Mean	11.6	11.4	11.9	10.7
	SD	2.3	1.6	2.4	3.0
	Sum	255	273	261	192
Liveborn [f]	Sum	255	271	261	192
Live Birth Index (%)		100.0	99.3	100.0	100.0
Stillborn day 0 [f]	Sum	0	2	0	0
Stillborn Index (%)		0.0	0.7	0.0	0.0

[k] - Kruskal-Wallis & Wilcoxon

[f] - Chi-Squared & Fisher's Exact

Litter: A = First litter

Live birth index: no. of liveborn pups *100/no. of total pups delivered

Stillborn index: no. of stillborn pups *100/no. of total pups delivered

 

Table 4. F0 Litter report

Sex: Female Day(s) Relative to Littering (Litter: A)		0 ppm	50 ppm	300 ppm	600 ppm
Live Pups/Litter day 0 [k]	Mean	11.5	11.3	11.9	10.6
	SD	2.4	1.7	2.4	3.0
	N	22	24	22	18
	Sum	254	271	261	191
Live Pups/Litter day 4 Pre [k]	Mean	11.5	11.2	11.7	10.4
	SD	2.5	2.1	2.3	3.0
	N	22	24	22	18
	Sum	252	268	258	188
Culled pups	Sum	43	39	46	27
Live Pups/Litter day 4 Post [k]	Mean	9.5	9.5	9.6	8.9
	SD	1.3	1.3	0.9	1.5
	N	22	24	22	18
	Sum	209	229	212	161
Live Pups/Litter day 7 [k]	Mean	9.5	9.5	9.6	8.9
	SD	1.3	1.3	0.9	1.5
	N	22	24	22	18
	Sum	209	229	211	161

[k] - Kruskal-Wallis & Wilcoxon

Litter: A = First litter

Viability index 0-4: no. of live pups on day 4 *100/no. of liveborn pups

Viability index 4-21: no. of live pups on day 21 * 100/no. of live pups post cull

Sex ratio day n: no. of live male pups on day n *100/ no. of live pups on day n

Post-implantation loss: no. of implant sites - no. of liveborn *100/no. of implant sites

 

Table 5. F0 Litter report

Sex: Female Day(s) Relative to Littering (Litter: A)		0 ppm	50 ppm	300 ppm	600 ppm
Live Pups/Litter day 14 [k]	Mean	9.5	9.5	9.6	8.9
	SD	1.3	1.3	0.9	1.5
	N	22	24	22	18
	Sum	209	229	211	161
Live Pups/Litter day 21 [k]	Mean	9.5	9.5	9.6	8.9
	SD	1.3	1.3	0.9	1.5
	N	22	24	22	18
	Sum	209	229	211	161
Dead, Missing, Cannibalized d0-d4		3	3	3	4
Dead, Missing, Cannibalized d5-d7		0	0	1	0
Dead, Missing, Cannibalized d8-d14		0	0	0	0
Dead, Missing, Cannibalized d15-d21		0	0	0	0
No. of litters lost entirely d0-d4 [f]	N+ve	0	0	0	0
	%	0.0	0.0	0.0	0.0
No. of litters lost entirely d5-d7 [f]	N+ve	0	0	0	0
	%	0.0	0.0	0.0	0.0
No. of litters lost entirely d8-d14 [f]	N+ve	0	0	0	0
	%	0.0	0.0	0.0	0.0
No. of litters lost entirely d15-d21 [f]	N+ve	0	0	0	0
	%	0.0	0.0	0.0	0.0
No. of litters lost entirely d0-d21 [f]	N+ve	0	0	0	0
	%	0.0	0.0	0.0	0.0
Viability Index 0-4 (%)		99	99	99	98
Viability Index 4-21 (%)		100	100	100	100
Live Males on Day 0	Mean	5.4	6.0	6.0	5.0
	SD	2.6	2.0	2.2	2.0
	N	22	24	22	18
	Sum	119	144	133	90
Sex Ratio Day 0 - Males (%)		46.9	53.1	51.0	47.1
Live Males on Day 21	Mean	4.4	4.9	4.9	4.4
	SD	1.5	1.3	1.0	1.4
	N	22	24	22	18
	Sum	96	118	108	79
Sex Ratio Day 21 - Males (%)		45.9	51.5	51.2	49.1
Implantation Sites Total	Mean	12.7	12.4	12.1	10.0
	SD	1.6	1.6	2.2	5.0
	N	22	24	22	21
	Sum	280	298	267	210
No. of lost implantations	Sum	25	27	6	18
Post-Implantation Loss % [k]	Mean	9.6	8.5	2.6	7.9
	SD	11.9	12.3	4.5	14.8

[k] - Kruskal-Wallis & Wilcoxon

[f] - Chi-Squared & Fisher's Exact

Litter: A = First litter

Viability index 0-4: no. of live pups on day 4 *100/no. of liveborn pups

Viability index 4-21: no. of live pups on day 21 * 100/no. of live pups post cull

Sex ratio day n: no. of live male pups on day n *100/ no. of live pups on day n

Post-implantation loss: no. of implant sites - no. of liveborn *100/no. of implant sites

 

Applicant's summary and conclusion

Conclusions:

In this GLP-compliant Extended One Generation Reproductive Toxicity Test, performed according to OECD 443, with the inclusion of Cohorts 1A and 1B, the following NOAECs were found:
- Parental - systemic toxicity: 50 ppm, based on decreased body weights (change), decreased food consumption, increased urinary volume, effects on organ weights, macroscopic observations in the kidneys and microscopic effects in several organs (kidneys, heart and thymus);
- Parental - reproductive toxicity: => 600 ppm (highest dose), based on the absence of any treatment-related adverse effects on any fertility and reproductive performance parameters;
- Offspring - systemic toxicity: 50 ppm, based on the increased relative organ weights (kidney, lung, liver, heart) and the histopathological changes in the kidney after macroscopic and microscopic examination;
- Offspring - sexual developmental toxicity: => 200 ppm, based on no treatment-related effects on sexual development at any exposure concentration in the F1-generation animals.

Executive summary:

The objective of this GLP-compliant study, performed according to OECD 443, was to provide data on the possible effects of the test substance on reproductive performance of Wistar rats and the development of pups consequent to daily inhalation exposure to various concentrations of the test substance (intended target concentrations: 0 ppm, 50 ppm, 300 ppm and 900 ppm) to male and female rats during a premating period of 10 weeks and during mating (max. 2 weeks), gestation and lactation until postnatal day (PN) 21. At weaning on postnatal day 21 (PN 21), pups were distributed to Cohorts 1A and 1B and were exposed to the test substance at lower concentrations as their parents during their growth into adulthood (intended target concentrations: 0 ppm, 50 ppm, 300 ppm and 600 ppm).

The overall average actual concentration (± standard deviation) of the test substance in the low-concentration test atmosphere (group 2; 50 ppm target concentration), as determined by total carbon analysis, was 49.3 (± 2.1) ppm. Exposure to the mid-concentration (group 3) was started at a target concentration of 300 ppm, which was reduced to 100 ppm as of 3 June 2018; the average actual concentrations were 300.3 (± 7.9) and 100.3 (± 1.2) ppm, respectively. Average concentrations in the high-concentration test atmosphere (group 4) were 900.3 (± 12.9), 601.0 (± 19.5) and 199.8 (± 2.4) ppm, in the periods 5-9 February, 10 February — 2 June, and 3 June — 29 August 2018 when target concentrations were 900, 600 and 200 ppm, respectively.

Three P0-females of the high-concentration group (900 ppm) and several animals of Cohorts 1A and 1B of the high-concentration groups (600 ppm) died during the first days of exposure. Accordingly, the target concentration of the high-concentration group was lowered during exposure of the P0-generation (from 900 ppm to 600 ppm) and the target concentrations of the mid- and high-concentration groups of the F1-generation were lowered before the start (day 0) of Cohort 1A and Cohort 18 (from 300 ppm to 100 ppm and from 600 ppm to 200 ppm, respectively). Animals that died were replaced by surplus animals and after lowering the target concentrations, no other animals died during the study.

Clinical observations revealed piloerection, hunched posture, respiration dyspnoea and/or muscle weakness in several animals of the high-concentration group during the first exposure days of the P0-generation and in the first exposure weeks of Cohort 1A and 1B. These signs were mainly observed in the high-concentration group (900 ppm) of the P0-generation and in the high-concentration group (600 ppm) of Cohorts 1A and 1B, before lowering the target concentrations and disappeared thereafter. No other treatment-related (detailed) clinical signs were observed during the study.

In the P0-generation, body weights of the male animals of the mid- and high-concentration groups and in females of the high-concentration group were statistically significantly lower than the corresponding control animals (maximally, during premating: P0-males 17%, P0-females 10%; P0-females during gestation 9%, P0-females during lactation 7%).

In Cohorts 1A and 1B of the F1-generation, body weights were statistically significantly lower in the males and females of the mid- and high-concentration groups (in Cohort 1A maximally 30% in males and 28% in females, respectively, and in Cohort 1B maximally 35% in males and 30% in females, respectively).

In the P0-generation, food consumption of the male and female animals of the high-concentration group was statistically significantly lower than of the control animals (maximally 44% in males and 39% in females). In male and females animals of the mid-concentration groups food consumption was statistically significantly lower compared to control animals during the first week(s) of the study.

In the F1-generation, food consumption of the male and females animals of the high-concentration group was statistically significantly lower than of controls (in Cohort 1A maximally 25% in males and 18% in females, respectively, and in Cohort 1B maximally 22% in males and 16% in females, respectively).

The observed effects on body weights were considered, at least in part, as transient and/or as related to the lower food consumption. The observed effects on body weights in the high-concentration groups of the P0- (600 ppm) and F1- (200 ppm) generations, however, were considered to be related to treatment.

Except for the increase in urea in male animals of the high-concentration group of the P0-generation (68% higher than controls) and in male animals of the high-concentration group of Cohort 1A of the F1-generation (18% higher than controls), no treatment-related effects were observed on haematology and clinical chemistry parameters.

Urinalysis revealed a higher urinary volume in males of the low-, mid-and high-concentration groups of the P0-generation and in females of the high-concentration group of Cohort 1A of the F1-generation.

In the P0-generation, the increased relative kidney weight in male and female animals of the mid-and high-concentration groups (300-600 ppm: ~91-130% increase in males and ~44­65% increase in females compared to controls, respectively) were considered to be adverse and related to treatment. Additionally, in P0-generation animals, terminal body weight was decreased in males of the mid- and high-concentration groups (300 and 600 ppm; maximally 17% below controls). Furthermore, the increased relative lung weight (~19-30% in males and ~14% in females), heart weight (~20-39% in males and ~22% in females), liver weight (~15-18% in males and ~14% in females), and spleen weight (~10-21% in males and ~13% in females) as observed in male animals of the mid- (300 ppm) and high-concentration (600 ppm) groups and in female animals of the high-concentration group (600 ppm) were considered to be related to treatment.

In Cohort 1A of the F1-generation, the increased relative kidney weight in male and female animals of the mid-and high-concentration groups (100-200 ppm: ~17-58% increase in males and ~20-51% increase in females compared to controls, respectively) were considered to be adverse and related to treatment. Additionally, in Cohort 1A animals, terminal body weight was decreased in high-concentration (200 ppm) male and female animals (decrease of 13% and 12%, respectively). Furthermore, the increased relative lung weight (~7-13% in males and ~6-10% in females) and relative liver weight (~9-13% in males and ~7-11% in females) as observed in males and females of the mid- (100 ppm) and high concentration (200 ppm) groups and the increased relative heart weight observed in high-concentration (200 ppm) males and mid- (100 ppm) and high-concentration (200 ppm) females (~13% and 15%, respectively) were considered to be related to treatment.

No effects were observed on the weight of the reproductive organs of Cohort 1B F1-generation animals.

At necropsy, macroscopic examination revealed pale discoloration, enlargement and/or a pitted surface of the kidneys in mid- and high-concentration animals of the P0-generation (300 ppm and 600 ppm) and in animals of Cohort 1A of the F1-generation (100 ppm and 200 ppm).

Microscopic examination of the sampled organs and tissues in the P0-generation animals revealed treatment-related histopathological changes in the heart (minimal to moderate ventricular muscle degeneration) in high-concentration females (600 ppm) and kidneys (minimal to moderate tubular dilatation, mononuclear inflammation, proteinaceous casts and/or basophilic tubules) in mid- and high-concentration animals (300 and 600 ppm, respectively).

Microscopic examination of the sampled organs and tissues of the animals of Cohort 1A of the F1-generation revealed treatment related histopathological changes in the kidneys (minimal to moderate tubular dilatation, mononuclear inflammation, proteinaceous casts and/or basophilic tubules) in mid- and high-concentration animals (100 and 200 ppm, respectively).

Other organs and tissues did not reveal treatment related histopathological changes.

No treatment-related effects were observed on the fertility and reproductive performance of male and female animals of the P0-generation.

No treatment-related effects were observed on estrous cycle related parameters in female animals of the P0-generation and in animals of Cohort 1A of the F1-generation.

No treatment-related effects were observed on epididymal and testicular sperm parameters in male animals of the P0-generation and in animals of Cohort 1A of the F1-generation.

No effects were observed on TSH and T4 analysis in animals of the P0-generation and in adult F1-generation animals of Cohort 1A.

No treatment-related effects were observed on number of live pups, number of implantation sites, implantation loss, stillborn pups, dead, missing and/or cannibalized pups, litter loss, pup viability indices and sex ratio.

No treatment-related effects were observed on clinical signs of pups nor on macroscopic observations at sacrifice and of dead pups in F1-generation pups.

Overall, in the mid- (300 ppm) and high-concentration group (600 ppm), the body weight of F1-generation pups during the lactation period was lower than of the corresponding control pups (~10-15% on postnatal day 21). This finding was considered to be related to treatment.

No direct effects were observed on anogenital distance on PN4 in F1-generation pups or on nipple retention in male F1-generation pups.

Preputial separation (control: 42.4 days, high dose 46.4 days) was delayed in male pups of the high-concentration group (200 ppm). However, these differences were not considered as delayed sexual development but as a consequence of delayed general development (lower pup weights).

At sacrifice of the selected pups at post-natal day 21, the terminal body weights of the pups of the mid-concentration (300 ppm) and high-concentration (600 ppm) groups calculated per litter were lower than of the control group. No direct effects were observed on organ weights of F1-generation pups sacrificed on post-natal day 21.

No treatment-related effects were observed on the development of the ovarian follicles from primordial small follicles into corpora lutea in Cohort 1A animals of the F1-generation.

No treatment-related effects were observed on splenic lymphocyte subpopulation analysis in Cohort 1A animals of the F1-generation.

Based on the decreased body weights (change), decreased food consumption, increased urinary volume, effects on organ weights, macroscopic observations in the kidneys and microscopic effects in several organs (kidneys, heart and thymus) the No Observed Adverse Effect Concentration (NOAEC) for parental toxicity was placed at the low-concentration (50 ppm) after exposure via inhalation for 6 hours/day, 7 days/week for up to 12 weeks for males (pre-mating through mating) and for 12 weeks for females (pre-mating through mating) and then through post-natal day (PND) 21.

There were no treatment-related adverse effects of exposure to the test substance on any of the multiple endpoints assessed with regards to fertility and reproductive performance in the parental generation (P0-animals). In addition, there also were no treatment-related adverse effects on the various reproductive systems endpoints assessed in the male and female F1-animals (Cohorts 1A and 1B). Based on the absence of any treatment-related adverse effects on any fertility and reproductive performance parameters in this OECD 443-compliant study, the study NOAEC for fertility and reproductive performance was placed at the high-concentration (600 ppm).

Based on the increased relative organ weights (kidney, lung, liver, heart) and the histopathological changes in the kidney after macroscopic and microscopic examination in mid-(100 ppm) and high-concentration (200 ppm) animals of Cohort 1A, the No Observed Adverse Effect Concentration (NOAEC) was based on these general toxicity observations and placed at the low-concentration (50 ppm). There were no treatment-related effects on sexual development at any exposure concentration in the F1-generation animals.