Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Inhalation

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. (20 ppm = 116.11 mg/m^3).

Link to relevant study records
Reference
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
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
no
GLP compliance:
yes
Limit test:
no
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.
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
Dose / conc.:
5 ppm (nominal)
Dose / conc.:
20 ppm (nominal)
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
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
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: 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.
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
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
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: 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.
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
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
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
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.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
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
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.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
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
Reproductive effects observed:
not specified
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.

Effect on fertility: via inhalation route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
116.11 mg/m³
Study duration:
subchronic
Species:
rat
Additional information

2 Generation inhalation study

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. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

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.

Range-finding inhalation study

This range finding study was designed to determine atmospheric concentrations of the test material for a developmental toxicity study in rats; a two-generation reproductive toxicity study in rats and for a 13-week sub-chronic toxicity study in rats. The study design consisted of three separate phases, a pre-mating exposure phase, a reproductive toxicity phase and a sub-chronic toxicity phase. The first and second phases will be discussed in this section (the latter phase has been discussed under Section 7.5.2). The study was performed under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The pre-mating exposure phase consisted of three groups, each composed of 10 male and 10 female rats, exposed to the test material for six hours daily for 28 days prior to pairing until the day prior to necropsy for the males and through gestation day 11 for the females (for a total exposure period of 52 days and 41 to 51 days, respectively). Target exposure concentrations were 25, 75 and 150 ppm. Due to excessive toxicity observed at the 150 ppm exposure level, exposure concentrations were lowered to 100 ppm on study day 8; this exposure level is presented as 150/100 ppm. The measured mean exposure concentrations were 25, 73 and 148/99 ppm. For comparative purposes, a control group of identical design was administered filtered air on a comparable regimen. All animals were observed daily for appearance, behaviour and pharmacotoxic signs; detailed physical examinations were conducted weekly. Body weights and food consumption were recorded twice weekly prior to the breeding period (males and females) and until evidence of mating was observed (females); maternal body weights and food consumption were recorded on gestation days 0, 6, 9, 12 and 13. Laparohysterectomies were performed on gestation day 13. Females with no evidence of mating were necropsied 13 days following the completion of the mating period. Males were necropsied following the completion of the laparohysterectomies. Selected organs for males and females were weighed at the scheduled necropsy; lungs and gross lesions were retained.

The reproductive toxicity phase consisted of three groups, each composed of 15 F0 female rats, exposed to the test material for six hours daily from gestation day 0 through gestation day 20; exposure was suspended during the expected parturition period (gestation day 21 through lactation day 4), and resumed on lactation day 5 for surviving females and continued through lactation day 20 (the day prior to necropsy), for a total of 37 days of exposure. Target exposure concentrations were 25, 75 and 100 ppm. The measured mean exposure concentrations were 25, 72 and 99 ppm. One F1 male pup and one F1 female pup from each control and 25 ppm group litter were selected for exposure from postnatal day (PND) 22 through PND 26. All animals were observed daily for appearance, behaviour and pharmacotoxic signs within one hour (approximately) following exposure; detailed physical examinations were conducted weekly. Maternal body weights and food consumption were recorded on gestation days 0, 6, 9, 12, 15, 18 and 20; lactation body weights and food consumption were recorded on lactation days 0, 4, 5 (pre-exposure), 7, 14, and 21. F0 females were necropsied on lactation day 21; selected organs were weighed; lungs and gross lesions were retained. F1 pups were observed daily for general appearance, behaviour and survival; detailed physical examinations were recorded on PND 0, 1, 4, 7, 14 and 21. Pup weights were recorded on PND 1, 4, 7, 14 and 21. After PND 21, body weights were recorded on PND 22, 24 and 26 (exposure days 0, 3 and 5, respectively). Pups not selected for exposure were necropsied on PND 21 or 22. Pups selected for exposure were necropsied on PND 27. This study was designed as a dose range-finding study for a subsequent 90-day study and histopathological investigations were not conducted at the low and intermediate dose levels to enable a NOAEC to be defined.

In the pre-mating exposure phase, six males and five females in the 150/100 ppm group (exposed to 150 ppm) were euthanised in extremis prior to reducing the exposure level to 100 ppm on study day 8. Following reduction of the exposure level, one male was found dead on study day 20, one male each was euthanised in extremis on study days 30 and 46, and one female each was euthanised in extremis on study days 23, 30, 36 and 45. The most prevailing clinical signs for the males and females exposed to 150 ppm included piloerection, a hunched appearance, hypoactivity, clonic tremors of the forelimbs, repeated head jerk, drooping eyelids, yellow urogenital material, clear material around the mouth and red material around the nose at the weekly examinations and/or one hour following exposure. Test material-related mean body weight losses were observed in the 150/100 ppm group males and females (exposed at 150 ppm) and 75 ppm groups. No test material-related effects on body weights were observed in the 25 ppm group males and females. Food consumption (g/animal/day) in the 150/100 and 75 ppm groups was reduced. No test material-related effects on food consumption were observed in the 25 ppm group males and females. Mean gestation body weight gains and food consumption (g/animal/day) in the 75 ppm group females were numerically reduced compared to the control group values throughout gestation. At the scheduled laparohysterectomy on gestation day 13, the mean number of implantation sites in the 75 ppm group was reduced and the litter proportion of pre-implantation loss was increased compared to the control group values. The differences were not statistically significant; however, the values were out of the range of the WIL historical control data, and were attributed to the test material. No test material-related macroscopic findings were observed in the animals that were found dead or euthanised in extremis or at the scheduled necropsies. Mean absolute brain, liver and kidney weights in the 75 ppm group males and females were reduced (statistically significant) compared to the control group values. Mean absolute thyroid weights in the 75 ppm group males were increased (statistically significant) compared to the control group values. Mean absolute uterus/cervix/oviduct weights were also slightly but not statistically lower in the 75 ppm group when compared with the control group. No test material-related effects on organ weights were observed in the 25 ppm group males and females.

In the reproductive toxicity phase, twelve females in the 100 ppm group were euthanised in extremis or found dead during gestation. One female in the 75 ppm group was found dead during lactation. The mortality/moribundity of these animals was attributed to the test material. One, one, one and two females in the control, 25, 75 and 100 ppm groups, respectively, were euthanised due to total litter loss. One female in the control group was euthanised due to premature delivery. Several changes in the general clinical condition of the dams exposed to atmospheres of 100 ppm occurred prior to death or removal from study. These included splayed or dragging hindlimbs, clonic convulsions, a hunched appearance, hypersensitivity to handling, piloerection, repetitive movement of the mouth and jaws, whole body tremors, lethargy, red material around the nose, half-closed eyes, decreased defecation, salivation, clear material around the mouth and red urogenital material at the weekly and/or one-hour post-exposure examinations. Test material-related reduced mean maternal body weight gains or body weight losses and inhibition of food consumption were observed in the 100 ppm group throughout gestation. Evaluation of maternal body weight gains and food intake during lactation was precluded by mortality and total litter loss in the 100 ppm group. No test material-related internal findings were observed at the necropsies of females. In the 75 ppm group females examined on lactation day 21, mean absolute liver, kidney and uterus/cervix/oviduct weights were statistically significantly reduced compared to the control group value. In the 75 ppm group, 68 pups were found dead or euthanised in extremis and 34 pups were missing and presumed cannibalised prior to weaning. Due to the physical condition of the pups in the 75 ppm group, all pups were euthanised on PND 22 prior to the initiation of exposure. Mean F1 pup weight gains in the 75 ppm group males and females were reduced (statistically significant) compared to the control group during the three-week lactation period. During PND 21-22, mean body weight losses (statistically significant) were observed in these pups in comparison to the control group gains. At the necropsies of pups found dead, euthanised in extremis, or at the scheduled euthanasia, no test material-related internal findings were observed.

This study was designed as a dose range-finding study for a subsequent 90-day study and histopathological investigations were not conducted at the low and intermediate dose levels to enable a NOAEC to be defined.

Effects on developmental toxicity

Description of key information

Prenatal Rabbit Study (WIL-418002)

Under the conditions of this study, the NOAEL (no-observed-adverse-effect level) for maternal toxicity via whole-body inhalation exposure was considered to be 10 ppm, and the NOAEL for prenatal developmental toxicity was considered to be 2 ppm.

Prenatal Rat Study (WIL-418010)

Under the conditions of this study, the NOAEL (no-observed-adverse-effect level) for maternal toxicity was considered to be 20 ppm, the NOAEL for developmental toxicity was considered to be 60 ppm.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 April 2001 to 11 July 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
Draft
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: JMAFF 12 NouSan No. 8147
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: six and one-half months old
- Weight at study initiation: 2989 to 4133 g
- Housing: individually housed in clean, stainless-steel wire-bottom cages suspended above ground corn cob bedding.
- Diet: ad libitum (restricted to 150 g/animal/day during the acclimation period)
- Water: ad libitum
- Acclimation period: 20 or 27 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18.9 to 22.1 °C (66.0 to 71.7 °F)
- Humidity: 44.1 to 70.9 %
- 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/12-hour dark photoperiod.
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
EXPOSURE METHODS
- Because of the limited amount of space available in the exposure chambers, animals in each group were divided into two replicates. The exposures of each replicate of animals were conducted separately, with the initial exposure of animals in the second replicate occurring four weeks following the initial exposure of animals in the first replicate.
- All animals in each replicate were exposed simultaneously in four 1.5 m^3 stainless steel and glass whole-body exposure chambers. One chamber was dedicated for each group for the duration of exposures.
-The test material and filtered air were administered as daily six-hour exposures during a period that included major organogenesis, gestation days 6 through 28 (from implantation to one day prior to laparohysterectomy). All rabbits were exposed at approximately the same time each day, and exposures were conducted seven days per week. Animal cage batteries were rotated through the different cage rack positions within the chamber on a daily basis to minimize the effects of any potential variation due to chamber position. The control group was exposed to filtered air under conditions identical to those used for the test material exposure groups.
- The rabbits were removed from their home cages, placed in exposure caging in the animal room and transported to the exposure chambers for exposure to the test material. The animals were exposed for the requisite duration and then returned to their home cages. Food and water were withheld during each daily exposure period.
-The daily mean chamber temperatures were 72-77 °F (22-25 °C). The daily mean chamber humidity ranged from 42-76 %.
- Exposure concentrations within each chamber were measured at least 10 times (approximately every 35 minutes) during each daily exposure period by a validated gas chromatographic method.
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 resevoir 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 = 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 summarises the GC conditions:
Instrument: Hewlett Packard 5890 Series II with a 3396 Series II integrator
Detector: Flame ionisation
Column: J & W DB-Wax Narrow Bore, 30 m x 0.25 mm I.D., 0.25- micron film thickness
Gases: (Pressure (psig) Flow Rate (mL/min.)): Carrier - Helium 46 15.4, Fuel - Hydrogen 18 31, Air 35 300
Temperatures (°C): Injector 250, Column 45, isothermal and Detector 250
Injection volume (mL) 0.25
Retention time (min.) Approximately 1.0 min.
Integrator Run Parameters: Chart Zero Offset 0, Chart Attenuation 1, Chart Speed 2.5 cm/min, Peak Area Rejection Value 0, Peak Threshold 2 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: 32 L of air was added to the following to prepare the standards: 1.3 ppm: 0.1 µL test material, 6 ppm: 0.5 µL test material, 13 ppm: 1.0 µL test material, 21 ppm: 1.7 µL test material and 29 ppm: 2.3 µL test material
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 was within ± 20 % for the 1.3 PPM standard, ± 15 % for the 6 PPM standard, and ± 10 % for the remaining standards 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 Upper Front, Right Lower Rear, Left Lower Front, Left Upper Rear identified as 1, 2, 3 and 4, respectively for Chamber 4. The test locations for Chambers 2 and 3 were Right Upper Rear, Left Lower Rear, Right Lower Front and Left Upper Front, identified as 1, 2, 3 and 4, respectively. 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 each test material exposure chamber
- Results indicated that homogeneity of exposure atmospheres were adequate for the purpose of this study. Maximum mean % from reference was – 9.5 %.

RESULTS
- Nominal Exposure Concentrations: 4.7, 16 and 28 ppm
- Actual Exposure Concentrations: 2, 10 and 20 ppm
Details on mating procedure:
- Semen was collected from eight resident male rabbits of the same strain and obtained from the same source as the females. The collection apparatus consisted of an artificial vagina filled with warm water (approximately 55°C) and fitted with a graduated collection tube. After collection, the gelatinous plug (if any) was removed from each sample and the ejaculate was evaluated for volume, motility and concentration.
- Motility was determined microscopically on a percentage basis from a drop slide preparation using low-power magnification. The concentration of sperm/mL was determined using a standard dilution in a red blood cell pipette and a haemocytometer under high-power magnification. Semen with greater than 50 % motility was diluted with 3.0 mL of 0.9 % USP physiological normal saline and was maintained at 34-37 °C in a water bath during the insemination procedure. The final concentration obtained was greater than 3 million motile sperm/mL.
- Diluted semen from one male was used to inseminate two or four females in each group (a maximum of two females in each replicate in each group was inseminated with semen from one male). A 0.25- to 0.50-mL aliquot of each diluted semen sample was deposited into the anterior vagina of each female with a glass insemination pipette. Immediately following insemination, each doe was administered an intravenous injection (100 USP Units) of human chorionic gonadotropin via the marginal ear vein to induce ovulation.
- The day of insemination was designated gestation day 0.
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
once daily
Duration of test:
Gestation days 6 to 28
Dose / conc.:
2 ppm (analytical)
Dose / conc.:
10 ppm (analytical)
Dose / conc.:
20 ppm (analytical)
No. of animals per sex per dose:
24 females per dose
Control animals:
yes
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- All rabbits were observed twice daily, once in the morning and once in the afternoon (at least seven hours apart), for moribundity and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Individual detailed clinical observations were recorded from day 0 through 29 of gestation (prior to test article administration during the exposure period).
- Animals were observed for signs of toxicity at the midpoint of exposure and within one hour following exposure; all significant clinical findings were recorded at these observation periods.

BODY WEIGHT: Yes
- Individual maternal body weights were recorded on gestation days 0 and 6-29 (daily). A group mean body weight was calculated for each of these days. Mean body weight changes were calculated for each corresponding interval and also for gestation days 6-9, 9-15, 15-21, 21-29, 6-29 and 0-29.
- Gravid uterine weight was collected and net body weight (the day 29 body weight exclusive of the weight of the uterus and contents) and net body weight change (the day 0-29 body weight change exclusive of the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy.

FOOD CONSUMPTION: Yes
- Individual food consumption was recorded on a daily basis throughout gestation. Food intake was reported as g/animal/day and g/kg/day for the corresponding body weight change intervals.

GESTATION DAY 29 LAPAROHYSTERECTOMY
- All surviving females were euthanised on gestation day 29 by an intravenous injection of sodium pentobarbital via the marginal ear vein. The thoracic, abdominal and pelvic cavities were opened by a ventral midline incision and the contents examined. In all instances, the post mortem findings were correlated with the ante mortem comments and any abnormalities were recorded. The uterus and ovaries were excised and the number of corpora lutea on each ovary was recorded. The trimmed uterus was weighed and opened, and the number and location of all foetuses, early and late resorptions and the total number of implantation sites were recorded. The individual uterine distribution of implantation sites was documented using the following procedure. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn.
- Maternal tissues were preserved in 10 % neutral-buffered formalin for possible future histopathological examination only as indicated by the gross findings. The carcass of each female was then discarded. Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10 % ammonium sulphide solution for detection of early implantation loss.
Ovaries and uterine content:
GESTATION DAY 29 LAPAROHYSTERECTOMY
- The uterus and ovaries were excised and the number of corpora lutea on each ovary was recorded. The trimmed uterus was weighed and opened, and the number and location of all foetuses, early and late resorptions and the total number of implantation sites were recorded. The individual uterine distribution of implantation sites was documented using the following procedure. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn.
- Maternal tissues were preserved in 10 % neutral-buffered formalin for possible future histopathological examination only as indicated by the gross findings. The carcass of each female was then discarded. Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10 % ammonium sulphide solution for detection of early implantation loss.
Fetal examinations:
- Each foetus was weighed, tagged for identification, and euthanised by an intrathoracic injection of sodium pentobarbital, if necessary.
- A detailed external examination of each foetus was conducted to include, but was not limited to, an examination of the eyes, palate and external orifices, and each finding was recorded. Crown-rump measurements were recorded for nonviable foetuses and late resorptions, if present, the degree of autolysis was recorded, and the tissues were discarded. The sex of each foetus was determined internally and each foetus was examined viscerally by a fresh dissection technique to include the heart and major vessels. The brain from each foetus was examined by a mid-coronal slice. foetal kidneys were examined and graded for renal papillae development.
- All carcasses were eviscerated and fixed in 100 % ethyl alcohol. Following fixation in alcohol, each foetus was macerated in potassium hydroxide and stained with Alizarin Red. External, visceral and skeletal findings were recorded as developmental malformations (those structural anomalies that alter general body conformity, disrupt or interfere with body function, or may be incompatible with life) or 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).

Statistics:
All analyses were conducted using two-tailed tests for minimum significance levels of 5 and 1 %, comparing each treated group to the vehicle control group:
- One-way ANOVA with Dunnett’s test: Corpora Lutea, Total Implantations, Viable Foetuses, Foetal Body Weights, Maternal Body Weights, Body Weight Changes, Net Body Weights, Net Body Weight Changes and Gravid Uterine Weights and Maternal Food Consumption.
- Kruskal-Wallis test with Mann-Whitney U test: Litter Proportions of Intrauterine Data (Considering the Litter, Rather than the Foetus, as the Experimental Unit), Litter Proportions of Foetal Malformations and Developmental Variations.
Indices:
Group Mean Litter Basis:
- Postimplantation Loss/Litter = [No. Dead Foetuses, Resorptions (Early/Late)/Group] / No. Gravid Females/Group
- Proportional Litter Basis: Summation per Group (%) = [ΣPostimplantation Loss/Litter (%)*] / No. of Litters/Group
*= [[No. Dead Foetuses, Resorptions (Early/Late)/Litter ] / No. Implantation Sites/Litter] x 100

- The foetal developmental findings were summarised by: 1) presenting the incidence of a given finding both as the number of foetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison and calculating the number of affected foetuses in a litter on a proportional basis as follows:
Summation per Group (%) = (Σ Viable Foetuses Affected/Litter (%)) / No. of Litters/Group
Viable Foetuses Affected/Litter (%) = [(No. Viable Foetuses Affected/Litter) / No. Viable Foetuses/Litter] x 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The only exposure-related clinical finding observed was wet clear matting around the nose, noted at increased frequencies during the daily examinations (conducted prior to daily exposures) in all exposure groups relative to the control group. The matting was generally slight and was noted predominantly during the last week of exposure. This finding was considered to be exposure-related, but was not considered to be a sign of toxicity. It is acknowledged that wet clear matting around the nose was also observed in the control group, but the finding was noted less frequently and in fewer animals than in the exposed groups. Other clinical findings noted in the exposed groups, including hair loss, scabbing and staining on various body surfaces, soft stool and decreased defecation, occurred infrequently and/or at similar frequencies in the control group.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
All females survived to the scheduled laparohysterectomy on gestation day 29.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Mean maternal body weight changes in the 20 ppm group were similar to the control group values for gestation days 6-9, 9-15 and 15-21. During gestation days 24-29, a statistically significant (p<0.05) mean body weight loss was observed in this group. When the entire exposure period (gestation days 6-29) was evaluated, mean body weight gain in the 20 ppm group was reduced relative to the control group value; the difference was statistically significant (p<0.05). Mean body weights throughout gestation, net body weight and net body weight gain were unaffected by exposure to the test material in the 20 ppm group.
- In the 10 ppm group, mean body weights and body weight gains throughout gestation were unaffected by exposure to the test material. Differences from the control group values were slight and were not statistically significant. Mean gravid uterine weight in the 10 ppm group was slightly reduced (not statistically significant) relative to the control group value. This reduction was attributed to a decrease in the mean number of viable foetuses in this group. Mean net body weight and net body weight gain in the 10 ppm group were unaffected by test material exposure.
- Mean body weights and body weight gains throughout gestation, gravid uterine weight, net body weight and net body weight gain in the 2 ppm group were unaffected by exposure to the test material. Differences from the control group were slight and were not statistically significant.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Group mean food consumption, evaluated as g/animal/day and g/kg/day, throughout gestation was unaffected by test material exposure at exposure levels of 2, 10 and 20 ppm. Food consumption was slightly reduced from gestation days 24-29 in a few of the 10 and 20 ppm maternal animals which had one or more late resorptions at the post mortem examination. These reductions were not of significant magnitude to effect the group mean value. Sporadic statistically significant (p<0.05 or p<0.01) increases in food consumption were noted in the 2 ppm group; however, because similar increases were not observed at exposure levels of 10 and 20 ppm, the increases were not attributed to exposure.
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
-No exposure-related internal findings were noted at the scheduled necropsy on gestation day 29. Cystic oviducts were noted in 3, 1, 7 and 4 animals in the control, 2, 10 and 20 ppm groups, respectively. Accessory spleens were observed in 4, 2, 4 and 4 females in the same respective dose groups. A major blood vessel variation (the left carotid artery arose from the brachiocephalic trunk) was observed in one female in the 20 ppm group. One female in the 2 ppm group had a mass in the median lobe of the liver. No other internal findings were observed at the scheduled necropsy.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
not examined
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
- The mean litter proportion of post-implantation losses was increased in the 20 ppm group relative to the control group values; the increases were statistically significant (p<0.05 or p<0.01). The mean litter proportion of post-implantation losses was also increased relative to the maximum mean values in the WIL historical control data. These increases in post-implantation losses resulted in statistically significant (p<0.01) reductions in the mean litter proportion and mean number of viable foetuses in the 20 ppm group.
- In the 10 ppm group, increases (not statistically significant) were observed in the mean litter proportion of post-implantation losses. The value was higher than the mean values in the WIL historical control data. The increase in post-implantation loss resulted in a reduction (not statistically significant) in the mean number of foetuses in this group.
- No effects of the test material were observed on intrauterine parameters in the 2 ppm group.
Total litter losses by resorption:
effects observed, treatment-related
Description (incidence and severity):
The mean litter proportion of total resorptions was increased in the 20 ppm group relative to the control group values; the increases were statistically significant (p<0.05 or p<0.01).
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
- The mean litter proportion of late resorptions was increased in the 20 ppm group relative to the control group values; the increases were statistically significant (p<0.05 or p<0.01). The mean litter proportions of late resorptions was also increased relative to the maximum mean values in the WIL historical control data.
- In the 10 ppm group, and increase (not statistically significant) was observed in mean litter proportion of late resorptions. The value were higher than the mean value in the laboratory's historical control data (the mean litter proportion of late resorptions was also higher than the maximum mean value in the WIL historical control data). It should be noted that late resorptions in the 10 ppm group were noted more frequently in the first replicate than in the second replicate of animals.
- No effects of the test material were observed on intrauterine parameters in the 2 ppm group.
Dead fetuses:
not examined
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
not examined
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
10 ppm (analytical)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Mean foetal body weights (male, female and combined) were reduced in the 20 ppm group relative to the concurrent control group values; the differences were statistically significant (p<0.01). The combined value was also reduced relative to the minimum mean value in the WIL historical control data.
- Mean foetal body weights (male, female and combined) were reduced in the 10 ppm group relative to the control group values; the difference for the female value was statistically significant (p<0.05). Mean numbers of foetuses and mean combined foetal body weights were also reduced relative to the mean values in the WIL historical control data (although the mean foetal body weight value was within the range of the historical control data). When evaluating the individual data, mean foetal weight values for surviving foetuses from litters which expressed a high degree of late embryonic resorption were often lower than the group mean value.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
Mean numbers of foetuses awere reduced relative to the mean values in the WIL historical control data.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No effect of test material exposure at an exposure level of 20 ppm was observed on the foetal sex ratio.
Changes in litter size and weights:
not specified
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- External malformations were observed in 1(1), 2(2) and 1(1) foetuses (litters) in the control, 10 and 20 ppm groups, respectively. One foetus in the 20 ppm group had tarsal flexure. One foetus in the 10 ppm group had a disseminated subcutaneous haemorrhage in the dorsal posterior area. One foetus in the 10 ppm group had a bent medial tail (only 11 caudal vertebrae were present, and caudal vertebrae nos. 7 through 10 were fused and/or misshapen). One foetus in the control group had a small nose (the rostral portion of the nasal bones was fused).
- No external developmental variations were observed for foetuses in this study.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- Skeletal malformations were observed in three foetuses in the 2 ppm group and in one foetus in the 10 ppm group. One foetus in each of these groups had vertebral anomalies with associated rib anomalies; these anomalies consisted of malpositioned arches and centra, fused centra and ribs, extra ribs, an extra arch, a small arch and an absent centrum. One foetus in the 2 ppm group had severe malalignment of sternebrae nos. 2 through 5. One foetus in this litter had fusion of sternebrae nos. 4 and 5.
- Skeletal developmental variations observed in all exposure groups included 13th full ribs, 13th rudimentary ribs, 27 presacral vertebrae and bent hyoid arches. Other skeletal developmental variations noted in the exposed groups occurred infrequently, at similar frequencies in the control group and/or in a manner that was not dose-related.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- No soft tissue malformations were observed for foetuses in this study.
- Soft tissue developmental variations that were observed at all exposure levels included major blood vessel variations (either the left carotid artery arose from the brachiocephalic trunk or the right carotid and subclavian arteries arose independently from the aortic arch [no brachiocephalic trunk was present]). Accessory spleens were also observed at all exposure levels; the mean litter proportion of accessory spleens in the 10 ppm group was reduced (statistically significant at p<0.05) relative to the control group value. The reduction did not occur in a manner that was dose-related, and it was not attributed to test material exposure. Four and five foetuses in the 2 and 10 ppm groups, respectively, had small gallbladders; the mean litter proportion of small gallbladders was significantly (p<0.05) increased in the 10 ppm group relative to the control group value. Because the increase did not occur in a manner that was dose-related, it was not attributed to test material exposure. One foetus in the 10 ppm group had an undeveloped right renal papilla. Three foetuses in the 2 ppm group had retrocaval ureters. One foetus each in the control and 2 ppm groups had small spleens. A haemorrhagic ring was observed around the right iris of a single control group foetus. Because the findings in the exposed groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related, they were not attributed to test material exposure.
- Several soft tissue findings were observed that were not classified as either malformations or developmental variations and were not included in any tabulation. Thyroid glands that were dark red and/or enlarged were observed in four foetuses in the 10 ppm group. Reddened thymus glands were observed in one foetus each in the 2 and 10 ppm groups. A cystic ovary was observed in one foetus in the 20 ppm group. One foetus in the 2 ppm group had a white area on the liver, and one foetus in the control group had a cystic liver. Because these findings occurred in a manner that was not dose-related or occurred primarily in single foetuses or litters, they were not attributed to test material exposure.
Other effects:
not examined
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Key result
Dose descriptor:
NOAEL
Effect level:
2 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
changes in litter size and weights
Abnormalities:
not specified
Developmental effects observed:
not specified

Table 1: Selected caesarean section data

(ppm)

0

2

10

20

 

No. in gp

24

24

24

24

 

 

 

 

 

Late resorptions

(%/litter) 

1.7(23)

3.1(20)

11.1(20)#

21.5(21)**#

Late resorptions (%/litter) laboratory’s historical control range = 0.0 – 6.2

 

Post implantation loss (%/litter)

13.8(23)

12.1(20)

17.9(20)

32.3(21)**#

Post implantation loss (%/litter) laboratory’s historical control range = 0.6 – 23.1

 

Proportion of viable foetuses (%/litter)

86.2(23)

87.9(20)

82.1(20)

67.7(21)**#

Viable foetuses (%/litter) laboratory’s historical control range = 76.9 – 99.4

 

Mean number of viable foetuses/dam

6.1

5.5

4.6

3.6**#

Viable foetuses/dam laboratory’s historical control range = 4.74 – 8.3

 

Mean foetal

weight (g)

 

47.0(22)

45.8(19)

43.3(20)

37.8(20)**#

Mean foetal weight (g) laboratory’s historical control range = 39.2 – 51.8

 

* Statistically significant p<0.05); **Statistically significant (p<0.01); # Outside of laboratory’s historical control range

Conclusions:
Under the conditions of this study it was concluded that the NOAEL for maternal toxicity was 10 ppm and the NOAEL for prenatal developmental toxicity was 2 ppm.
Executive summary:

The potential maternal toxicity and prenatal developmental toxicity of the test material were evaluated in accordance with the standardised guidelines OECD 414, OPPTS 870.3700 and JMAFF 12 Nousan No. 8147, under GLP conditions.

The test material was administered via whole-body inhalation as a vapour to three groups of 24 artificially inseminated New Zealand White rabbits once daily for a period of six hours per day from gestation days 6 through 28. A concurrent control group, composed of 24 artificially inseminated rabbits, was exposed to clean, filtered air on a comparable regimen. Because of the limited amount of space available in the exposure chambers, each group was divided into two replicates. Artificial insemination of animals in each replicate was conducted over two days, with four weeks between replicates. The targeted exposure concentrations were 2, 10 and 20 ppm. The overall test atmosphere concentrations were measured using gas chromatography and were found to be 2, 10 and 20 ppm for each replicate. Clinical observations, body weights and food consumption were recorded. On gestation day 29, a laparohysterectomy was performed on all rabbits. The uteri and ovaries were examined and the numbers of foetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and the net body weight changes were calculated. Foetuses were weighed, sexed and examined for external, soft tissue and skeletal malformations and developmental variations.

All females survived to the scheduled laparohysterectomy on gestation day 29. Wet clear matting around the nose was observed at increased frequencies during the daily examinations in all exposure groups relative to the control group; while this finding was considered to be exposure-related, it was not considered to be a sign of toxicity. No exposure-related internal findings were noted at the scheduled necropsy.

In the 20 ppm group, mean body weight changes were similar to the control group values for gestation days 6-9, 9-15 and 15-21. During gestation days 24-29, a statistically significant mean body weight loss was observed in this group. Mean body weights throughout gestation, net body weight and net body weight gain in the 20 ppm group were unaffected by test material exposure. In the 10 ppm group, a slight (not statistically significant) reduction was observed in the mean gravid uterine weight. In the 2 and 10 ppm groups, mean body weights and body weight gains throughout gestation, net body weight and net body weight gain were unaffected by test material exposure. No effect of test material exposure was observed on mean gravid uterine weight in the 2 ppm group. Group mean food consumption, evaluated as g/animal/day and g/kg/day, was unaffected by test material exposure at all exposure levels throughout gestation. Food consumption was slightly reduced from gestation days 24-29 in a few of the 10 and 20 ppm maternal animals which had one or more late resorptions at the post mortem examination. These reductions were not of significant magnitude to effect the group mean value.

In the 10 and 20 ppm groups, increases were noted in mean litter proportions of late resorptions and postimplantation losses; the increases in the 20 ppm group were statistically significant relative to the control group values. The increases in mean litter proportions of late resorptions in both of these groups were higher than the maximum mean values in the WIL historical control data. These increases resulted in reduced (statistically significant for the 20 ppm group) mean numbers of foetuses in the 10 and 20 ppm groups. Mean foetal body weights (male, female and combined) were reduced in the 10 and 20 ppm groups; the reductions were statistically significant in both sexes in the 20 ppm group but only in female foetuses at 10 ppm. The mean foetal weight in the 10 and 20 ppm groups were 8 and 20 % lower, respectively, than the concurrent control value. The decrement in foetal body mass at term in the high exposure group was below the range of the WIL historical control data sets. In contrast, the value for the 10 ppm group was within the range of the historical control data. Thus, the effect at 10 ppm was interpreted as treatment-related, but the toxicological significance is considered equivocal. No effects of test material exposure were observed on intrauterine parameters in the 2 ppm group.

Foetuses (litters) available for morphological evaluation numbered 140(22), 109(19), 91(20) and 76(20) in the control, 2, 10 and 20 ppm groups, respectively. Malformations were observed in 1(1), 3(2), 3(3) and 1(1) foetuses (litters) from the same respective dose groups, and were considered to be spontaneous in origin. foetal developmental variations in the exposed groups occurred infrequently or at similar frequencies in the control group; no relationship to test material exposure was evident.

In conclusion, maternal toxicity was expressed in the 20 ppm group by reduced mean body weight gains or mean body weight losses. Prenatal developmental toxicity was expressed at exposure levels of 10 and 20 ppm by increases in post implantation losses (primarily late resorptions), reduced mean numbers of viable foetuses and/or reduced mean foetal body weights. Under the conditions of this study, the NOAEL (no-observed-adverse-effect level) for maternal toxicity via whole-body inhalation exposure was considered to be 10 ppm, and the NOAEL for prenatal developmental toxicity was considered to be 2 ppm.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 April 2001 to 23 July 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 12 weeks
- Weight at study initiation: 213 to 300 g on day 0 of gestation
- Housing: Upon arrival and until pairing, all rats were individually housed in clean, wire-mesh cages suspended above cage-board. The rats were paired for mating in the home cage of the male. Following positive identification of mating, the females were returned to an individual suspended wire-mesh cage
- Diet: ad libitum (during the exposure periods, food was withheld)
- Water: ad libitum (during the exposure periods, water was withheld)
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature: 67.2 to 68.3 °F
- Humidity: 47.5 to 63.3 %
- 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/12-hour dark photoperiod.
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
- All animals were exposed simultaneously in four 2.0 m^3 stainless steel and glass whole-body inhalation chambers. One chamber was dedicated for each group for the duration of exposures.
- Animals were individually caged m a single cage-battery that was rotated around the cage rack positions within the chamber on a daily basis to minimise the potential effects of any variation due to chamber position. The chambers were operated under dynamic conditions at air flows ensuring approximately 12 to 15 air changes per hour.
- The females were exposed to the test atmospheres for daily six-hour exposures (seven days per week) from gestation days 6 through 19. The control group was exposed to filtered air under conditions identical to those used for the test material-exposure groups.
- The rats were removed from their home cages, placed in exposure caging in the animal room and transported to the exposure chambers for exposure to the test material. The animals were exposed for the requisite duration and then returned to their home cages. Food and water were withheld during each daily exposure period.
- The daily mean chamber temperature was 18-25 °C. The daily mean chamber humidity ranged from 37-59 %.
- Vapours of the test material are generated using an ambient temperature bubbler-type vaporisation system, in which carrier gas (air) is dispersed (bubbled) through the liquid test material. In this process, the carrier gas picks up and vaporises the test material as the fine air bubbles pass through the liquid and into the vapour phase above the liquid. Glass gas washing bottles were used as bubblers. A 250 mL bottle with a 25 mm bottom fritted disc for gas dispersion was used for chambers 2 and 3 and a 250 mL bottle with a 50 mm fritted glass disc for gas dispersion was used for chamber 4. Liquid test material was added to the bubblers prior to daily exposures, as needed, and the bubblers were wrapped with aluminurn foil to protect the test material from light. Regulated compressed air controlled by metering needle valves was dispersed by the fritted disc and carried the test material vapours to the chamber inlet through delivery tubing. The generator airflow rate was monitored by compact rotarneters; 1-280 mL/min. rotameter for chamber 2 and 20-2100 mL/min. rotameter for
chambers 3 and 4. These rotameters had been pre-calibrated prior to animal exposures using a mini Buck Flow Calibrator. The concentrated vapours were piped from the gas washing bottles to a 2" I.D. glass chamber inlet, where the concentration was diluted to the target level by the chamber ventilation air flow. Compressed air was also metered to the control chamber to maintain consistency between the control and exposure system.
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
Column: J & W DB-Wax Narrow Bore, 30 m x 0.32 mm I.D., 0.25- micron film thickness
Gases: (Pressure (psig) Flow Rate (mL/min.)): Carrier - Helium 64 10.0, Fuel - Hydrogen 18 32, Air 35 300
Temperatures (°C): Injector 250, Column 45, isothermal and Detector 250
Injection volume (mL) 0.25
Retention time (min.) Approximately 0.944 min.
Integrator Run Parameters: Chart Zero Offset 0, Chart Attenuation 1, Chart Speed 2.5 cm/min, Peak Area Rejection Value 0, Peak Threshold 2 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 66 ppm: 5.2 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 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, Right Upper Rear, Left Lower Rear, Left Upper Front identified as 1, 2, 3 and 4, respectively. 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 each test material exposure chamber.
- Results indicated that homogeneity of exposure atmospheres were adequate for the purpose of this study. Maximum mean % from reference was –6.2 %.

RESULTS
- Nominal Exposure Concentrations: The overall mean nominal concentrations were 7.6 ppm, 26 ppm and 70 ppm for the 5 ppm, 20 ppm and 60 ppm groups, respectively.
- Actual Exposure Concentrations: The overall mean analysed concentrations were 5 ppm, 20 ppm and 60 ppm for the 5 ppm, 20 ppm and 60 ppm groups, respectively.
Details on mating procedure:
- M/F ratio per cage: Animals were paired on a 1:1 basis in a suspended wire-mesh cage with a resident male from the same strain and source for breeding.
- Resident males were untreated, sexually mature rats utilised exclusively for breeding. These rats were maintained under similar laboratory conditions as the females. The selected females were approximately 12 weeks old when paired for breeding.
- 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. Each mating pair was examined daily.
- The day on which evidence of mating was identified was termed day 0 of gestation. The animals were then separated.
Duration of treatment / exposure:
6 hours per day
Frequency of treatment:
Once daily
Duration of test:
Gestation days 6 - 19
Dose / conc.:
5 ppm (analytical)
Dose / conc.:
20 ppm (analytical)
Dose / conc.:
60 ppm (analytical)
No. of animals per sex per dose:
24 females per dose
Control animals:
yes
Details on study design:
- Dose selection rationale: Dose levels were selected based on the results of a previous range-fmding study. In that study, excessive maternal toxicity (manifested by mortality/moribundity, changes in the clinical condition of the animals, mean gestation body weight losses or reduced gains and reduced food consumption) was observed at an exposure level of 100/150 ppm. Maternal toxicity at an exposure level of 75 ppm was manifested by reductions in mean body weight gain and food consumption. The degree of suppression of body weight gain and food consumption at this exposure level was considered too excessive for selection of 75 ppm as an exposure level in the definite developmental toxicity study. Embryonic toxicity was exhibited at exposure levels of 75 and I 00/150 ppm by a reduction in the mean number of implantation sites. No maternal or embryonic toxicity was observed at an exposure level of 25 ppm.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Individual detailed clinical observations were recorded from day 0 through 20 of gestation (prior to test article exposure during the exposure period). Findings were also recorded within one hour following completion of daily exposures.

BODY WEIGHT: Yes
- Individual maternal body weights were recorded on gestation days 0 and 6-20 (daily). Group mean body weights were calculated for each of these days. Mean body weight changes were calculated for daily intervals and intervals 6-9, 9-12, 12-16, 16-20 and also for gestation days 6-20 and 0-20.
- Gravid uterine weight was collected and net body weight (the day 20 body weight minus the weight of the uterus and contents) and net body weight change (the day 0-20 body weight change minus the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy.

FOOD CONSUMPTION: Yes
- Individual food consumption was recorded on gestation days 0 and 6-20 (daily). Food intake was reported as g/animal/day and g/kg/day for the corresponding body weight change intervals. On the occasions when food intake could not be measured for one of the days in a given interval ( due to spillage, etc.), the values were calculated using the appropriate number of days for that interval.

POST-MORTEM EXAMINATIONS: Yes
- GESTATION DAY 20 LAPAROHYSTERECTOMY:All females were euthanised by intravenous injection of sodium pentobarbital via a tail vein on gestation day 20. The thoracic, abdominal and pelvic cavities were opened by a ventral midline incision, and the organs were examined. In all instances, the post mortem findings were correlated with the ante mortem comments, and any abnormalities were recorded.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- The uterus was excised and its adnexa trimmed. Gravid uterine weights were recorded. Corpora lutea were counted and recorded. The number and location of all in utero foetuses, early and late resorptions and the total number of implantation sites were recorded. The individual uterine distribution of implantation sites was documented using the following procedure. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn.
- Maternal tissues were preserved in 10 % neutral-buffered formalin for possible future histopathological examination only as indicated by the gross findings. Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10 % ammonium sulphide solution for detection of early implantation loss.
Fetal examinations:
MORPHOLOGICAL EXAMINATION
- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: Yes

- Each foetus was sexed, weighed and tagged for identification. A detailed examination of each foetus was conducted to include, but was not limited to, the eyes, palate and external orifices, and each finding was recorded. Crown-rump measurements were recorded for late resorptions, if present, and the tissues were discarded.
- Each foetus was examined viscerally by a modification of the fresh dissection technique to include the heart and major vessels.
- The sex of each foetus was verified by an internal examination.
- Foetal kidneys were examined and graded for renal papillae development.
- Heads from approximately one-half of the foetuses in each litter were placed in Bouin's fixative for subsequent soft-tissue examination. The heads from the remaining one-half of the foetuses were examined by a mid-coronal slice. The following cephalic specimens were separated from their identification tags at the time of examination, all of the specimens had no remarkable cephalic findings.
- All carcasses were eviscerated and fixed in 100 % ethyl alcohol. Following fixation in alcohol, each foetus was macerated in potassium hydroxide and stained with Alizarin Red S and Alcian Blue. The skeletal examination was conducted utilizing low power magnification provided by a stereomicroscope. External, visceral and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effects on animal health or body conformity, representing slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with body function or may be incompatible with life).
Statistics:
All analyses were conducted using two-tailed tests for minimum significance levels of 5 and 1 %, comparing each exposed group to the vehicle control group:
- One-way ANOVA with Dunnett's test: Corpora Lutea, Total Implantations, Viable Foetuses, foetal Weights, Maternal Body Weights and Weight Changes, Maternal Net Body Weights and Net Body Weight Changes, Gravid Uterine Weights and Maternal Food Consumption.
- Kruskal-Wallis test with Mann-Whitney U-test: Litter Proportions of Intrauterine Data (Considering the Litter, Rather than the Foetus, as the Experimental Unit), Mean Litter Proportions of Malformations and Variations
Indices:
Intrauterine data were summarised using two methods of calculation. An example of each method of calculation follows:
- Group Mean Litter Basis: Postimplantation Loss/Litter = (No. Dead Foetuses, Resorptions (Early/Late)/Group)/ No. Gravid Females/Group
- Proportional Litter Basis: Summation per Group (%) = ∑ (Postimplantation Loss/Litter (%)) / No. of Litters/Group
- Postimplantation Loss/Litter (%) = [(No. Dead Foetuses, Resorptions (Early/Late)/Litter) / (No. Implantation Sites/Litter)] x 100

The foetal developmental findings were summarised as: 1) presenting the number of foetuses and litters affected by a given finding and 2) considering the litter as the basic unit for comparison and calculating the number of affected foetuses in a litter on a proportional basis as follows:
- Summation per Group(%)= [∑ (Viable Foetuses Affected/Litter(%)*)] / No. of Litters/Group
Where * = [ (No. Viable Foetuses Affected/Litter) / No. Viable Foetuses /Litter ] x 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test material-related clinical signs were observed at the daily examinations or within one hour following exposure. Clinical findings were present prior to initiation of the exposure period, were observed similarly in the control group, were noted in single animals only and/or did not occur in an exposure-related manner.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
All animals survived to the scheduled laparohysterectomy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- A test material-related reduction in mean maternal body weight gain was observed in the 60 ppm group (0 grams) during gestation days 6-9 due primarily to a mean body weight loss of 11 grams on the first day of exposure (gestation days 6- 7). The differences from the control group values were statistically significant (p<0.01) at both intervals. Mean body weight gain in this group was similar to that in the control group during gestation days 9-12 and 12-16. During gestation days 16-20 and when the entire exposure period (gestation days 6-20) was evaluated, mean body weight gains in the 60 ppm group were reduced compared to the control group values; the differences were statistically significant (p<0.01 ). Mean body weights in the 60 ppm group were slightly reduced compared to the control group values from gestation days 7 through 20. The differences were statistically significant (p<0.05) on gestation days 7 and 20. Mean net body weight and net body weight gain in these females were reduced compared to the control group values; the differences were statistically significant (p<0.05 and p<0.01, respectively). Mean gravid uterine weight in the 60 ppm group females was similar to the control group value.
- Mean body weights, body weight gains, net body weights, net body weight gains and gravid uterine weights in the 5 and 20 ppm groups were unaffected by exposure to the test material.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- Food consumption, evaluated as g/anirnal/day and g/kg/day, was reduced in the 60 ppm group during gestation days 6-9; the differences from the control group values were statistically significant (p<0.01). Food consumption in these females was similar to that in the control group during gestation days 9-12 and 12-16. During gestation days 16-20, food consumption in the 60 ppm group was slightly reduced compared to the control group values (the g/animal/day difference was statistically significant at p<0.01). When the entire exposure period (gestation days 6-20) was evaluated, food consumption in this group was reduced (statistically significant at p<0.01) compared to that in the control group. The reductions in food consumption were attributed to the test material at this exposure level.
- Food consumption in the 5 and 20 ppm groups was similar to that in the control group throughout the study; no statistically significant differences were noted.
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- No test material-related internal findings were observed at the scheduled necropsy on gestation day 20.
- One female in the 5 ppm group had mottled lungs. No other internal findings were observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Description (incidence and severity):
No statistically significant differences were noted between the control and exposure groups.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
No statistically significant differences were noted between the control and exposure groups.
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Dead fetuses:
no effects observed
Description (incidence and severity):
No statistically significant differences were noted between the control and exposure groups.
Changes in pregnancy duration:
not specified
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified
Changes in number of pregnant:
not specified
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
20 ppm (analytical)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Fetal body weight changes:
no effects observed
Description (incidence and severity):
No statistically significant differences were noted between the control and exposure groups.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No statistically significant differences were noted between the control and exposure groups.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No statistically significant differences were noted between the control and exposure groups.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No statistically significant differences were noted between the control and exposure groups.
Changes in postnatal survival:
not specified
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- One foetus in the 60 ppm group had a cleft palate (entire length). The mean litter proportion of this malformation (0.3 %) was not statistically significantly different from the control group value (0.0%), and the value was within the range of the WIL historical control data (0.0-0.3% per litter). No relationship to exposure was evident. No other external malformations and no developmental variations were observed in foetuses at any exposure level.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- No skeletal malformations were observed in foetuses at any exposure level.
- Skeletal developmental variations that were noted in all exposure groups, including the control group, consisted of ossification of cervical centrum no. 1, un-ossified hyoid, 14th rudimentary ribs and sternebra(e) nos. 5 and/or 6 un-ossified. The incidences of these findings in the exposure groups were generally similar to the control group incidences. No statistically significant differences were noted. Other skeletal developmental variations noted in the test material-exposed groups, including 7th cervical rib(s), reduced ossification of the 13 th rib(s), 25 presacral vertebrae, 27 presacral vertebrae, bent rib(s), reduced ossification of the vertebral arches, 14 th full ribs and malaligned sternebra(e), were observed infrequently and/or were not observed in an exposure-related manner. The mean litter proportion for 7 th cervical ribs in the 5 ppm group (0.0 %) was statistically significant (p<0.05) when compared to the control group value (1.6 %). However, no statistically significant differences from the control group were noted for this finding in the 20 and 60 ppm groups; therefore, the difference in the 5 ppm group was attributed to biological variation.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- No soft tissue malformations were observed in foetuses at any exposure level.
- One foetus each in the control and 20 ppm groups had a major blood vessel variation, consisting of the right carotid and right subclavian arteries arising independently from the aortic arch (no brachiocephalic trunk). No exposure-related response was evident; therefore, this developmental variation was not attributed to test material exposure.
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
60 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No test material-related effects
Abnormalities:
not specified
Developmental effects observed:
not specified

Table 1: Mean body weight changes during gestation

Day

Mean body weight changes (g)

0 ppm

5 ppm

20 ppm

60 ppm

0-6

35

39

37

34

6-7

2

2

3

-11**

7-8

2

3

3

7*

8-9

2

3

3

4

9-10

4

4

3

5

10-11

5

5

4

4

11-12

5

5

5

5

12-13

6

4

5

6

13-14

6

7

6

4

14-15

8

8

7

8

15-16

9

9

10

9

16-17

12

14

12

11

17-18

17

15

15

14

18-19

15

15

14

13

19-20

17

14

16

12**

6-9

7

9

9

0**

9-12

15

15

12

13

12-16

29

27

27

27

16-20

60

58

56

49**

6-20

111

109

104

90**

0-20

145

147

141

124**

* = Significantly different from the control group at 0.05 using Dunnett's test

** = Significantly different from the control group at 0.01 using Dunnett's test

Table 2: Mean food consumption during gestation

Day

Mean food consumption (g)

0 ppm

5 ppm

20 ppm

60 ppm

0-6

23

23

23

23

6-7

25

23

23

13**

7-8

23

23

23

20**

8-9

23

23

23

21*

9-10

23

23

23

22

10-11

24

23

23

22

11-12

24

25

24

23

12-13

25

25

24

24

13-14

25

25

24

23

14-15

25

25

24

24

15-16

26

25

26

25

16-17

26

27

26

25

17-18

27

27

27

25*

18-19

27

27

26

25*

19-20

25

25

25

22**

6-9

24

23

23

18**

9-12

24

24

23

22

12-16

25

25

23

24

16-20

26

26

25

24**

6-20

25

25

26

23**

0-20

24

24

24

23**

* = Significantly different from the control group at 0.05 using Dunnett's test

** = Significantly different from the control group at 0.01 using Dunnett's test

Conclusions:
Under the conditions of this study, the NOAEL (no-observed-adverse-effect level) for maternal toxicity was considered to be 20 ppm, the NOAEL for developmental toxicity was considered to be 60 ppm.
Executive summary:

The developmental toxicity of the test material to rats was investigated in accordance with the standardised guidelines OECD 414 and OPPTS 870.3700, under GLP conditions.

The test material was administered via whole-body inhalation as a vapour to three groups of 24 bred female rats as a single daily exposure for a period of six hours per day from gestation days 6 through 19. The targeted exposure concentrations were 5, 20 and 60 ppm. The overall test atmosphere concentrations were measured using gas chromatography and were found to be 5, 20 and 60 ppm in the 5, 20 and 60 ppm groups, respectively. A concurrent control group composed of 24 bred female rats was exposed to clean, filtered air on a comparable regimen. On gestation day 20, a laparohysterectomy was performed on all rats. Gravid uterine weights were recorded, and net body weight changes were calculated. The uteri and ovaries were examined and the numbers of foetuses, early and late resorptions, total implantations and corpora lutea were recorded. Foetuses were weighed, sexed, and examined for external, visceral and skeletal malformations and developmental variations.

All maternal animals survived to the scheduled laparohysterectomy; no test material-related internal findings were observed at necropsy. No test material-related clinical observations were observed at any exposure level during the daily examinations or within one hour following the completion of exposure. Mean maternal body weight gain and food consumption in the 60 ppm group were reduced (generally statistically significant) relative to the control group during gestation days 6-9 and 16-20 and when the entire exposure period (gestation days 6-20) was evaluated. Mean body weight gain and food consumption in this group were similar to those in the control group during gestation days 9-12 and 12-16. Mean body weights in the 60 ppm group were slightly reduced during gestation days 7-20 (statistically significant on gestation days 7 and 20). Mean net body weight and net body weight gain in the 60 ppm group were reduced compared to the control group values; the differences were statistically significant. Mean gravid uterine weight in this group was similar to that in the control group. Mean body weights, body weight gains, net body weights, net body weight gains, gravid uterine weights and food consumption in the 5 and 20 ppm groups were unaffected by exposure to the test material.

Intrauterine growth and survival were unaffected by exposure to the test material at all exposure levels. Parameters included post-implantation loss, viable litter size, feta! body weights, feta! sex ratios and the numbers of corpora lutea and implantation sites. The numbers of foetuses (litters) available for foetal morphological evaluation were 367(24), 354(23), 337(23) and 326(22) in the control, 5, 20 and 60 ppm groups, respectively. A single malformation was observed in one foetus in the 60 ppm group; this malformation was considered to be spontaneous in origin. Developmental variations in the test material-exposed groups were noted infrequently, were not observed in an exposure-related manner or were observed at frequencies similar to those in the control group, and were not considered to be related to test material exposure.

In conclusion, maternal toxicity was expressed at an exposure level of 60 ppm by reductions in mean body weight gain, body weights, net body weight, net body weight gain and food consumption. No maternal toxicity was exhibited at exposure levels of 5 and 20 ppm. No developmental toxicity was manifested at any exposure level. Under the conditions of this study, the NOAEL (no-observed-adverse-effect level) for maternal toxicity was considered to be 20 ppm, the NOAEL for developmental toxicity was considered to be 60 ppm.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
11.61 mg/m³
Study duration:
subacute
Species:
rabbit
Additional information

Prenatal Rabbit Study (WIL-418002)

The potential maternal toxicity and prenatal developmental toxicity of the test material were evaluated in accordance with the standardised guidelines OECD 414, OPPTS 870.3700 and JMAFF 12 Nousan No. 8147, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was administered via whole-body inhalation as a vapour to three groups of 24 artificially inseminated New Zealand White rabbits once daily for a period of six hours per day from gestation days 6 through 28. A concurrent control group, composed of 24 artificially inseminated rabbits, was exposed to clean, filtered air on a comparable regimen. Because of the limited amount of space available in the exposure chambers, each group was divided into two replicates. Artificial insemination of animals in each replicate was conducted over two days, with four weeks between replicates. The targeted exposure concentrations were 2, 10 and 20 ppm. The overall test atmosphere concentrations were measured using gas chromatography and were found to be 2, 10 and 20 ppm for each replicate. Clinical observations, body weights and food consumption were recorded. On gestation day 29, a laparohysterectomy was performed on all rabbits. The uteri and ovaries were examined and the numbers of foetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and the net body weight changes were calculated. Foetuses were weighed, sexed and examined for external, soft tissue and skeletal malformations and developmental variations.

All females survived to the scheduled laparohysterectomy on gestation day 29. Wet clear matting around the nose was observed at increased frequencies during the daily examinations in all exposure groups relative to the control group; while this finding was considered to be exposure-related, it was not considered to be a sign of toxicity. No exposure-related internal findings were noted at the scheduled necropsy.

In the 20 ppm group, mean body weight changes were similar to the control group values for gestation days 6-9, 9-15 and 15-21. During gestation days 24-29, a statistically significant mean body weight loss was observed in this group. Mean body weights throughout gestation, net body weight and net body weight gain in the 20 ppm group were unaffected by test material exposure. In the 10 ppm group, a slight (not statistically significant) reduction was observed in the mean gravid uterine weight. In the 2 and 10 ppm groups, mean body weights and body weight gains throughout gestation, net body weight and net body weight gain were unaffected by test material exposure. No effect of test material exposure was observed on mean gravid uterine weight in the 2 ppm group. Group mean food consumption, evaluated as g/animal/day and g/kg/day, was unaffected by test material exposure at all exposure levels throughout gestation. Food consumption was slightly reduced from gestation days 24-29 in a few of the 10 and 20 ppm maternal animals which had one or more late resorptions at the post mortem examination. These reductions were not of significant magnitude to effect the group mean value.

In the 10 and 20 ppm groups, increases were noted in mean litter proportions of late resorptions and postimplantation losses; the increases in the 20 ppm group were statistically significant relative to the control group values. The increases in mean litter proportions of late resorptions in both of these groups were higher than the maximum mean values in the WIL historical control data. These increases resulted in reduced (statistically significant for the 20 ppm group) mean numbers of foetuses in the 10 and 20 ppm groups. Mean foetal body weights (male, female and combined) were reduced in the 10 and 20 ppm groups; the reductions were statistically significant in both sexes in the 20 ppm group but only in female foetuses at 10 ppm. The mean foetal weight in the 10 and 20 ppm groups were 8 and 20 % lower, respectively, than the concurrent control value. The decrement in foetal body mass at term in the high exposure group was below the range of the WIL historical control data sets. In contrast, the value for the 10 ppm group was within the range of the historical control data. Thus, the effect at 10 ppm was interpreted as treatment-related, but the toxicological significance is considered equivocal. No effects of test material exposure were observed on intrauterine parameters in the 2 ppm group.

Foetuses (litters) available for morphological evaluation numbered 140(22), 109(19), 91(20) and 76(20) in the control, 2, 10 and 20 ppm groups, respectively. Malformations were observed in 1(1), 3(2), 3(3) and 1(1) foetuses (litters) from the same respective dose groups, and were considered to be spontaneous in origin. foetal developmental variations in the exposed groups occurred infrequently or at similar frequencies in the control group; no relationship to test material exposure was evident.

In conclusion, maternal toxicity was expressed in the 20 ppm group by reduced mean body weight gains or mean body weight losses. Prenatal developmental toxicity was expressed at exposure levels of 10 and 20 ppm by increases in post implantation losses (primarily late resorptions), reduced mean numbers of viable foetuses and/or reduced mean foetal body weights. Under the conditions of this study, the NOAEL (no-observed-adverse-effect level) for maternal toxicity via whole-body inhalation exposure was considered to be 10 ppm, and the NOAEL for prenatal developmental toxicity was considered to be 2 ppm.

Prenatal Rat Study (WIL-418010)

The developmental toxicity of the test material to rats was investigated in accordance with the standardised guidelines OECD 414 and OPPTS 870.3700, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was administered via whole-body inhalation as a vapour to three groups of 24 bred female rats as a single daily exposure for a period of six hours per day from gestation days 6 through 19. The targeted exposure concentrations were 5, 20 and 60 ppm. The overall test atmosphere concentrations were measured using gas chromatography and were found to be 5, 20 and 60 ppm in the 5, 20 and 60 ppm groups, respectively. A concurrent control group composed of 24 bred female rats was exposed to clean, filtered air on a comparable regimen. On gestation day 20, a laparohysterectomy was performed on all rats. Gravid uterine weights were recorded, and net body weight changes were calculated. The uteri and ovaries were examined and the numbers of foetuses, early and late resorptions, total implantations and corpora lutea were recorded. Foetuses were weighed, sexed, and examined for external, visceral and skeletal malformations and developmental variations.

All maternal animals survived to the scheduled laparohysterectomy; no test material-related internal findings were observed at necropsy. No test material-related clinical observations were observed at any exposure level during the daily examinations or within one hour following the completion of exposure. Mean maternal body weight gain and food consumption in the 60 ppm group were reduced (generally statistically significant) relative to the control group during gestation days 6-9 and 16-20 and when the entire exposure period (gestation days 6-20) was evaluated. Mean body weight gain and food consumption in this group were similar to those in the control group during gestation days 9-12 and 12-16. Mean body weights in the 60 ppm group were slightly reduced during gestation days 7-20 (statistically significant on gestation days 7 and 20). Mean net body weight and net body weight gain in the 60 ppm group were reduced compared to the control group values; the differences were statistically significant. Mean gravid uterine weight in this group was similar to that in the control group. Mean body weights, body weight gains, net body weights, net body weight gains, gravid uterine weights and food consumption in the 5 and 20 ppm groups were unaffected by exposure to the test material.

Intrauterine growth and survival were unaffected by exposure to the test material at all exposure levels. Parameters included post-implantation loss, viable litter size, feta! body weights, feta! sex ratios and the numbers of corpora lutea and implantation sites. The numbers of foetuses (litters) available for foetal morphological evaluation were 367(24), 354(23), 337(23) and 326(22) in the control, 5, 20 and 60 ppm groups, respectively. A single malformation was observed in one foetus in the 60 ppm group; this malformation was considered to be spontaneous in origin. Developmental variations in the test material-exposed groups were noted infrequently, were not observed in an exposure-related manner or were observed at frequencies similar to those in the control group, and were not considered to be related to test material exposure.

In conclusion, maternal toxicity was expressed at an exposure level of 60 ppm by reductions in mean body weight gain, body weights, net body weight, net body weight gain and food consumption. No maternal toxicity was exhibited at exposure levels of 5 and 20 ppm. No developmental toxicity was manifested at any exposure level. Under the conditions of this study, the NOAEL (no-observed-adverse-effect level) for maternal toxicity was considered to be 20 ppm, the NOAEL for developmental toxicity was considered to be 60 ppm.

Prenatal Rabbit Study (WIL-418001)

During a prenatal developmental toxicity study, three groups (6 animals/group) of timed-pregnant female rabbits were exposed via whole body inhalation to the test material once daily for a period of 6 hours/day from gestation days (GD) 6-28. Target exposure concentrations were 10, 25 and 75 ppm, with a concurrent control receiving filtered air. Due to the degree of maternal toxicity observed, on GD 18, the maximum exposure was lowered to 50 ppm.

 Due to the toxicity observed in animals exposed at the maximum dose, 3 females were euthanised in extremis between GD 24-27. For animals which survived to the scheduled necropsy, test material related decreased defecation was observed in the 25 and 75/50 ppm groups.

 Food consumption (GD 10-21), mean net body weights (GD 14-29) and gravid uterine weights in the 75/50 ppm group were all reduced. When the entire exposure period was evaluated only statistically significant decreases in food consumption (p<0.05) were observed in this treatment group. Mean litter proportion of post implantation loss was significantly increased (p<0.05 or p<0.01) in both the 25 and 75/50 ppm groups. Similar findings were reported for the low (10 ppm) dose group, however this was not statistically significant, but considered potentially related to test material exposure.

 No external developmental variations were observed in foetuses in this study.

The objective of this study was to select dose levels for the definitive prenatal developmental toxicity study in rabbits. Based on this study, exposure levels of 2, 10 and 20 ppm were selected for the main study.

 

Rabbit Phased Exposure Supporting Study

The objective of this supplemental study was to determine the critical period of exposure during gestation that produced the embryo-fetotoxicity observed in a previous prenatal developmental toxicity study in rabbits. The study was performed in accordance with the standardised guidelines OECD 414, OPPTS 870.3700 and MAFF 12 Nousan No. 8147, under GLP conditions.

The test material, at the target exposure concentration of 20 ppm, was administered via whole-body inhalation as a vapour to six groups of 24 artificially inseminated female New Zealand White rabbits once daily for a period of six hours per day as follows. Group 2 animals were exposed during gestation days 6-28, Group 3 animals were exposed during gestation days 6-14, Group 4 animals were exposed during gestation days 15-22, Group 5 animals were exposed during gestation days 23-24, Group 6 animals were exposed during gestation days 25-26 and Group 7 animals were exposed during gestation days 27-28. For reporting purposes, these groups were designated the 20 ppm 6-28, 20 ppm 6-14, 20 ppm 15-22, 20 ppm 23-24, 20 ppm 25-26 and 20 ppm 27-28 groups, respectively. A concurrent control group, composed of 24 artificially inseminated rabbits, was exposed to clean, filtered air on a comparable regimen during gestation days 6-28. Because of the limited amount of space available in the exposure chambers, each group was divided into two replicates consisting of 12 animals each and artificial insemination of animals in each replicate was conducted over three days, with four weeks between replicates. The overall test atmosphere concentrations were measured using gas chromatography and were found to be 20 ppm for Group 2, 21 ppm for Group 3 and 21 ppm for Groups 4, 5, 6 and 7, respectively, for the first and second replicates. Clinical observations, body weights and food consumption were recorded. On gestation day 29, a laparohysterectomy was performed on all surviving rabbits. The uteri and ovaries were examined and the numbers of foetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and body weight changes were calculated. Foetuses were weighed, sexed and examined for external and soft tissue malformations and developmental variations.

The exposure windows were for different lengths of time varying from 2-23 days in duration throughout gestation (gestation days 6-28), early gestation (gestation days 6-14), mid gestation (gestation days 15-22) or during two-day specific intervals late in gestation (gestation days 23-24, 25-26 or 27-28). No exposure-related clinical or internal findings were noted for any female exposed in this study. Mean body weights, body weight gains, net body weights, net body weight gains and food consumption were not significantly affected by test material exposure. Gravid uterine weights for females exposed on gestation days 6-14, 15-22, 23-24, 25-26 and 27-28 were similar to the control group. Intrauterine parameters were unaffected when females were exposed on gestation days 6-14, 15-22 and 27-28. No exposure-related external or visceral malformations or developmental variations were observed in foetuses in this study. Test material-related effects noted in the group exposed during the entire gestation period from days 6-28 and in the groups exposed on days 23-24 and 25-26 consisted of increased number of late resorptions (statistically significant only in the 20 ppm 6-28 group). Test material-related effects noted in the group exposed during the entire gestation period from days 6-28 and in the group exposed for two days late in gestation on days 25-26 consisted of: Increased number of post-implantation losses (not statistically significant), decreased number of viable foetuses (statistically significant only in the 20 ppm 6-28 group) and decreased mean foetal weight (not statistically significant). Test material-related effects noted in the group exposed during the entire gestation period from gestation days 6-28 consisted of decreased gravid uterine weight (not statistically significant).

No maternal or developmental toxicity was observed for females exposed during gestation days 6-14, 15-22 or 27-28. However, exposure-related developmental effects (an increase in the number of late resorptions with or without a corresponding decrease in viable foetuses) were noted for females exposed during gestation days 6-28, 23-24 and 25-26. Although the developmental effects noted when animals were exposed on gestation days 23-24 and 25-26 were not statistically significant as noted when animals were exposed on gestation days 6-28, it can be deduced that the window of gestation days 23-26 was the most susceptible time of exposure for eliciting developmental toxicity when animals were exposed at a concentration of 20 ppm.

Toxicity to reproduction: other studies

Additional information

In order to illicit the mode of action, critical period during gestation which produced the embryo-foetotoxicity and what time this corresponded to during foetal development (observed in the prenatal developmental study in rabbits) three studies were designed and conducted in order to provide these answers.

 

In study TMN-603 the most susceptible period of exposure for eliciting developmental toxicity during gestation was deemed to be between GD 23 -26.

 

Study TMN-592 sodium iodide was confirmed to induce identical effects on foetal thyroid structure and function as the test material, iodomethane; thus identify iodide as the putative agent responsible for the disruption of the hypothalamic-pituitary-thyroid axis in foetal rabbits.

 

In study TMN-593 it was confirmed that foetal susceptibility to iodomethane during GD 23 -26 corresponded directly with the timing of key developmental events involving the rabbit foetal thyroid. Exposure to pregnant dams during this specific gestational period was found to cause significant disruption of the pituitary-thyroid axis in the developing foetus.

Justification for classification or non-classification

Additional information