Iodomethane

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 May 2001 to 07 June 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Iodomethane

EC Number:

200-819-5

EC Name:

Iodomethane

Cas Number:

74-88-4

Molecular formula:

CH3I

IUPAC Name:

iodomethane

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): Iodomethane
- Physical state: liquid
- Analytical purity: 99.7%
- Impurities (identity and concentrations): 0.2 % water and <0.1 % methanol
- Composition of test material, percentage of components: not stated
- Isomers composition: not stated

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
- Age at study initiation: 6 weeks
- Weight at study initiation: males: 181 to 236 g, females: 148 to 182 g
- Housing: Following the initial acclimation period (F0) or selection (F1) and until pairing, all F0 and F1 parental test animals were individually housed in clean, wire-mesh cages suspended above cage-board. The cage board was changed three times per week.
- Diet: ad libitum (except during exposure)
- Water: ad libitum (except during exposure)
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19.3 to 22.44 °C (66.7 to 72.4 °F)
- Humidity: 34.7 to 61.24 %
- Air changes: Air handling units were set to provide approximately 10 fresh air changes per hour.
- Photoperiod : Light timers were calibrated to provide a 12-hour light (6 a.m. to 6 p.m.)/12-hour dark photoperiod.

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Each group of animals was exposed in a 2.0 m^3 stainless-steel and glass whole-body inhalation chamber.
- Temperature, humidity, pressure in air chamber: Instrumentation was set to maintain the temperature inside the exposure chamber between 18 and 26 °C and relative humidity between 30 and 70 %.
- Air flow rate: The chambers were operated under dynamic conditions at air flows of at least 12 to 15 air changes per hour.
- The F0 and F1 males and females were exposed to the test atmosphere for daily six-hour exposures (seven days a week) for a minimum of 70 consecutive days prior to mating. Exposure of the F0 and F1 males continued throughout mating and through the day prior to euthanasia. The F0 and F1 females continued to be exposed throughout mating and gestation through gestation day 20. To prevent confounding effects on nursing, exposures were not conducted from gestation day 21 through lactation day 4. Exposure of the F0 and F1 females was re-initiated on lactation day 5 and continued through the day prior to euthanasia. During lactation (except where indicated above), the dams were removed from their litters during each daily six-hour exposure period. Due to unforeseen circumstances (human error), the exposure periods were extended 48 minutes on November 15, 2001 in chamber no. 2, 34 minutes on December 8, 2001 in chamber no. 4 and 27 minutes on January 4, 2002 in chamber no. 2; however, it is believed that animals only received a total 6-hour exposure to test material.
- The control group was exposed to clean, filtered air under conditions identical to those used for the groups exposed to the test material.
- The rats were removed from their home cages in the animal room, placed into exposure cages and transported to the inhalation chambers for the six-hour exposure period. The animals were exposed to a vapour atmosphere of the test material or filtered air at approximately the same time each day. Following exposure, the animals were returned to their home cages.
- Each chamber was dedicated to one exposure group. Groups for the companion 13-week toxicity study (WIL-4180151) were exposed to the test material simultaneously in the same chambers during the F0 generation exposure period. In order to minimise any potential variation occurring due to positioning within the chamber, the cages were sequentially rotated around the available rack positions within the chamber on a daily basis throughout the study, in accordance with the standard operating procedures.
- Exposure levels were selected by the sponsor based upon an inhalation range-finding study.
- The offspring of the F0 generation (F1 litters) were potentially exposed to the test material in utero and through nursing during lactation days 5-21 (there was no direct maternal exposure from lactation days 0-4, inclusively). The selected F1 pups were supposed to have been directly exposed to the test material via whole-body inhalation following weaning (beginning on PND 22). However, due to mortalities in the F1 weanlings after initiation of direct inhalation exposure on PND 22 in all groups (including the control group), test material exposures were suspended and re-initiated on PND 28 and the pups remained group-housed until PND 35. Three, four, two and one males and three, five, four and two females in the control, 5, 20 and 50 ppm groups, respectively, died or were euthanised in extremis and were replaced with pups from the same group (representing the maximum number of litters in an exposure group) during PND 23-35. A record of the animals affected and those used for replacement is maintained in the study records.
- The F1 pups selected for breeding (30/sex/group) were exposed to the test material for six hours daily for at least 70 days prior to mating and throughout mating, gestation and lactation (with the exception of gestation day 21 through lactation day 4) until the day prior to necropsy. The offspring of the F1 generation (F2 litters) were potentially exposed to the test material in utero and through nursing during lactation days 5-21.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Cohabitation: Each female was housed in the home cage of the male for up to 14 days.
- Proof of pregnancy: Positive evidence of mating was confirmed by the presence of a copulatory plug or the presence of sperm in a vaginal smear. Once mating was confirmed, this was termed day 0 of gestation and the female was housed individually.
- Each generation was mated once to produce one litter/generation (the F1 and F2 litters).
- If no evidence of copulation was obtained after 14 days, the animals were separated without further opportunity for mating, and the female was placed in a plastic cage containing nesting material.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

NOMINAL EXPOSURE CONCENTRATIONS
- A nominal exposure concentration was calculated for each daily exposure for each chamber from the total amount of test material used during the exposure and the total volume of air passed through the chamber during that day's exposure. The amount of test material used was obtained by weighing the gas-washing bottle containing the test material for each chamber prior to and after each daily exposure. The total volume of air passed through each chamber was calculated from the daily average chamber ventilation flow rate in litres per minute (LPM) and the exposure duration. The nominal concentration was calculated as follows:
ppm Iodomethane = (Wt. Iodomethane · Mol. Vol. · 10^6) / (MW · Ch. Flow · Exp. Dur.)
Where:
Wt. Iodomethane = weight of test material in grams
Mol. Vol. = Molar volume at 730 mmHg and 21 °C, 25.11 L/mole
10^6 = ppm conversion factor
MW = Iodomethane molecular weight, 141.94 g/mole
Ch. Flow = Daily average chamber flowrate for a given day, in LPM.
Exp. Dur. = Duration of a given day's exposure, in minutes

ACTUAL EXPOSURE CONCENTRATIONS
- Actual exposure concentrations were measured using a gas chromatograph (GC). Samples of the exposure atmospheres from each chamber were automatically collected at approximately 35-minute intervals using a sample loop and computer-controlled gas-sampling and multiposition valve. The following table summarises the GC conditions:
Instrument: Hewlett Packard 5890 Series II with a 3396 Series II integrator
Detector: Flame ionisation (FID)
Column: J & W DB-5, 30 m x 0.32 mm I.D., 0.25- micron film thickness
Gases: (Pressure (psig) Flow Rate (mL/min.)): Carrier - Helium 35 8.7, Fuel - Hydrogen 20 30, Air 40 300
Temperatures (°C): Injector 100, Column 60 and Detector 100
Injection volume (mL) 0.25
Retention time (min.) Approximately 0.84 min.
Integrator Run Parameters: Chart Zero Offset 0, Chart Attenuation 0, Chart Speed 1.5 cm/min, Peak Area Rejection Value 1200-1800, Peak Threshold 0 and Peak Width 0.24-0.25
- During the animal exposure on 7/22/01 the gas chromatograph malfunctioned during the fourth round of sampling. Following the animal exposure, the gas chromatograph was replaced with a similar one using the original DB-5 column. However, analytical problems continued on 7/23/01 and two samples were collected from chambers 2, 3 and 5 in Tedlar gas sample bags to permit later determination of the exposure concentrations. Following the 7/23/01 exposure, the DB-5 column was replaced with a DB-Wax column and the gas chromatograph was calibrated by preparation of a prime calibration curve based on a single set of gas standards. Using this prime calibration curve, the samples collected on 7/23/01 were analysed. During the animal exposure period on 07/24/01, analytical problems continued due to retention time shifts that resulted in improper integration of multiple room air and chamber samples during the LabVIEW-controlled automated sampling rounds. It was again necessary to collect and analyse chamber samples collected in Tedlar gas sample bags. After conditioning the DB-Wax column and adjustment of specific GC run parameters, a complete prime calibration curve was created using three sets of standards prepared and analysed on 7/25/01 and 7/26/01. This prime curve was put in place starting on 7/27/01. For the animal exposures on 7/25/01 and 7/26/01, the chamber concentrations were calculated using calibration curves based on one set of gas standards and two sets of gas standards, respectively. Specifics concerning these changes and documentation of manual samples and gas chromatograph parameters appear in the study records. After making adjustments to the run parameters the final GC parameters are the following:
Instrument: Hewlett Packard 5890 Series II with a 3396 Series II integrator
Detector: Flame ionization (FID)
Column: J & W DB-Wax, 30 m x 0.25 mm I.D., 0.25-micron film thickness
Gases: (Pressure (psig) Flow Rate (mL/min.)): Carrier - Helium 35 8.7, Fuel - Hydrogen 20 30, Air 40 300
Temperatures (°C): Injector 65, Column 40 and Detector 75
Injection volume (mL) 0.25
Retention time (min.) Approximately 0.84 min.
Integrator Run Parameters: Chart Zero Offset 0, Chart Attenuation 0, Chart Speed 1.0 cm/min, Peak Area Rejection Value 0, Peak Threshold 0 and Peak Width 0.04.
- The chromatograph was standardised using 40-liter Tedlar® gas bags prepared to contain known concentrations of the test material. The standard bags were prepared by injecting known volumes of test material into a 500 mL glass vaporisation bulb. A continuous flow of air carried the vaporised test material to a 40 L bag. The total volume of air was measured by a dry test meter (Model DTM-200A, American Meter Co., Nebraska City, PA). Concentrations of the gas-phase standards were calculated as follows: Concentration = (VOL · R · T · D · 10^-3 · 10^6) / (L · GMW · P)
Where:
Conc. is in ppm
VOL = volume of test material vaporised into bag in μL
R = universal gas constant, 62.36 L mmHg/mole K
T = nominal laboratory temperature in K (273 + 21 °C = 294 K)
D = density of the test material, 2.280 g/mL
L = volume of air used to prepare bag, 32 L
GMW = gram molecular weight, 141.94 g/mole
P = nominal laboratory barometric pressure, 730 mmHg
10^-3 = μL to mL conversion factor
10^6 = conversion factor to ppm

- Standards prepared for this study: 3.8 ppm: 0.3 µL test material and 32 L air, 19 ppm: 1.5 µL test material and 32 air, 39 ppm: 3.1 µL test material and 32 L air, 58 ppm: 4.6 µL test material and 32 L air and 78 ppm: 6.2 µL test material and 32 L air. Each standard was prepared in triplicate prior to the exposure period and analysed with the GC. A least-squares line was fitted to the resulting peak areas. Concentrations were then calculated using the slope and intercept of this prime calibration curve. On a daily basis, the integrity of the prime calibration curve was checked by analysing one freshly prepared standard. On a rotational basis, a different one of the five standards was used each day. If the analysed concentration were within ± 10 % of the known concentration, the GC was considered within calibration specifications.

DETERMINATION OF HOMOGENEITY OF EXPOSURE ATMOSPHERES
- Evaluation of the homogeneity of exposure concentrations was accomplished during the method development phase of the study prior to animal exposures. Four test locations and a reference location were used for these determinations. The test locations were Right Lower Front (RLF), Right Upper Rear (RUR), Left Lower Rear (LLR), Left Upper Front (LUF), Left Lower Front (LLF), Right Lower Rear (RLR), Right Upper Front (RUF) and Left Upper Rear (LUR). Samples were Samples were collected as rapidly as possible always collecting a sample from the reference location and then from one of the four test locations. For each test location, the measured concentration was calculated as a percent difference from the reference location. The homogeneity determination was performed in triplicate for chamber 2, 3 and 4.
- Results indicated that homogeneity of exposure atmospheres were adequate for the purpose of this study. Maximum mean % from reference was – 7.4 %.

RESULTS
- Nominal Exposure Concentrations: The overall mean nominal concentrations for the F0 generation were 7.0 ppm, 25 ppm and 59 ppm for the 5 ppm, 20 ppm and 50 ppm groups, respectively. The overall mean nominal concentrations for the F1 generation were 6.0 ppm, 24 ppm and 56 ppm for the 5 ppm, 20 ppm
and 50 ppm groups, respectively.
- Actual Exposure Concentrations: The overall mean concentrations for the F0 generation were 5 ppm, 20 ppm and 50 ppm for the 5 ppm, 20 ppm and 50 ppm groups, respectively. The overall mean concentrations for the F1 generation were 5 ppm, 21 ppm and 49 ppm for the 5 ppm, 20 ppm and 50 ppm groups, respectively.

Duration of treatment / exposure:

- 70 consecutive days prior to mating, then daily throughout breeding and post-natal period until euthanasia (F0 and F1).
- Approximately 126 to 132 days of exposure for the F0 generation and approximately 146 to 155 days of exposure for the F1 generation.

Frequency of treatment:

6 hours per day

Details on study schedule:

- F1 parental animals not mated until 16 weeks of age
- Selection of parents from F1 generation on PND 21

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5 ppm (nominal)

Doses / concentrations 2

Dose / conc.:

20 ppm (nominal)

Doses / concentrations 3

Dose / conc.:

50 ppm (nominal)

No. of animals per sex per dose:

F0 and F1 generation: 30 animals per sex per dose

Control animals:

yes

Details on study design:

- Dose selection rationale: Exposure levels were selected by the sponsor based upon an inhalation range-finding study

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- All animals were observed twice daily (at least seven hours apart) for moribundity and mortality. The animals were also observed daily at the midpoint of exposure (only animals visible in the chambers) and within one hour following exposure. The observations included, but were not limited to, evaluation for changes in appearance of skin and fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous system function, somatomotor activity and behaviour patterns. Special attention was paid to the degree of salivation and lacrimation, presence or absence of urination and defecation (including polyuria and diarrhoea), pupil size, degree of palpebral closure, presence of convulsions, tremors or abnormal movements, presence of posture and gait abnormalities, the presence of any unusual or abnormal behaviours and any repetitive actions (stereotypies). Females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labour, delayed labour) or other difficulties.

DETAILED CLINICAL OBSERVATIONS: Yes
- Detailed physical examinations were recorded weekly for all parental animals throughout the study period.

BODY WEIGHT: Yes
- Individual F0 and F1 male body weights were recorded weekly throughout the study and prior to the scheduled necropsy. Individual F0 and F1 female body weights were recorded weekly until evidence of copulation was observed.
- Body weights for the F1 generation were recorded beginning on interval 17 when the animals were 28 days or older. Group mean weekly body weights and body weight changes were calculated for each interval. Once evidence of mating was observed, female body weights were recorded on gestation days 0, 4, 7, 11, 14 and 20 and on lactation days 1, 4, 7, 14 and 21. Group mean body weights and body weight changes were calculated for each of these intervals and for the entire gestation and lactation intervals (days 0-20 and 1-21, respectively). After weaning (lactation day 21 for the F1 pups and lactation day 21 for the F2 pups), weekly body weights were recorded for these females until the scheduled necropsy.

FOOD CONSUMPTION:
- Individual F0 and F1 male and female food consumption was measured weekly until pairing. Because the F1 generation remained group-housed until PND 35, food consumption for the F1 generation did not begin until interval 19. Food intake was not recorded during the mating period as the animals were cohabitated. Male food consumption was measured after mating on a weekly basis until the scheduled necropsy. Female food consumption was recorded on gestation days 0, 4, 7, 11, 14 and 20 and lactation days 1, 4, 7, 14 and 21. Food consumption was calculated and reported as g/animal/day and g/kg/day for the corresponding body weight change intervals. Food efficiency (body weight gained as a percentage of food consumed) was also calculated and reported for these intervals.

Oestrous cyclicity (parental animals):

- Vaginal smears were prepared daily to determine the stage of oestrus for each female, beginning 21 days prior to pairing and continuing until evidence of mating was observed.
-The F0 generation was paired on the 21st day of oestrous smears and the F1 generation was paired on the 21st or 22nd day of oestrous smears. For females with no evidence of mating, smearing was continued until termination of the mating period.
- The average cycle length was calculated for complete oestrous cycles (i.e. the total number of returns to met-oestrus [M] or di-oestrus [D] from oestrus [E] or pro-oestrus [P] beginning 21 days prior to initiation of the mating period and until the detection of evidence of mating). Oestrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in a subsequent cycle. The cycle during which evidence of mating was observed for a given animal was not included in the mean individual oestrous cycle length calculation. In addition, females exhibiting one or no stage of oestrus were also excluded from the mean. Vaginal smears were also performed on the day of necropsy to determine the stage of oestrus.

Sperm parameters (parental animals):

SPERMATOGENIC ENDPOINT EVALUATIONS:
- Immediately upon euthanasia, the reproductive tract of each F0 and F1 male was exposed via a ventral mid-line incision. The right epididymis was excised and weighed. An incision was made in the distal region of the right cauda epididymis, which was then placed in Dulbecco’s phosphate-buffered saline (maintained at approximately 37 °C) with 10 mg/mL bovine serum albumin (BSA). After a ten-minute incubation period, a sample of sperm was loaded into a 100 μm cannula for determination of sperm motility. Because sperm motility can be affected by temperature shock, all cannulas and diluents were pre-warmed in an incubator, and motility determinations were performed under constant temperature (approximately 37 °C) using the Hamilton-Thorne HTM-IVOS Version 10 computer-assisted sperm analysis (CASA) system. Analysis of a minimum of 200 motile and nonmotile spermatozoa per animal (if possible) in all exposure groups was performed by the analyzer.
- The motility score (percent) was reported: Percent Motile Sperm = (Number of Motile Sperm / Total Number of Sperm Counted) x 100
- Sperm morphology was evaluated by light microscopy via a modification of the wetmount evaluation technique. Abnormal forms of sperm (double heads, double tails, microcephalic or megacephalic, etc.) from a differential count of 200 spermatozoa per animal, if possible, were recorded. The left testis and epididymis from all F0 and F1 males from all exposure groups were stored frozen, homogenised and evaluated for determination of homogenization-resistant spermatid count and sperm production rate. For determination of homogenisation-resistant spermatid count and sperm production rate, the samples were thawed and homogenised, and a sample was retained for subsequent analysis. An aliquot of the sample was added to a solution containing a DNA-specific fluorescent dye (the dye stains DNA that is present in the head of the sperm). For analysis, each sample was mixed, and an aliquot was placed on a slide with a 20-μm chamber depth. Illumination from a xenon lamp within the HTM-IVOS analyser allowed for the visualization and quantitation of the sperm. A minimum of 200 cells, if possible, or 20 fields were counted for each sample.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- To reduce variability among the litters, eight pups per litter, four per sex when possible, were randomly selected on PND 4. All selections were performed by computerised randomisation. The remaining offspring were weighed, euthanised by intraperitoneal injection of sodium pentobarbital and discarded on PND 4. Standardisation of litter size was not performed on litters with fewer than eight pups.

PARAMETERS EXAMINED
- On the day parturition was judged complete (PND 0), pups were sexed and examined for gross malformations, and the numbers of stillborn and live pups were recorded. Individual gestation lengths were calculated using the date delivery started.
- Each litter was examined twice daily for survival, and all deaths were recorded. A daily record of litter size was maintained.
- Litters were examined daily for survival and any adverse changes in appearance or behaviour. Each pup received a detailed physical examination on PND 1, 4, 7, 14 and 21. The anogenital distance (in mm) of all pups was measured using Mitutoyo calipers (or equivalent) from the caudal portion of the anus to the cephalad portion of the genital tubercle on PND 1. Any abnormalities in nesting and nursing behaviour were recorded.
- Pups were individually weighed on PND 1, 4, 7, 14 and 21. Mean pup weights were presented by sex for each litter and by exposure group.
- Pups were individually sexed on PND 0, 4, 7, 14 and 21.

GROSS EXAMINATION OF DEAD PUPS:
- Intact offspring dying from PND 0 to 4 were necropsied using a fresh dissection technique. Findings were recorded as either developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity, representing slight deviations from normal) or malformations (those anomalies that alter general body conformity, disrupt or interfere with body function, or may be incompatible with life).
- A detailed gross necropsy was performed on any pup dying after PND 4 and prior to weaning; tissues were preserved for possible future histopathologic examination only as deemed necessary by the gross findings.

DEVELOPMENTAL LANDMARKS
- BALANOPREPUTIAL SEPARATION: Each male pup was observed for balanopreputial separation beginning on PND 35. The day on which balanopreputial separation was first observed was recorded for each pup. Examination of the pups continued daily until balanopreputial separation was present. Body weights were recorded on the day of acquisition of this landmark.
- VAGINAL PATENCY: Each female pup was observed for vaginal perforation beginning on PND 25 as described. The day on which the vaginal lumen was first observed to open was recorded for each pup. Examination of the females was continued daily until vaginal patency was present. Body weights were recorded on the day of acquisition of this landmark.

Postmortem examinations (parental animals):

SACRIFICE
- All F0 adults were euthanised following the selection of the F1 generation and completion of a detailed clinical observation. All surviving F1 adults were euthanised following weaning of the F2 pups.
- A complete necropsy and selective histopathologic examination were conducted following approximately 126 to 132 days of exposure for the F0 generation and approximately 146 to 155 days of exposure for the F1 generation.

MACROSCOPY
- A complete necropsy examination was conducted on all F0 and F1 parental animals found dead, euthanised in extremis or at termination (weaning of offspring for F1 and F2 generations). All animals were euthanised by isoflurane inhalation. The necropsy included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal and pelvic cavities including viscera.
- At the time of necropsy, the following F0 and F1 parental tissues and organs were collected and were placed in 10 % neutral-buffered formalin: Adrenals, Ovaries and oviducts, Aorta, Cervix, Bone with marrow (sternebrae), Pancreas, Brain (forebrain, midbrain, hindbrain), Peripheral nerve (sciatic), Coagulating gland, Pituitary, Eyes with optic nerve, Prostate, Gastrointestinal tract, Salivary gland [mandibular], Oesophagus, Seminal vesicles, Stomach, Skeletal muscle (rectus femoris), Duodenum, Skin with mammary gland, Jejunum, Spinal cord (cervical), Ileum, Spleen, Cecum, Colon, Testes with epididymides and vas deferens, Rectum, Thymus, Heart, Kidneys, Thyroids [with parathyroids, if present, Liver (sections of two lobes), Trachea, Lungs (including bronchi fixed by inflation with fixative), Urinary bladder, Uterus with vagina, Lymph node (mesenteric) and All gross lesions.

ORGAN WEIGHTS
- The following organs from all F0 and F1 parental animals euthanized at scheduled termination were weighed: Adrenal glands, Seminal vesicles with coagulating glands (with accessory fluids), Brain, Kidneys, Spleen, Liver, Testes and epididymides (total and cauda), Ovaries, Thymus gland, Pituitary, Uterus (with oviducts and cervix) and Prostate.

HISTOLOGICAL PROCEDURES AND MICROSCOPIC EXAMINATION
- Microscopic evaluations were performed on the following tissues for all F0 and F1 parental animals (30/sex/group) from the control and high exposure groups and for all adult animals found dead or euthanized in extremis: Adrenal glands, Brain, Cervix, Epididymis (right): caput, corpus and cauda, Kidneys, Liver, Lung, Nasal cavities, Ovaries, Pituitary, Prostate, Seminal vesicles with coagulating gland, Spleen, Testis (right), Thymus, Uterus (with oviducts), Vagina, Vas deferens and All gross (internal) lesions.
- Initially, histopathological examination of tissues from 10 F0 rats/sex/group from control and high dose groups were evaluated by light microscopy in accordance with EPA OPPTS Guideline 870.3800. To fully comply with the concurrent OECD guideline 416 for 2-generation reproductive toxicity studies, additional microscopic evaluation was required to increase the number of tissues examined in the F0 and F1 generation adults to 30 animals/sex/group. Because the nasal tissue was identified as a target tissue, all of the animals in the 5 and 20 ppm groups were examined in addition to the initial examination of the control and 50 ppm groups.
- After fixation, protocol-specified tissues were trimmed according to standard operating procedures and the protocol. Trimmed tissues were processed into paraffin blocks, sectioned at 4-8 microns, mounted on glass microscope slides and stained with haematoxylin and eosin.

Postmortem examinations (offspring):

- Prior to weaning, 30 F1 pups/sex/group were randomly selected for the F1 parental generation and for evaluation of developmental landmarks. Additional F1 pups (8, 15, 17 and 8 in the control, 5, 20 and 50 ppm groups, respectively) were retained as potential replacement animals; the animals that were not used for replacement were euthanised by CO2 inhalation and necropsied on PND 34 or 35. In addition, one F1 and one F2 pup/sex/litter (when available) were selected from the F1 and F2 weanlings for complete necropsy on PND 21 and only one F1 pup/sex/litter (when available) was selected for complete necropsy on PND 35. Brain, spleen and thymus gland weights were recorded for the selected PND 21 (F1 and F2) and surplus PND 35 (F1) pups.
- All remaining non-selected F1 and F2 weanlings were euthanised by CO2 inhalation and necropsied on PND 21 or 35, with emphasis on developmental and reproductive system morphology. All gross lesions from F1 and F2 weanlings were preserved in 10 % neutral-buffered formalin for possible future histopathologic examination; all other tissues were discarded.

Statistics:

All analyses were conducted using two-tailed tests for a minimum significance level of 5 %, comparing each test material-treated group to the control group.
- Parental mating and fertility indices were analysed using the Chi-square test with Yates’ correction factor.
- Mean parental (weekly, gestation and lactation) and offspring body weight data, parental food consumption and food efficiency data, oestrous cycle data (mean length of oestrous cycle), pre-coital intervals, gestation lengths, implantation sites, live litter sizes, unaccounted sites, numbers of pups born, balanopreputial separation (day of acquisition and body weight), vaginal patency (day of acquisition and body weight), anogenital distance, absolute and relative organ weights, sperm production rate and epididymal and testicular sperm numbers were subjected to a parametric one-way analysis of variance (ANOVA)13 to determine intergroup differences. If the ANOVA revealed statistical significance (p<0.05), Dunnett’s test was used to compare the test material-treated groups to the control group.
- Mean litter proportions (percent per litter) of postnatal pup survival, pup sexes at birth (percentage of males per litter), percentage of motile sperm, and percentage of sperm with normal morphology were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistical significance (p<0.05), the Mann-Whitney U-test was used to compare the test material-treated groups to the control group.
- Histopathologic findings in the test material-treated groups were compared to the control group using a two-tailed Fisher’s Exact test.

Reproductive indices:

MATING AND FERTILITY INDICIES:
- Male (Female) Mating Index (%): (No. of Males (Females) with Evidence of Mating (or Confirmed Pregnancy) / Total No. of Males (Females) Used for Mating) x 100
- Female Fertility Index (%): (No. of Females with Confirmed Pregnancy / Total No. of Females Used for Mating) x 100
- Male Fertility Index (%): (No. of Males Siring a Litter / Total No. of Males Used for Mating) x 100

Offspring viability indices:

LITTER INDICIES
- Live Litter Size = Total Viable Pups Day 0 / No. Litters With Viable Pups Day 0
- Postnatal Survival Between Birth and PND 0 or PND 4 (Pre-Selection) (% Per Litter) = [ Σ (Viable Pups Per Litter on PND 0 or PND 4/No. of Pups Born Per Litter) / No. Litters Per Group] x 100
- Postnatal Survival for All Other Intervals (% Per Litter) = [ Σ (Viable Pups Per Litter at End of Interval N/Viable Pups Per Litter at Start of Interval N) / No. Litters Per Group] x 100
Where: N = PND 0-1, 1-4 (Pre-Selection), 4 (Post-Selection)-7, 7-14, 14-21 or 4 (Post-Selection)-21

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related clinical findings were noted during the generation at the weekly detailed physical examinations or at the midpoint exposure and one-hour following exposure observations.
- Findings noted in the treated groups, including hair loss, swelling, scabbing and material findings on various body surfaces, decreased defecation, soft stool and malaligned incisors, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not exposure-related.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

All F0 parental animals in the control, 5, 20 and 50 ppm groups survived to the scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Mean weekly body weight gains in the F0 males in the 50 ppm group were statistically significantly (p<0.01) reduced during study weeks 0-1 and 1-2 when compared to the control group. These reductions were considered to be related to exposure. Mean body weight gains in the 5 and 20 ppm group F0 males were unaffected by exposure during these intervals. Other statistically significant reductions were noted in the F0 males in the 20 ppm group during study weeks 14-15 (p<0.05) and in the 50 ppm group during study weeks 9-10 (p<0.01) and 15-16 (p<0.05). These decreases were considered to be slight and/or transient and were, therefore, not related to exposure. Increases in mean body weight gain were noted in the 20 ppm group F0 males during study weeks 5-6 (p<0.05) and all male groups during study weeks 13-14 (p<0.05 or p<0.01). The only statistically significant difference noted in mean body weight was a decrease (p<0.05) in the 50 ppm group males during study week 3 as a result of decreased (4.8%) mean body weight gains related to exposure on study weeks 0-1 and 1-2. No other statistically significant differences in mean body weight were noted in this group. Mean body weights in the 5 and 20 ppm F0 males were unaffected by test material exposure.
- Mean body weight gains in the 5, 20 and 50 ppm group F0 females were not statistically significantly different from the control group throughout the study. Mean body weights in the 5, 20 and 50 ppm group F0 females were similar to the control group during the first seven weeks of the study. No statistically significant differences from the control group were noted. Exposure-related statistically significant decreases (p<0.05 or p<0.01) in mean body weight were noted in the 50 ppm group F0 females during study weeks 8, 9, 10 and 18 (4.5, 4.4, 5.9 and 5.2 %, respectively). No other statistically significant differences from the control group were noted.
- Cumulative mean body weight gains in the 50 ppm F0 male group were statistically significantly (p<0.05 or p<0.01) decreased when compared to the control group between study weeks 0-1 and 0-5 as a result of the aforementioned decreased mean body weight gains during study weeks 0-1 and 1-2. Cumulative mean body weight gains in these males were unaffected by exposure throughout the remainder of the study (study weeks 0-6 through 0-18); the differences from the control group were not statistically significant. In the 50 ppm group F0 females, cumulative mean body weight changes were similar to the control group during study weeks 0-1 through 0-4. Cumulative mean body weight gains in this group were reduced throughout the remainder of the pre-mating period and during the post-lactation period; the differences were generally statistically significant (p<0.05 or p<0.01). Cumulative mean body weights in the 5 and 20 ppm group F0 males and females were unaffected by exposure throughout the study; no statistically significant differences were noted.

GESTATION
- A statistically significant (p<0.05) decrease in mean body weight gain was noted in the 50 ppm group F0 females during gestation days 14-20 and resulted in a decreased mean body weight gain during gestation days 0-20 that was not statistically significant when compared to the control group. Mean body weights in the 50 ppm group F0 females were decreased throughout the gestation period; the differences from the control group were statistically significant (p<0.05 or p<0.01) at all intervals and were considered related to exposure.
- Mean body weights and body weight gains in the 5 and 20 ppm group F0 females were unaffected by exposure. A statistically significant (p<0.05) decrease in mean body weight gain was noted in the 5 ppm group during gestation days 0-4; however, because no exposure-response relationship was observed, the decrease was not attributed to the test material.

LACTATION
- Mean body weight gains in the 5, 20 and 50 ppm group F0 females were similar to the control group during the lactation period; none of the differences from the control group were statistically significant. Statistically significant decreases (p<0.01) in mean body weight were noted in the 50 ppm group F0 females on lactation days 1, 4 and 7. Mean body weights in the 5 and 20 ppm group F0 females were unaffected by exposure; none of the differences from the control group were statistically significant.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

- Weekly food consumption (evaluated as g/animal/day and g/kg/day) in the 5, 20 and 50 ppm group F0 males and females was unaffected by exposure to the test material. The only statistically significant (p<0.01) g/animal/day food consumption decrease in the 50 ppm group F0 females was noted during study week 17-18. Other statistically significant (p<0.05 or p<0.01) increases in the g/kg/day food consumption values were noted in the 50 ppm group F0 males and females during study weeks 1-2 through 8-9, 13-14, 14-15 and/or 17-18. The increase observed in the male group during study week 1-2 was related to the decrease observed in mean body weight. The g/animal/day values were also increased (p<0.05 or p<0.01) for these males during study weeks 13-14 and 14-15 and for these females during study week 1-2. In the 5 and 20 ppm F0 male and female groups, increased (p<0.05 or p<0.01) g/kg/day food consumption values were noted during study weeks 4-5, 7-8 and/or 8-9. No other statistically significant differences from the control group were noted.

GESTATION
Food consumption (evaluated as g/animal/day and g/kg/day) in the 5, 20 and 50 ppm group F0 females was unaffected by test material exposure during gestation. None of the differences from the control group were statistically significant.

LACTATION
Food consumption (evaluated as g/animal/day and g/kg/day) in the 5, 20 and 50 ppm group F0 females was unaffected by test material exposure during lactation. None of the differences from the control group were statistically significant.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

- Food efficiency was statistically significantly (p<0.05 or p<0.01) decreased during study week 14-15 in the 20 ppm group F0 males and during study weeks 0-1, 1-2, 9-10 and 15-16 in the 50 ppm group F0 males. The decreases observed in the 50 ppm group during study weeks 0-1 and 1-2 were related to the decreases observed in mean body weight gain. Increased food efficiency was noted in the 20 ppm group during study week 5-6 and in the 5, 20 and 50 ppm group F0 males during study week 13-14. Food efficiency in the 5, 20 and 50 ppm group F0 females was similar to the control group. The only statistically significant (p<0.05) difference from the control group was a decrease in the 50 ppm group during study week 2-3.

GESTATION
- Food efficiency in the 5, 20 and 50 ppm group F0 females was unaffected by test material exposure during gestation. None of the differences from the control group were statistically significant.

LACTATION
- Food efficiency in the 5, 20 and 50 ppm group F0 females was unaffected by test material exposure during lactation. None of the differences from the control group were statistically significant.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Test material-related microscopic findings were noted in the nasal tissues of F0 males and females in the 50 ppm group. Degeneration (minimal to moderate) of the olfactory epithelium was observed at all levels of the nasal tissues examined in which olfactory epithelium was present (Levels II, III and IV). The numbers of males affected in this exposure group at nasal tissue levels II, III and IV were 14/30, 11/30 and 10/30, respectively, and the numbers of females affected were 11/30, 10/30 and 6/29, respectively; all of the differences were statistically significant (p<0.05) when compared to the control group. Degeneration was characterised by loss of sustentacular cells, vacuolation and desquamation of neuroepithelial cells resulting in decreased height of olfactory epithelium. Basal cells and underlying structures such as Bowman’s glands and ducts, olfactory nerve bundles, and connective tissue were not affected. Degenerative changes were noted on the nasal septum, in the dorsal meatus and on turbinates. In some cases, lesions showed evidence of attempts at regeneration, characterised by the replacement of olfactory epithelium by ciliated columnar epithelium. A high incidence of perivascular mononuclear cell infiltrates, subacute inflammation, and/or alveolar macrophages in the lungs was observed in animals of both sexes in the control and high-exposure groups of the F0 generation; the differences from the control group were not statistically significant. These lesions are morphologically similar in appearance to idiopathic pulmonary lesions in rats which have recently been reported in several publications. The cause of these lesions has not yet been elucidated. The presence of the lesions in the animals of this study was not thought to affect the validity of the study results.
- All other microscopic changes were consistent with normal background lesions in clinically normal rats of the strain and age used in this study, and were considered to be spontaneous and/or incidental in nature and unrelated to test material exposure.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

GESTATION LENGTH AND PARTURITION
- The mean lengths of gestation in the F0 female groups were unaffected by test material exposure. Mean lengths were 21.8, 21.6 and 22.1 days in the 5, 20 and 50 ppm groups, compared to 21.6 days in the control group and 21.8 days in the WIL historical control data. The difference in the 50 ppm group was statistically significant at p<0.01. However, the duration of gestation was increased by only one-half day, and the value was within the WIL historical control data range (21.6-22.3 days). In addition, no statistically significant differences in mean gestation length were noted in the F1 generation; therefore, no relationship to exposure was apparent. No signs of dystocia were noted at any exposure level.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

- Individual variation in the oestrous cycle occurred in all study groups. The regularity and duration of oestrus were not affected by test article exposure. The mean lengths of oestrous cycles were 5.3, 5.3 and 5.5 days in the 5, 20 and 50 ppm F0 female groups, respectively, compared to 4.9 days in the control group and 4.3 days in the WIL historical control data. Although the values exceeded the maximum value in the WIL historical control data (5.1 days), the differences from the control group were not statistically significant and the apparent increases in mean duration of the oestrous cycle in the 5, 20 and 50 ppm groups were due to three, two and three females, respectively, having oestrous cycle lengths of ≥ 10 days.-In addition, no effects of exposure were observed on the mean numbers of days between pairing and coitus nor were any significant effects noted in the oestrous cycles in the F1 generation. Differences between the control and test article-exposed group values were slight and were not statistically significant. The number of females for which oestrous cycle length could not be determined (due to single occurrences of oestrus or the lack of occurrences of oestrus) were one, one and five females in the 5, 20 and 50 ppm groups, respectively.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

- Administration of the test material at exposure levels of 5, 20 and 50 ppm had no effects on F0 spermatogenic endpoints (mean testicular and epididymal sperm numbers, sperm production rate, sperm motility and sperm morphology). Differences between the control and exposure groups were slight and not statistically significant.

Reproductive performance:

no effects observed

Description (incidence and severity):

- Male and female mating indices were 100.0, 96.7, 100.0 and 93.3 % and male and female fertility indices were 96.7, 93.3, 90.0 and 90.0 in the control, 5, 20 and 50 ppm F0 groups, respectively. None of the differences in mating and fertility indices from the control group were statistically significant.
- The actual numbers of fertile females were 29, 28, 27 and 27 in the same respective groups. Males that did not sire a litter numbered one, two, three and three in the same groups, respectively. For females in the control, 5, 20 and 50 ppm groups, one, one, three and one, respectively, had physical evidence of mating but did not deliver; all of these females were nongravid. Two, one, two and one females in the control, 5, 20 and 50 ppm groups, respectively, had no evidence of mating, but delivered. One and two females in the 5 and 50 ppm groups, respectively, had no evidence of mating, did not deliver and were nongravid.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

parental systemic toxicty

Effect level:

5 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

food consumption and compound intake

food efficiency

2

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No exposure-related clinical findings were noted during the generation at the weekly detailed physical examinations or at the midpoint exposure and
one-hour following exposure observations. Findings noted in the treated groups, such as hair loss, swelling, scabbing and material findings on various body surfaces, soft stool, lacrimation, salivation and malaligned incisors occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not exposure-related.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

- One, one and two F1 males in the control, 5 and 50 ppm groups, respectively, were found dead between study weeks 18 and 36. Because of the mortality observed in the control group and the absence of clinical findings prior to death, the mortalities observed in the F1 males were not considered related to exposure. The cause of death for these males was undetermined. Additionally, one control group F1 female (no. 70129-12) was euthanised in extremis during study week 29 because of a mass on the right lateral abdominal area that was observed five times over a period of a month.
- All other F1 parental animals in the control, 5, 20 and 50 ppm groups survived to the scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Mean weekly body weight gains in the 50 ppm F1 male group were reduced (p<0.01) during study weeks 19-20 through 20-21. Throughout the remainder of the generation, male body weight gains in the 50 ppm group were similar to the control group (with statistically significant (p<0.05) increases during study weeks 25-26 and 33-34). By the end of the generation (study week 37-38), a statistically significant (p<0.05) decrease was noted in the 50 ppm F1 male group. Mean weekly body weight in these males were reduced from study week 17 through study week 38; the differences were generally statistically significant at p<0.05 or p<0.01 when compared to the control group. These reductions were considered to be related to exposure. Mean body weights and body weight gains in the 5 and 20 ppm group F0 males were unaffected by exposure. Intermittent, statistically significant (p<0.05 or p<0.01) increases and decreases in body weight gain were observed in the 20 ppm male group during the generation when compared to the control group. However, these differences were not attributed to exposure since the differences were not large and no trends were apparent.
- Mean weekly body weights in the 50 ppm F1 female group were decreased (7.5-13.6%) during study weeks 17-19 compared to the control group; the difference during study week 18 was statistically significant at p<0.05. Mean body weights in the 50 ppm F1 female group were generally similar to the control group during the remainder of the study; statistically significant (p<0.05) decreases were noted during study week 25 and 28. Mean weekly body weights in the 5 and 20 ppm F1 female groups and body weight gains in the 5, 20 and 50 ppm groups were unaffected by exposure to the test material. Statistically significant (p<0.05) increases in mean body weight gains during study weeks 19-20 and 26-27 and in mean body weight during study week 37 were noted in the 5 ppm F1 female group.
- Cumulative mean body weight gains in the 50 ppm F1 male groups were statistically significantly (p<0.05 or p<0.01) decreased when compared to the control group during study weeks 20-21 through 20-23 and during study week 20-38. Although not statistically significant, cumulative mean body weight gains in these males remained slightly decreased throughout the remainder of the study (study weeks 20-24 through 20- 37). A statistically significant (p<0.05 or p<0.01) decrease in cumulative mean body weight were noted in the 20 ppm group F1 males during study weeks 20-21 and 20-22 that was considered slight, transient and not related to exposure. During the remainder of the study (study weeks 20-24 through 20-38), cumulative mean body weights in the 20 ppm F1 male group were similar and not statistically significant compared to the control group. Cumulative mean body weights in the 5 ppm group F1 males and 5, 20 and 50 ppm F1 females were similar to the control group throughout the generation. The only statistically significant (p<0.05) differences noted in cumulative mean body weight gain were an increase in the 5 ppm group F1 females for study week 20-37 and a decrease in the 50 ppm group for study week 20-25.

GESTATION
- An exposure-related statistically significant (p<0.01) decrease in mean body weight gain was noted in the 50 ppm group F1 females during gestation days 14-20 and resulted in a decreased (p<0.01) mean body weight gain during gestation days 0-20 when compared to the control group. Mean body weights in the 50 ppm group F1 females were decreased (4.6-8.1 %) from gestation days 4 through 20; the differences from the control group were statistically significant at p<0.05 and p<0.01 during gestation days 11 and 20, respectively. The decreased body weight gain observed during gestation days 14-20 correlated with the decreased litter size and number of implantations observed in the 50 ppm F1 generation.
- Mean body weights and body weight gains in the 5 and 20 ppm group F1 females were unaffected by exposure.

LACTATION
- Mean body weights in the 50 ppm females were decreased throughout the lactation period; differences from the control group were statistically significant (p<0.05 or p<0.01) on lactation days 4, 7 and 14. Mean body weights in the 5 and 20 ppm groups and body weight gains in the 5, 20 and 50 ppm group F1 females were unaffected by exposure to the test material; none of the differences were statistically significant.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Weekly food consumption (evaluated as g/animal/day) in the 5, 20 and 50 ppm group F1 males and females was similar to the control group throughout exposure with the test material. The only statistically significant (p<0.05) difference from the control group was an increase during study week 19-20 in the 50 ppm group F1 females. Increased g/kg/day food consumption values, as a result of decreased mean body weights, were noted in the 50 ppm F1 male group throughout the generation and in the 50 ppm female group through study week 29-30 (prior to mating). The differences from the control group were generally statistically significant at p<0.01. Other differences in g/kg/day food consumption values noted in the 5 and 20 ppm groups did not occur in an exposure related manner, and no relationship to exposure was apparent.

GESTATION
- Food consumption (evaluated as g/animal/day and g/kg/day) in the 5, 20 and 50 ppm group F1 females was unaffected by test material exposure during gestation. Food efficiency in the 50 ppm group F1 females was slightly decreased during gestation days 0-4, 4-7, 14-20 and 0-20. The gestation day 0-20 value was statistically significant at p<0.01.

LACTATION
- Food consumption (evaluated as g/animal/day and/or g/kg/day) in the 20 and 50 ppm F1 groups was decreased (p<0.05 or p<0.01) during lactation days 1-4 (50 ppm group only), 7-14, 14-21 and 1-21. Food consumption in the 5 ppm group was similar to the control group during lactation; however, a statistically significant (p<0.05) decrease was noted in the g/kg/day value during lactation days 7-14.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

- Intermittent, statistically significant (p<0.05 or p<0.01) increases and decreases in food efficiency were observed in the 20 and 50 ppm group F1 males and females during the generation when compared to the control group. However, these differences were not attributed to exposure since the differences were not large and no trends were apparent. Food efficiency in the 5 ppm group was similar to the control group.

GESTATION
- Food efficiency in the 5 and 20 ppm group F1 females was unaffected by test material exposure during gestation. Food efficiency in the 50 ppm group F1 females was slightly decreased during gestation days 0-4, 4-7, 14-20 and 0-20. The gestation day 0-20 value was statistically significant at p<0.01.

LACTATION
- No statistically significant differences were noted in food efficiency among the control and test material exposure groups.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- Exposure-related effects on adrenal gland and thymus weights were observed in the 20 and/or 50 ppm groups. Mean adrenal gland weights (absolute and relative to final body weights) in the F1 males and females in the 50 ppm group were decreased when compared to the control group; the differences were statistically significant at p<0.01. The absolute adrenal gland values for the F1 males and females were 20.7 and 21.6 % lower than the control value. Increased (p<0.05 or p<0.01) thymus weights (absolute and relative to final body weight) were noted in the 20 and 50 ppm group males. The adrenal gland weight effects in the 50 ppm F1 males and females and thymus weight effects noted in the 50 ppm F1 males were considered related to exposure. However, no macroscopic or microscopic findings were noted in the F1 animals to correlate with the weight differences observed; therefore, the changes were not considered adverse.
- Mean absolute testicular and epididymal (total and cauda) weights in the 20 and 50 ppm groups were decreased compared to the control group; differences were often statistically significant (p<0.05 or p<0.01). Relative (to final body weight) left testicular weight in the 20 ppm group was statistically significantly (p<0.05) decreased compared to the control; no other differences from the control group were noted on the reproductive organs when evaluated relative to final body weight. However, the values were within the WIL historical control data ranges for these parameters, correlating microscopic changes were lacking and no effects on seminology parameters indicated that the reductions observed were secondary to the decreased body weight effects. Additionally, in a 13-week inhalation toxicity study in albino rats administered at 5, 20 and 70 ppm, there were no testicular or epididymal effects noted when evaluated for weight or microscopically at the exposure level of 70 ppm. Other organ weight changes that were not considered related to exposure included decreased (p<0.05 or p<0.01) mean absolute kidney and brain weights in the 50 ppm group F1 males and females, decreased relative kidney weight in the 5 and 20 ppm female groups, and increased (p<0.01) relative (to final body weight) liver weight in the 50 ppm group F1 males and females.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- One, one and two F1 males in the control, 5 and 50 ppm groups, respectively, were found dead between study weeks 18 and 36. Internal findings noted in the 50 ppm group included dark red areas on all lobes of the lung in a male and an enlarged liver in another male. The cause of death for these males was undetermined. A control group female was euthanised in extremis during study week 29 and had a skin mass on the right lateral abdominal area and an enlarged spleen. No other internal findings were noted for found dead F1 animals.
- At the scheduled F1 male and female necropsies, no test article-related internal findings were observed at any exposure level. A slight increase in the number of animals with a dilated renal pelvis was noted in the 5, 20 and 50 ppm F1 males and 20 and 50 ppm F1 females compared to the control group. However, the findings were usually unilateral and often could not be confirmed microscopically; therefore, no relationship to exposure was apparent. Other macroscopic findings observed in the exposure groups occurred infrequently, at similar frequencies in the control group and/or in a manner that was not exposure-related.
- Statistically significant (p<0.01) exposure-related decreases in the number of pups born (11.0 pups/dam) and the number of former implantation sites (11.7 sites/dam) were noted in the 50 ppm F1 females when compared to the control group (14.3 pups and 15.2 sites/dam, respectively). The number of unaccounted sites was similar between the control and 50 ppm groups. No treatment-related effects were observed on the number of pups born, the number of former implantation sites and the number of unaccounted sites in the 5 and 20 ppm F1 groups; the differences from the control group were slight and not statistically significant.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Test material-related microscopic findings were noted in the nasal tissues of F1 males and females in the 50 ppm group. Degeneration (minimal and/or mild) of the olfactory epithelium was seen at all levels of the nasal tissues examined in which olfactory epithelium was present (Levels I, II, III and IV). The numbers of males affected in this exposure group at nasal tissue levels I, II, III and IV were 1/28, 5/28, 4/28 and 2/28, respectively, and the numbers of females affected were 0/30, 7/30, 7/30 and 3/30, respectively. The differences from the control group were statistically significant (p<0.05) for the males at the nasal tissue level II and for females at nasal tissue levels II and III. Degeneration was characterised by loss of sustentacular cells, vacuolation and desquamation of neuroepithelial cells resulting in decreased height of olfactory epithelium. Basal cells and underlying structures such as Bowman’s glands and ducts, olfactory nerve bundles, and connective tissue appeared to be spared. Degenerative changes were noted on the nasal septum, in the dorsal meatus and on turbinates. In some cases, lesions showed evidence of attempts at regeneration, characterised by the replacement of olfactory epithelium by ciliated columnar epithelium. A high incidence of subacute inflammation and/or alveolar macrophages in the lungs was observed in animals of both sexes in the control and high exposure groups in the F1 generation; the differences from the control group were not statistically significant. These lesions are morphologically similar in appearance to idiopathic pulmonary lesions in rats which have recently been reported in several publications. The cause of these lesions has not yet been elucidated. The presence of the lesions in the animals of this study was not thought to affect the validity of the study results.
- All other microscopic changes (including statistically significant differences) were consistent with normal background lesions in clinically normal rats of the strain and age used in this study, and were considered to be spontaneous and/or incidental in nature and unrelated to test material exposure.
- An exposure-related statistically significant (p<0.05) increase in the number of primordial ovarian follicle counts was noted in the 50 ppm group (158.4) when compared to the control group value (132.9). In addition, a corresponding decrease (p<0.05) in the number of corpora lutea was also noted in this group. Although the primordial ovarian follicle counts in both the control and 50 ppm groups were elevated relative to the WIL historical control data (54.4-98.1), due to ease of visualisation of the primordial follicles because of the decreased numbers of corpora lutea, these changes were attributed to test material exposure. These findings correlated to the observed decreases in the number of implantation sites and F2 pups born.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

GESTATION LENGTH AND PARTURITION
The mean lengths of gestation in the F1 female groups were unaffected by test material exposure. Mean lengths were 21.7, 21.9 and 22.0 days in the 5, 20 and 50 ppm F1 groups, respectively, compared to 21.8 days in the control group and WIL historical control data. None of the differences from the control group were statistically significant. No signs of dystocia were noted.

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Individual variation in the oestrous cycle occurred in all study groups. The regularity and duration of oestrus were not affected by test article exposure. The mean lengths of oestrous cycles were 4.7, 5.1 and 4.6 days in the 5, 20 and 50 ppm F1 female groups, respectively, compared to 5.5 days in the control group and 4.3 days in the WIL historical control data. No effects of exposure were observed on the mean numbers of days between pairing and coitus. Differences between the control and treated-group values were slight and were not statistically significant.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Administration of the test material at exposure levels of 5, 20 and 50 ppm had no effects on F1 spermatogenic endpoints (mean testicular and epididymal sperm numbers, sperm production rate, sperm motility and sperm morphology). Differences between the control and exposure groups were slight and not statistically significant.

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating indices were 96.6, 100.0, 86.7 and 92.9 % in the control, 5, 20 and 50 ppm F1 male groups, respectively, and 96.6, 100.0, 86.7 and 93.3 % in the same respective F1 female groups. Male fertility indices were 86.2, 86.7, 76.7 and 82.1 % and female fertility indices were 86.2, 86.7, 76.7 and 80.0 % in the control, 5, 20 and 50 ppm F1 groups, respectively. The lack of a dose-relationship and absence of statistical significance indicate no effect of exposure on fertility indices. The actual numbers of fertile females were 26, 23 and 24 in the 5, 20 and 50 ppm groups, respectively, compared to 25 females in the control group. Males in the F1 generation that did not sire a litter numbered four, four, seven and five in the control, 5, 20 and 50 ppm groups, respectively. For F1 females in the control, 5, 20 and 50 ppm groups, three, four, three and five, respectively, had evidence of mating but did not deliver. One F1 female in the control group had no physical evidence of mating, but delivered. One, four and two F1 females in the control, 20 and 50 ppm groups, respectively, had no evidence of mating, did not deliver and were nongravid.

Effect levels (P1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

parental systemic toxicity

Effect level:

50 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

food consumption and compound intake

food efficiency

2

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

20 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

3

Key result

Dose descriptor:

LOAEL

Remarks:

reproductive toxicity

Effect level:

50 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

-The general physical condition of the F1 pups in the treated groups was similar to that in the control group during the postnatal period. An increased incidence of uneven hair growth was noted in the 50 ppm group; however, this finding was observed primarily in eight pups of a single dam.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

- In the 50 ppm group, the mean live litter size (12.5 pups/dam) was reduced in comparison to the mean values for the concurrent control group and WIL historical control data (both 14.2 pups/dam). Although the difference was not statistically significant, it was considered related to exposure as a result of the reduced viability in the pups in this group on PND 0. The mean number of pups born and percentage of males per litter at birth were unaffected by parental exposure at a exposure level of 50 ppm. The mean live litter size, mean number of pups born and percentage of males per litter at birth in the 5 and 20 ppm groups were unaffected by treatment.
- Exposure-related reductions in postnatal survival (percent per litter) were noted in the 50 ppm group on PND 0, PND 0 to 1, PND 1 to 4 and birth to PND 4 (pre-selection). The differences from the control group values were statistically significant (p<0.05 or p<0.01). The postnatal survival value for birth to PND 4 (pre-selection) in the 50 ppm group (74.3% per litter) was below the minimum value in the WIL historical control data (91.3% per litter). Three of 27 females in this group had total litter loss between lactation days 0 and 4, contributing to the early decrease in postnatal survival. Postnatal survival in this group was similar to that in the control group from PND 4 to 7, PND 7 to 14, PND 14 to 21 and from PND 4 (post-selection) to 21; differences from the control group were slight and were not statistically significant.
- Pup survival was not affected by exposure at parental exposure levels of 5 and 20 ppm. A statistically significant (p<0.05) decrease in postnatal survival was noted in the 5 ppm group during PND 1 to 4 (pre-selection). However, an exposure-response relationship was not observed; therefore, no relationship to treatment was apparent.
- Pups that were found dead or euthanised in extremis prior to weaning (PND 21) numbered 24, 40, 19 and 74 in the control, 5, 20 and 50 ppm groups, respectively. In the same respective groups, 11, 10, 12 and 29 pups were missing and presumed to have been cannibalised. Due to mortalities in the F1 weanlings after initiation of direct inhalation exposure on PND 22 in all groups (including the control group), test article exposures were suspended and re-initiated on PND 28, and the pups remained group-housed until PND 35. Three, four, two and one males and three, five, four and two females in the control, 5, 20 and 50 ppm groups, respectively, were found dead or euthanised in extremis and were replaced during PND 23-35. A record of the found dead and replacement animals is maintained in the study records. The numbers of pups found dead or euthanised in extremis following weaning to PND 35 were six, nine, six and three in the same respective groups; in addition, one pup in the 50 ppm group was missing.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean male and female pup body weight changes in the 50 ppm group were similar to the control group during PND 1-4 and 4-7. Mean offspring body weight gains in the 50 ppm group F1 males and females were decreased during PND 7-14 and 14-21 when compared to the control group, but only the 50 ppm F1 male difference during PND 7-14 was statistically significant (p<0.05). Male and female mean F1 offspring body weights in the 50 ppm group were similar to the control group values for PND 1, 4, 7 and 14. On PND 21, F1 male and female weights in this group were decreased (not statistically significant) by 10.0 and 7.8 %, respectively, when compared to the control group. Mean pup body weights and body weight changes in the 5 and 20 ppm group F1 males and females were unaffected by the test material throughout the postnatal period. No statistically significant differences were noted.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

effects observed, treatment-related

Description (incidence and severity):

BALANOPREPUTIAL SEPARATION
The mean ages of acquisition of balanopreputial separation were 45.1, 46.4 and 47.0 days in the 5, 20 and 50 ppm groups, respectively, when compared to 46.5 days in the control group. The differences from the control group were not statistically significant, and the values were within the WIL historical control data range (41.6-49.0 days). A statistically significant (p<0.01) decrease (7.3%) in mean male body weight on the day of acquisition was observed in the 50 ppm group. Mean body weights on the day of acquisition in the 5 and 20 ppm groups were unaffected by parental exposure.

VAGINAL PATENCY
The mean ages of acquisition of vaginal patency were 37.9, 38.8 and 40.0 days in the 5, 20 and 50 ppm groups, respectively, when compared to 37.0 days in the control group. The differences in the 20 and 50 ppm groups were statistically significant (p<0.05 and p<0.01, respectively) and the values were equal to or exceeded the maximum value in the WIL historical control data (38.8 days); therefore, the increases were considered exposure-related. Mean body weights on the day of acquisition in the 20 and 50 ppm groups were increased by 8.7 and 7.1 %, respectively, when compared to the control group; the differences were not statistically significant. The mean body weight on the day of acquisition in the 5 ppm group was unaffected by parental exposure.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

PND 21: ORGAN WEIGHTS
- Mean absolute thymus gland weights were decreased in the 20 and 50 ppm group F1 males (10.2 and 12.8 %, respectively) and females (9.9 and 16.9 %, respectively) when compared to the control group; the differences were not statistically significant. The relative (to final body weight) values for thymus weight for males and females in these groups were not statistically significantly different from the control group and/or did not occur in a dose-related manner. Mean thymus weights in the 5 ppm F1 male and female groups were similar to the control group.
- Mean brain and spleen weights (absolute and relative to final body weights) in the 5, 20 and 50 ppm group F1 males and females examined at the PND 21 necropsy were similar to the control group. Any differences from the control group were slight, not statistically significant and/or did not occur in an exposure-related manner.

PND 35: ORGAN WEIGHTS
- Mean brain, spleen and thymus gland weights (absolute and relative to final body weights) in the 5, 20 and 50 ppm group F1 males and females examined at the PND 35 necropsy were similar to the control group. Any differences from the control group were slight and/or did not occur in an exposure-related manner.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

NECROPSIES OF PUPS FOUND DEAD
- The numbers of pups (litters) found dead or euthanised in extremis from postnatal day 0 through the selection of the F1 generation numbered 30(14), 49(16), 25(14) and 77(20) in the control, 5, 20 and 50 ppm groups, respectively. No internal findings that could be attributed to parental exposure to the test material were noted at the necropsies of pups that were found dead or euthanised in extremis. Aside from the presence or absence of milk in the stomach, internal findings included a dilated renal pelvis in one pup in the 5 ppm group, red adrenal glands in one pup in this group, and a developmental variation consisting of a haemorrhagic ring around the iris in a pup in the 50 ppm group. No other internal findings were noted.

NECROPSIES OF PUPS NOT SELECTED FOR ORGAN WEIGHTS
- No internal findings that could be attributed to parental exposure with the test material were noted at the necropsy of pups euthanised on PND 21 or 35. A dilated renal pelvis was noted in a pup in the 20 ppm group. No other internal findings were noted in the 5, 20 and 50 ppm groups.

NECROPSIES OF PUPS SELECTED FOR ORGAN WEIGHTS
PND 21: MACROSCOPIC EXAMINATION
- At the PND 21 necropsy of F1 weanlings selected for organ weights, no internal findings were observed at any exposure level.

PND 35: MACROSCOPIC EXAMINATION
- At the PND 35 necropsy of F1 weanlings selected for organ weights, no internal findings were observed at any exposure level.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

The anogenital distances (absolute, relative to pup body weight and relative to the cube root of pup body weight) in the 5, 20 and 50 ppm groups were similar to the control group values. Any differences from the control group were slight and not statistically significant.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

5 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

sexual maturation

organ weights and organ / body weight ratios

Results: F2 generation

General toxicity (F2)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

- The general physical condition of the F1 pups in the treated groups was similar to that in the control group during the postnatal period. An increased incidence of uneven hair growth was noted in the 50 ppm group; however, this finding was observed only in the pups of a single dam.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

- In the 50 ppm F1 group, the mean live litter size (10.5 pups/dam) and the number of pups born (11.0 pups/dam) were reduced in comparison to the concurrent control group (13.9 and 14.3 pups/dam, respectively) and the minimum WIL historical control values (11.6 and 12.0 pups/dam, respectively). The differences were statistically significant (p<0.01) and were considered related to exposure. The percentage of F2 males per litter at birth was unaffected by F1 parental exposure at an exposure level of 50 ppm. The mean live litter size, mean number of pups born and percentage of males per litter at birth in the 5 and 20 ppm F1 groups were unaffected by treatment.
- Exposure-related reductions in F2 postnatal survival (percent per litter) were noted in the 50 ppm group for PND 0 to 1 and birth to PND 4 (pre-selection); the differences from the control group values were statistically significant (p<0.05 or p<0.01). The postnatal survival value for birth to PND 4 (pre-selection) in the 50 ppm group (86.1 % per litter) was below the minimum value in the WIL historical control data (91.3% per litter). Postnatal survival in the 50 ppm group was similar to that in the control group on PND 0, PND 1-4, PND 4 to 7, PND 7 to 14, PND 14 to 21 and from PND 4 (post-selection) to 21; differences from the control group were slight and not statistically significant. Pup survival was not affected by exposure at F1 parental exposure levels of 5 and 20 ppm; none of the differences from the control group were statistically significant.
- F2 pups that were found dead or euthanized in extremis prior to weaning (PND 21) numbered 15, 15, 8 and 31 in the control, 5, 20 and 50 ppm groups, respectively. In the same respective groups, 4, 4, 2 and 18 pups were missing and presumed to have been cannibalised. The increased number of pups found dead or euthanised in extremis in the 50 ppm group was considered related to exposure.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean male and female F2 pup body weight changes in the 20 and 50 ppm groups were similar to the control group during PND 1-4. Mean offspring body weight gains in the 20 and 50 ppm group F2 males and females were decreased (p<0.05 or p<0.01) during PND 4-7, 7-14 and 14-21 when compared to the control group. Mean F2 male and female offspring body weights during PND 1 and 4 were similar between the control, 5, 20 and 50 ppm groups. A statistically significant (p<0.05) decrease was noted in the 5 ppm female group on PND 1; however, no exposure-response relationship was observed. During the remainder of the pre-weaning period, mean F2 male and female body weights were reduced in the 50 ppm group on PND 7 (p<0.05 for the males only) and in the 20 and 50 ppm groups on PND 14 and 21 (p<0.01 for both sexes). A statistically significant (p<0.05) decrease in F2 female body weight was noted in the 5 ppm group on PND 14; however, because the decrease did not persist to PND 21 and was not observed in the males in this group, no relationship to exposure was apparent. No other differences were noted in pup body weight between the control and 5 ppm groups.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

PND 21: ORGAN WEIGHTS
Exposure-related reductions in mean absolute and relative (to final body weight) spleen and thymus weights were noted in the 20 and 50 ppm group F2 males and females when compared to the control group. The differences in absolute weights were generally statistically significant at p<0.01. The relative (to final body weight) thymus gland weight values in the 20 and 50 ppm female groups were also reduced (p<0.05) compared to the control group. Mean brain weights in the 50 ppm F2 male and female groups were also reduced compared to the control group; the differences in the male group were statistically significant at p<0.05. Mean relative (to final body weight) brain weight in the 20 ppm female and 50 ppm male and female groups were statistically significantly (p<0.05) increased compared to the control group as a result of the reductions (p<0.05 or p<0.01) in final body weight in these groups.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

NECROPSIES OF PUPS FOUND DEAD
- The numbers of pups (litters) found dead or that were euthanised in extremis during the postnatal period (prior to and following weaning) numbered 15(6), 15(10), 8(8) and 31(12) in the control, 5, 20 and 50 ppm groups, respectively. No internal findings that could be attributed to parental exposure to the test material were noted at the necropsies of pups that were found dead or euthanised in extremis. Aside from the presence or absence of milk in the stomach, nodules were noted in the right and left distal humeri in a pup from a dam in the control group. No other internal findings were noted.

NECROPSIES OF PUPS NOT SELECTED FOR ORGAN WEIGHTS
- No internal findings that could be attributed to F1 parental exposure with the test material were noted at the necropsy of F2 pups euthanized on PND 21. A dilated renal pelvis was noted in three, five, two and three pups in the control, 5, 20 and 50 ppm groups, respectively. No other internal findings were noted.

NECROPSIES OF PUPS SELECTED FOR ORGAN WEIGHTS
PND 21: MACROSCOPIC EXAMINATION
- At the PND 21 necropsy of F2 weanlings selected for organ weights, no internal findings that could be attributed to F1 parental exposure with the test material were noted. A dilated renal pelvis was noted in one male each in the 5 and 20 ppm groups and in one female in the 20 ppm group. In addition, one female in the 50 ppm did not have a right eye.

Histopathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

ANOGENITAL DISTANCE
- The anogenital distances (absolute, relative to pup body weight and relative to the cube root of pup body weight) in the 5, 20 and 50 ppm F2 groups were similar to the control group values. The only statistically significant (p<0.05) difference noted was an increase in the anogenital distance relative to pup body weight in F2 females in the 20 ppm group.
- Because a similar increase was not observed in the 50 ppm group, a relationship to exposure was not apparent.

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

5 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

sexual maturation

organ weights and organ / body weight ratios

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study the NOAEL for F0 and F1 parental systemic toxicity was 5 ppm. The NOAEL for reproductive toxicity was 50 ppm for the F0 generation. The NOAEL for F1 reproductive toxicity was 20 ppm. The NOAEL for F1 and F2 neonatal toxicity was 5 ppm.

Executive summary:

The objective of this study was to determine the potential adverse effects of the test material on reproduction in a two-generation study, in accordance with the standardised guidelines OECD416 and OPPTS 870.3800, under GLP conditions. This included determining the effects of the test material on male and female reproductive processes including gonadal function, oestrous cyclicity, mating behaviour, conception, gestation, parturition, lactation, weaning and on growth and development of the offspring. One litter was produced in each generation.

This study was conducted to evaluate the potential adverse effects of whole-body inhalation exposure of F0 and F1 parental animals to the test material on the reproductive capabilities of the F0 and F1 generations and F1 and F2 neonatal survival, growth, and development. Four groups of male and female rats (30/sex/group) were exposed to either clean filtered air or vapour atmospheres of the test material, for six hours daily for at least 70 consecutive days prior to mating.

F0 animals were approximately six weeks of age at the beginning of exposure. Offspring selected to become the F1 animals were initially exposed on postnatal day (PND) 22; however, due to the number of mortalities observed in the control and exposure groups, test material exposure was suspended and re-initiated on PND 28. Exposure of the F0 and F1 males continued throughout mating, and through the day prior to euthanasia. The F0 and F1 females continued to be exposed throughout mating and gestation through gestation day 20. After parturition, exposure of the F0 and F1 females was re-initiated on lactation day 5 and continued through the day prior to euthanasia. Target test material concentrations were 5, 20 and 50 parts per million (ppm) for the F0 and F1 generations. Mean measured exposure concentrations were 5, 20 and 50 ppm for the F0 generation. For the F1 generation, mean measured exposure concentrations were 5, 21 and 49 ppm.

All animals were observed twice daily for appearance and behaviour. Clinical observations, body weights, and food consumption were recorded at appropriate intervals prior to mating and during gestation and lactation. Daily vaginal smears were performed for determination of oestrous cycles beginning 21 days prior to pairing. All F0 and F1 females were allowed to deliver and rear their pups until weaning on lactation day 21. For the F1 and F2 generations, eight pups per litter (four per sex, when possible) were selected on PND 4 to reduce the variability among the litters. Following selection, F1 weanlings were exposed to the test material beginning on PND 28. Acquisition of developmental landmarks (balanopreputial separation and vaginal patency) was evaluated for the selected F1 rats. Unselected F1 pups were necropsied on PND 21 or 35, and F2 pups were necropsied on PND 21. Selected organs were weighed for both F1 and F2 pups. All surviving F0 and F1 parental animals received a complete detailed gross necropsy following the completion of weaning of the F1 and F2 pups, respectively; selected organs were weighed. Spermatogenic endpoints (sperm motility, morphology and numbers) were recorded for all F0 and F1 males, and ovarian primordial follicle counts and corpora lutea counts were recorded for all F1 females in each of the control and high-exposure groups. Designated tissues from all F0 and F1 parental animals were examined microscopically.

The adverse reproductive effects observed at 50 ppm in the F1 generation were a result of prolonged exposure over a period of two generations that lasted for 38 weeks, and were manifested by an increased mean number of primordial ovarian follicles with corresponding decreases in mean number of corpora lutea, implantation sites and live litter size (number of pups born). Mean live litter size was decreased in the 50 ppm group in both the F1 and F2 generations; however, the effect was more pronounced in the F2 pups. Postnatal survival of the 50 ppm group F1 pups was also decreased compared to the control group on PND 0, PND 0-1, PND 1-4 and birth to PND 4 (pre-selection), while only the PND 0-1 and birth to PND 4 intervals were affected for F2 pup survival in that same group.

Reductions in body weight, body weight gain and cumulative body weight gain were noted in the F0 and F1 generations of the 50 ppm group. Reductions in weekly mean body weight gain were noted in the 50 ppm F0 males primarily during the first two weeks (study weeks 0-1 and 1-2) of the study with corresponding decreases in food efficiency during these intervals. The weekly body weight gain reductions noted in the 50 ppm F0 females occurred later in the study (study weeks 8, 9, 10 and 18), and continued body weight reductions were noted throughout gestation and during lactation days 1, 4 and 7.

Cumulative body weight gain effects were noted in the F0 males and females from study weeks 0-1 through 0-5 and 0-5 through 0-18, respectively. Reduced offspring body weights as a result of F0 parental exposure were carried on in the 50 ppm F1 generation as exhibited by decreased pup body weight gains (both sexes) beginning on PND 7 and continuing through PND 21. Following weaning of the F1 generation, F1 male body weight was decreased on the day of acquisition of balanopreputial separation in the 50 ppm group and increases in body weight and age on the day of acquisition of vaginal patency were noted in the 20 and 50 ppm F1 females. Weekly F1 male body weight gain and cumulative body weights were generally reduced through study week 20-21 and weekly female body weight was slightly decreased during study weeks 17-19 with more significant decreases noted during gestation days 4-20 and throughout lactation. Reductions in body weight (PND 14 and 21) and body weight gain (PND 4-7, 7-14 and 14-21) extended into the F2 males and females in the 20 and 50 ppm groups. Corresponding decreases in food efficiency and food consumption were generally noted during the gestation (50 ppm F1 females) and lactation (20 and 50 ppm F1 females) periods.

Degeneration of the olfactory epithelium was noted in the nasal tissues (Levels I, II, III and/or IV) of the males and females in the 50 ppm group in both generations. Reductions in mean absolute and relative adrenal gland weights were noted in the 50 ppm males and females of both the F0 and F1 generations and increased thymus weight was noted in the 20 and 50 ppm males in both generations. Although these differences were considered test material-related, they were not considered adverse due to the absence of correlating microscopic changes. Reductions in thymus gland weights in the 20 and 50 ppm F1 pups and thymus gland and spleen weights in the 20 and 50 ppm F2 pups were noted for both sexes.

Based on the results of administration of the test material via whole-body inhalation exposure to rats, an exposure level of 5 ppm was considered to be the NOAEL (no-observed-adverse-effect level) for F0 and F1 parental systemic toxicity. The NOAEL for reproductive toxicity was 50 ppm for the F0 generation. The LOAEL (lowest observed- adverse-effect level) for reproductive toxicity was 50 ppm for the F1 generation. The NOAEL for reproductive toxicity was 20 ppm for the F1 generation dependent on microscopic ovarian quantification of corpora lutea and primordial follicles for the 5 and 20 ppm groups. The NOAEL for F1 and F2 neonatal toxicity was 5 ppm administered by inhalation exposure to rats.