Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19th October 1999 - 24th May 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Purity is 99.86 %

Test animals

Species:
rat
Strain:
other: CrI:CD (SD)IGS BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, North Carolina
- Age at start of acclimation period (F0) 29 days
- Age at start of dosing: (F0) 44 days; (F1) 22 days
- Weight at study initiation: (F0) Males: 171 - 229 g; Females: 141 - 184 g; (F1 mean dosage group weights) Males: 76.0 - 88.1 g; Females: 71.8 - 79.5 g
- Fasting period before study: NDA
- 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. Cage board was changed three times per week, with the following exception. Cage board was documented as being changed only twice during the week of March 20, 2000. This deviation did not affect the outcome of the study. The animals were paired for mating in the home cage of the male. Following positive evidence of mating, the males were housed in suspended wire-mesh cages until the scheduled necropsy of the parental generations, and the females were transferred to plastic maternity cages with nesting material. The dams were housed in these cages until weaning on lactation day 21. Following weaning of the litters in each generation (F1 and F2), the maternal females were individually housed in suspended wire-mesh cages until the scheduled necropsy, and the weaned F1 pups were housed together by litter for one week. Beginning on PND 28, the F1 pups were individually housed in suspended wire-mesh cages until the start of the mating period. Maternal females for which there was no evidence of mating were placed in plastic maternity cages with nesting material upon completion of a 14-day mating period. If these animals did not deliver after 25 days, they were returned to individual suspended wire-mesh cages.
- Use of restrainers for preventing ingestion (if dermal): N/A
- Diet (e.g. ad libitum): ad libitum except during exposure
- Water (e.g. ad libitum): ad libitum except during exposure
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2 ± 2 ºC
- Humidity (%): 30 - 70 %
- Air changes (per hr): ca. 10
- Photoperiod (hrs dark / hrs light): 12/12 from 6.am to 6.pm

IN-LIFE DATES: From: 19th October 1999 To: 18th July 2000

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 2.0 m^3 stainless steel and glass whole-body inhalation chamber
- Method of holding animals in test chamber: NDA
- System of generating Vapours: Vapours of the test article was generated using a glass-bead column type vapourisation system. Test article and vapourisation nitrogen are metered to the column and nitrogen enters below the glass beads while test article is deposited on the beads. Vapourisation occurs at the test article flows down over the beads and nitrogen moves up through the beads. The concentration vapours are piped to the chamber inlet where the concentration is reduced to the desired level by mixing with the chamber ventilation air. The vapourisation columns can be heated if necessary. The atmosphere for the control chamber was generated using using the same procedure except that no test article was added.

TEST ATMOSPHERE
- Brief description of analytical method used: Exposure concentrations within each chamber were measured 9 to 10 times (approximately every 35 minutes) during each daily exposure period by a validated gas chromatographic method. At least one standard was analysed each day prior to exposure to confirm gas chromatographic calibration. Chamber temperature, relative humidity, ventilation rate, and negative pressure within the chambers were monitored continuously and were recorded approximately every 35 minutes. Oxygen content within the chamber was measured during pre-study method development and at least once per month during the exposure phase, with the following exception. Oxygen content was not determined during June, 2000. This deviation did not affect the outcome of the study.
- Samples taken from breathing zone: N/A
Details on mating procedure:
- M/F ratio per cage: 1:1 during the mating period. Sexes were seperated at other times.
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug or evidence of sperm in vaginal smear.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility. Only one mating attempt
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually in a plastic cage.
- The day when evidence of successful mating was termed as gestation day 0.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Duplicate 10-mL retention samples (collected prior to the initiation of exposure) and residual samples (collected following completion of exposures) were collected; these samples were analysed for purity. The purity of the test article was a minimum of 99.8%. One 10-mL sample was collected from the first drum (at the end of the drum) and from the second drum (prior to use). The deviation from the number of samples to be collected (duplicate) did not affect the outcome of the study since these samples were back-up samples. The analysis of the residual samples demonstrated that the test article was stable throughout the course of the study.
Duration of treatment / exposure:
6 hours a day
Frequency of treatment:
daily
Details on study schedule:
- F1 parental animals not mated until 13 - 15 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: 16 - 19 weeks
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
100, 250, 500 and 750 ppm
Basis:
nominal conc.
750 ppm not used for F1 generation.
Remarks:
Doses / Concentrations:
99, 252, 505 and 750 ppm for the F0 generation
Basis:
analytical conc.
mean measured
Remarks:
Doses / Concentrations:
100, 252 and 504 ppm for the F1 generation.
Basis:
analytical conc.
mean measured
No. of animals per sex per dose:
25 per sex per dose
Control animals:
yes
Details on study design:
- Dose selection rationale: Target test article concentrations were 100, 250, 500 and 750 ppm (parts per million) for the F0 generation. Target test article concentrations for the F1 generation were 100, 250 and 500 ppm because infertility in the F0 750 ppm group precluded having an F1 750 ppm group.
- Rationale for animal assignment (if not random): At the conclusion of the acclimation period, all available F0 animals were weighed and examined in detail for physical abnormalities. At the discretion of the study director, animals judged to be in good health and meeting acceptable body weight requirements were selected for use in the computerised randomisation procedure.
Positive control:
Not used.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations checked were for moribundity and mortality. In addition, the animals were observed for appearance, behavior and pharmacotoxic signs within one hour after completion of exposure. 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
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Individual male body weights were recorded weekly throughout the study and prior to the scheduled necropsy. Individual female body weights were recorded weekly until evidence of copulation was observed.

FOOD CONSUMPTION: Yes
- Time schedule: Individual male and female food consumption was measured weekly until pairing. Food intake was not recorded during the mating period. 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 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 estrus for each female, beginning 21 days prior to pairing and continuing until evidence of mating was observed. The F0 generation was paired on the 22nd day of estrous smears and the F1 generation was paired on the 21st day of estrous smears. This deviation did not affect the outcome of the study. For females with no evidence of mating, smearing was continued until termination of the mating period. The average cycle length was calculated for complete estrous cycles (i.e. the total number of returns to metestrus [M] or diestrus [D] from estrus [E] or proestrus [P] beginning 21 days prior to initiation of the mating period and until the detection of evidence of mating). Estrous 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 estrous cycle length calculation. Vaginal smears were also performed on the day of necropsy to determine the stage of estrus.
Sperm parameters (parental animals):
Parameters examined in [F0/F1] male parental generations:
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. The right cauda epididymis was then placed in Dulbecco's phosphate buffered saline (maintained at approximately 37 ºC) 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 ºC) 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 analyser. 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 wet-mount 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, homogenized and evaluated for determination of homogenisation-resistant spermatid count and sperm production rate, using the method described by Blazak et al. and the Hamilton-Thorne CASA system.
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-NQS analyser allowed for the visualisation 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
- If yes, maximum of 4/sex/litter as nearly as possible; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2] offspring:
Mortality, clinical observations, body weights, sex determination, live litter size, post natal survival.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for animals found dead after PND 4
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals necropsied following selection of the F1 generation.
- Maternal animals: All surviving animals necropsied following selection of the F1 generation.

GROSS NECROPSY
- A complete necropsy examination was conducted on all parental animals (F0 and F1) euthanised in extremis or at termination. All animals were euthanised by an injection of sodium pentobarbital. 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
Aorta
Bone with marrow (sternebrae)
Brain (forebrain, midbrain, hindbrain)
Coagulating gland
Eyes with optic nerve
Gastrointestinal tract including:
Esophagus
Stomach
Duodenum
Jejunum
Ileum
Cecum
Colon
Rectum
Heart
Kidneys
Liver (sections of two lobes)
Lungs (including bronchi, fixed by inflation with fixative)
Lymph node (mesenteric)
Ovaries* and oviducts
Pancreas
Peripheral nerve (sciatic)
Pituitary
Prostate
Salivary gland [mandibular]
Seminal vesicles
Skeletal muscle (vastus medialis)
Skin with mammary gland
Spinal cord (cervical)
Spleen
Testes with epididymides** and vas deferens
Thymus
Thyroids [with parathyroids, if present]
Trachea
Urinary bladder
Uterus with vagina
All gross lesions

* = Five sections of each ovary from F0 and F1 females were examined.
** = The right testis and epididymis were fixed in Bouin's solution. Both testes and epididymides from animals euthanised in extremis were fixed in Bouin's solution.

ORGAN WEIGHTS

The following organs from all F0 and F1 parental animals euthanised at scheduled termination were weighed:

Adrenal glands
Brain
Epididymidesa (total and cauda)
Kidneys
Liver
Lungs
Ovaries
Pituitary
Prostate
Seminal vesicles with coagulating glands (with accessory fluids)
Spleen
Testes*
Thymus gland
Uterus with oviducts and cervix

* = These paired organs were weighed separately.

Except as noted, paired organs were weighed together. Absolute weights and organ to final body weight ratios were reported.

HISTOPATHOLOGY

Microscopic evaluations were performed on the following tissues for all F0 and F1 parental animals from the control and high exposure groups and for all adult animals euthanised in extrernis:

Adrenal glands: cortex and medulla
Brain
Epididymis (right): caput, corpus and cauda
Coagulating gland
Kidneys
Liver
Lungs
Ovaries
Oviducts
Pituitary
Prostate
Seminal vesicles
Spleen
Testis (right)
Thymus
Uterus
All gross (internal) lesions
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age.
- These animals were subjected to postmortem examinations 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 (except as noted below) for a minimum significance level of 5% comparing each treated group to the control group. Each mean was presented with the standard deviation (S.D.) and the number of animals (N) used to calculate the mean. Data obtained from nongravid animals were excluded from statistical analyses following the mating period. Statistical analyses were not performed when weekly food or body weight data for one or more animals were not available because the animals remained in the lactation phase. Statistical tests were performed using appropriate computing devices or programs.
Reproductive indices:
Mating and fertility indices were calculated as follows:

Male (Female) Mating Index (%) = (No. of Males (Females) with Evidence of Mating) / (Total Number 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 parameters were defined as follows:

Live Litter Size = Total Viable Pups Day 0 / Number of Litters With Viable Pups Day 0

Postnatal Survival Between Birth and PND 0 or PND 4 (% Per Litter) = (Σ(Viable Pups Per Litter on PND 0 or PND 4 / Number of Pups Per Litter) / Number of 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 if Interval N) / Number of Litters Per Group) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Reproductive function: sperm measures:
effects observed, treatment-related
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

F0 GENERATION

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)

Female no. 32821 in the 500 ppm group was euthanised in extremis on study day 119 (study week 17) due to an enlarged, glaucomatous eye with a dry cornea and lids that were unable to close. The eye abnormality of this animal was not attributed to the test article. All other F0 males and females survived to the scheduled necropsy.
There were no exposure-related clinical observations in the F0 males and females at the weekly examinations or one hour following the completion of exposure. Clinical findings were observed infrequently, with similar incidence in the control group and/or did not occur in an exposure-related manner.

BODY WEIGHT(PARENTAL ANIMALS)

WEEKLY - Mean body weight gains were reduced in the 750 ppm group F0 males during weeks 0-1 to 4-5 when compared to the control group values. The differences for weeks 0-1, 1-2 and 4-5 were statistically significant (p<0.05 or p<0.01). Throughout the remainder of the generation (weeks 5-6 to 18-19), mean body weight gains in these males were similar to the control group values, with the exception of statistically significant (p<0.05 or p<0.01) decreases (weeks 7-8, 12-13 and 16-17) or increases (week 13 - 14). Cumulative body weight gains (from study week 0) in the 750 ppm group males were reduced throughout the generation. The differences from the control group values were statistically significant (p<0.01). Mean body weights in these males were 5.4%-12.7% lower than the control group values from week 3 through the end of the generation (study week 19). The differences were statistically significant (p<0.01) from week 5 through week 19.
Mean weekly body weight gains in the 750 ppm group F0 females were reduced during study week 0-1; the difference from the control group value was statistically significant (p<0.01). Throughout the remainder of the pre-mating period (weeks 1-2 to 9-10), mean body weight gains in these females were similar to the control group values, with the exception of a statistically significant (p<0.01) decrease during week 4-5. Following weaning (week 18-19), mean body weight gain was reduced; the difference was statistically significant (p<0.01). Cumulative body weight gains in the 750 ppm group females were reduced when compared to the control group values throughout the pre-mating period. The differences were generally statistically significant (p<0.05 or p<0.01). Mean weekly body weights in the 750 ppm group females were comparable to the control group values during weeks 0-4. Mean body weights in these females were 5.0%-5.8% lower than the control group values during weeks 5-10. The differences were statistically significant (p<0.05) on study weeks 7 and 8.
Mean body weight gains in the 500 ppm group males were slightly reduced during weeks 0-1 to 4-5; the differences from the control group values were not statistically significant. Throughout the remainder of the generation, mean body weight gains in these males were generally similar to the control group values. The only statistically significant (p<0.05) difference from the control group values was a decrease during week 7-8. Cumulative body weight gains in the 500 ppm group males were similar to the control group values during weeks 0-1 to 0-7, but were slightly reduced throughout the remainder of the generation (weeks 0-8 to 0-19); the differences were not statistically significant. Mean body weights in these males were 4.7% - 6.3% lower than the control group values during study weeks 12-19. The differences were not statistically significant.
No exposure-related effects on mean weekly body weights, body weight gains and cumulative body weight gains were observed in the 100 and 250 ppm group males and the 100, 250 and 500 ppm group females. The only statistically significant differences from the control group values were reductions (p<0.01) in mean body weight gain in the 250 ppm group males during study week 1-2 and in cumulative body weight gain in the 250 ppm group females during study week 0-3. Similar reductions were not observed in the 500 ppm group. Therefore, these transient reductions were attributed to biological variation.

GESTATION - There were no gravid females in the 750 ppm group, precluding evaluation of gestation body weight data.
Maternal body weight gains in the 250 and 500 ppm groups were comparable to the control group values during gestation days 0-4, 4-7 and 7-11. Mean body weight gains in these groups were reduced compared to those in the control group during gestation days 11-14, 14-20 and when the overall gestation period (days 0-20) was evaluated. The differences were statistically significant (p<0.05 or p<0.01) during gestation days 14-20 and 0-20 (500 ppm group only). Mean gestation body weights in the 250 and 500 ppm group females were similar to the control group values on gestation days 0, 4, 7 and 11. Mean gestation body weights in these females were reduced and statistically significant (p<0.05 or p<0.01) compared to the control group values on gestation days 14 (500 ppm group only) and 20. The reductions in body weights and body weight gains late in gestation were attributed to the reduced litter sizes in these groups.
Mean gestation body weights and body weight gains in the 100 ppm group females were similar to those in the control group. No statistically significant differences were noted.

LACTATION - Evaluation of lactation body weight data in the 750 ppm group was precluded by infertility.
Maternal body weight gain in the 500 ppm group females was slightly reduced (not statistically significant) during lactation days 1-4 and 7-14.
Mean body weight gain in this group was similar to the control group value during lactation days 4-7. A mean body weight loss in the 500 ppm group females during lactation days 14-21 was less than the loss in the control group. When the entire lactation period (lactation days 1-21) was evaluated, mean body weight gain in the 500 ppm group females was similar to the control group value. Mean body weights in this group were reduced throughout lactation. The differences from the control group were statistically significant (p<0.05 or p<0.01) on lactation days 4, 14 and 21.
Mean body weight changes in the 250 ppm group females were similar to those in the control group during lactation, with the following exception. Mean body weight gain in this group was reduced (not statistically significant) during lactation days 7-14. This transient reduction during the middle of the lactation period was not attributed to the test article. When the entire lactation period (days 1-21) was evaluated, mean body weight gain in these females was similar to the control group value. Mean body weights in the 250 ppm group females were similar to the control group values throughout the lactation period. The differences were not statistically significant.
Mean lactation body weights and body weight changes in the 100 ppm group females were similar to those in the control group. No statistically significant differences were observed.

FOOD CONSUMPTION (PARENTAL ANIMALS)

WEEKLY - Food consumption, evaluated as g/animal/day and g/kg/day, in the 750 ppm group males and females was not adversely affected by test article exposure during the pre-mating (weeks 0-10) and/or post-mating (weeks 13-19) periods. Differences from the control group values were generally slight and not statistically significant, with the following exceptions. Slight reductions (p<0.05 or p<0.01) in g/animal/day food consumption were noted for the males in this group during the last three weeks of the generation (weeks 16-17, 17-18 and 18-19) and for the females during the last week of the generation (week 18-19). These slight reductions were not attributed to the test article. Slightly increased g/kg/day food consumption values were noted in the 750 ppm group beginning during week 1-2 and continuing until week 9-10 (females) or week 14-15 (males); the differences from the control group values were generally statistically significant (p<0.01 or p<0.05). The increases in g/kg/day food consumption were attributed to the reduced mean body weight gains in these animals during the pre-mating period, and were not considered to be direct effects of the test article. Food efficiency in the 750 ppm group males was slightly reduced relative to the control group values throughout the pre-mating period; the differences during weeks 0-1, 1-2, 4-5 and 7-8 were statistically significant (p<0.01 or p<0.05). During the post-mating period, food efficiency in these males was unaffected by test article exposure; the only statistically significant (p<0.05) differences from the control group were an increase during week 13-14 and a decrease during week 16-17. Food efficiency in the 750 ppm group females was slightly reduced in comparison to the control group values during weeks 0-1 through 4-5; the differences for weeks 0-1 and 4-5 were statistically significant (p<0.01). Throughout the remainder of the pre-mating period, food efficiency in these females was similar to the control group values. Food efficiency in the 750 ppm group females was reduced (p<0.05) during the last week of the generation (week 18-19).
Weekly food consumption (g/animal/day and g/kg/day) and food efficiency in the 500 ppm group males and females were not adversely affected by treatment. Differences from the control group values were generally slight and not statistically significant, with the following exceptions. A slightly decreased (p<0.01) glanimal/day value during week 18-19, slightly increased (p<0.05 or p<0.01) g/kg/day values during weeks 3-4, 4-5, 9-10 and 13-14, and a decreased (p<0.05) food efficiency value during week 7-8 were noted in the 500 ppm group males. In the 500 ppm group females, g/animal/day food consumption during week 34 and g/kg/day food consumption during weeks 1-2 through 5-6, 8-9 and 9-10 were increased (p<0.05 or p<0.01). Similar increases were not observed in the 750 ppm group females during this period. Therefore, these increases were not attributed to the test article.
Food consumption (g/animal/day and g/kg/day) and food efficiency in the 100 and 250 ppm group males and females were similar to the control group values during the pre-mating and/or post-mating periods. Differences were slight and were not statistically significant, with the following exceptions. During study week 16-17, g/animal/day food consumption in the 100 ppm group males was decreased (p<0.05). The g/kg/day food consumption in the 250 ppm group females was increased (p<0.05 and p<0.01) during study weeks 4-5 and 5-6, respectively. Food efficiency values for the 250 ppm group males and females during study weeks 1-2 and 0-1, respectively, were reduced (p<0.05). No exposure-relationship was noted for these transient changes in food consumption and food efficiency in the 100 and 250 ppm groups, and the differences were attributed to biological variation. No other differences from the control group values were statistically significant.

GESTATION - Evaluation of gestation food consumption data in the 750 ppm group was precluded by infertility.
Maternal food consumption, evaluated as g/animal/day, in the 500 ppm group females was slightly reduced (not statistically significant) throughout gestation (days 0-4, 4-7, 11-14 and 14-20) in comparison to the control group values. These slight reductions resulted in a statistically significant (p<0.0I) reduction in g/animal/day food consumption in these females when the overall gestation period (days 0-20) was evaluated. The g/animal/day food consumption during gestation days 7-11 and the g/kg/day food consumption throughout gestation in the 500 ppm group females were generally similar to the control group values; none of the differences were statistically significant. Food efficiency in these females was similar to the control group values during gestation days 0-4, 4-7, 7-11 and 11-14. During gestation days 14-20 and when the overall gestation period (days 0-20) was evaluated, food efficiency in the 500 ppm group females was reduced relative to the control group values; the differences for gestation days 14-20 and 0-20 were statistically significant (p<0.01). The reduction in food efficiency late in gestation was attributed to the reduced number of fetuses developing in utero.
Food consumption (g/animal/day and g/kg/day) in the 250 ppm group females was comparable to the control group values throughout gestation, with the following exception. During the last week of gestation (days 14-20), g/animal/day food consumption in the 250 ppm group females was slightly reduced (statistically significant at p<0.05) relative to the control group value. Food efficiency in these females was similar to the control group values throughout gestation.
Gestation food consumption and food efficiency in the 100 ppm group females was unaffected by exposure to the test article.

LACTATION - Evaluation of lactation food consumption data in the 750 ppm group females was precluded by infertility.
Food consumption, evaluated as g/animal/day and g/kg/day, in the 250 and 500 ppm group females was reduced (often statistically significant at p<0.05 or p<0.01) throughout the entire lactation period (days 1-4, 4-7, 7-14 and 14-21) and when the overall lactation period (days 1-21) was evaluated. Food efficiency in these females during lactation was similar to the control group values. None of the differences were statistically significant. The reductions in food consumption during lactation in the 250 and 500 ppm groups were attributed to decreased maternal lactational demand as a result of the reductions in mean litter sizes.
Lactation food consumption and food efficiency in the 100 ppm group females were similar to the control group values. No statistically significant differences were observed.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)

The mean lengths of estrous cycles were 4.2, 4.5, 4.7, 5.5 and 5.6 days in the control, 100, 250, 500 and 750 ppm groups, respectively. The values for the 500 and 750 ppm groups were above the range in the WIL historical control data (4.1-5.1 days). In addition, the length of estrous cycles could not be determined for 2 and 3 females in the 500 and 750 ppm groups, respectively, because no complete estrous cycles occurred in these females. Therefore, the increases in the 500 and 750 ppm groups were considered to be test article-related.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)

Mean epididymal sperm numbers (369.6 million cells/gram of tissue), sperm motility (53.2%) and the percentage of morphologically normal sperm (90.6%) in the 750 ppm group F0 males were reduced when compared to the control group values (471.9 million cells/gram of tissue, 86.8% and 99.7%, respectively); the differences were statistically significant (p<0.01). Morphologically abnormal sperm in observations in the 750 ppm group consisted of normal head separated from flagellum and normal flagellum with absent head. Mean testicular sperm numbers and sperm production rate in the 750 ppm group males were similar to those in the control group.
In the 500 ppm group males, sperm motility (71.9%) and the percentage of morphologically normal sperm (98.2%) were reduced and statistically significant (p<0.01) when compared to the above-mentioned control group values. Morphologically abnormal sperm observations in the 500 ppm group consisted of normal head separated from flagellum and normal flagellum with absent head. Mean testicular and epididymal sperm numbers and sperm production rate in the 500 ppm group males were similar to those in the control group.
In the 100 and 250 ppm group males, no exposure-related effects on spermatogenic endpoints were observed. The only statistically significant difference from the concurrent control group values was a slight decrease (p<0.05) in the percentage of morphologically normal sperm (99.3%) for the 250 ppm group males. The value was greater than the mean value of the WIL historical control data (99.0%), and the negligible difference from the control group value (99.7%) was not considered to be exposure-related.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)

Test article-related reductions in reproductive performance were observed in the 500 and 750 ppm group males and females. Male and female fertility indices were 92.0%, 100%, 88.0%, 52.0% and 0.0% in the control, 100, 250, 500 and 750 ppm groups, respectively. The 500 and 750 ppm group differences were statistically significant (p<0.01) when compared to the control group values. The mean values for male and female fertility in the WIL historical data are 89.3% and 90.4%, respectively. Male and female mating indices were 96.0%, 100%, 100%, 84.0% and 68.0% in the same respective groups. The 750 ppm group value was statistically significant (p<0.05) in comparison to the control group value. In the WIL historical control data, the mean male and female mating indices are 96.0% and 97.0%, respectively. Males that did not sire a litter numbered 2, 0, 3, 12 and 25 in the control, 100, 250, 500 and 750 ppm groups, respectively. Females that had evidence of mating but did not deliver numbered 1, 0, 3, 10 and 17 in the same respective groups. All of these females were nongravid, with the exception of female nos. 32903 and 32925 in the 500 ppm group. These females had evidence of mating, did not deliver, and were gravid (two and one former implantation sites, respectively).
The mean numbers of days between pairing and coitus were increased in the 500 and 750 ppm groups (4.3 and 4.8 days, respectively) compared to that in the control group. (3.4 days). The differences were not statistically significant. However, the values were above the range in the WIL historical control data (2.0-3.5 days). In addition, pre-coital intervals could not be determined for 1, 0, 0, 4 and 8 females in the control, 100, 250, 500 and 750 ppm groups, respectively, because these animals had no evidence of mating and were determined to be nongravid. Therefore, the increases in pre-coital intervals in the 500 and 750 ppm groups were considered to be test article related. No effects of exposure were observed on the mean number of days between pairing and coitus in the 100 and 250 ppm groups.

ORGAN WEIGHTS (PARENTAL ANIMALS)

Organ weight changes were observed in the brain (250 ppm group males and 500 and 750 ppm group males and females), ovary (500 and 750 ppm group females), epididymis (250, 500 and 750 ppm group males), seminal vesicles (500 and 750 ppm group males), prostate (250, 500 and 750 ppm group males) and liver (500 and 750 ppm group males and 750 ppm group females) when compared to the control group values. It should be noted that the physiological condition of the 750 ppm group females was different from the other F0 females due to the lack of pregnancy and subsequent demands of lactation.
Mean absolute brain weights were reduced compared to the control group values in the 250, 500 and 750 ppm group males and in the 500 and 750 ppm group females. The differences were statistically significant (p<0.05 or p<0.01). There were no corresponding macroscopic or microscopic findings, and brain weights relative to final body weights in these animals were similar to the control group values. The decreases in the males did not occur in an exposure-related manner. Mean absolute and relative brain weights in the 100 ppm group males and females and in the 250 ppm group females were similar to those in the control group.
Mean absolute and relative ovary weights were decreased in an exposure-related manner in the 250, 500 and 750 ppm group females. The differences in the 750 ppm group were statistically significant (p<0.01) when compared to the control group values. The reductions in the 500 and 750 ppm group females were considered to be test article-related due to the correlation with the microscopic finding of decreased corpora lutea.
Mean absolute left and right epididymal and left and right cauda epididymal weights in the 250, 500 and 750 ppm group males were decreased compared to the control group values, in an exposure-related manner. The differences from the control group values were generally statistically significant (p<0.05 or p<0.01) in the 500 and 750 ppm groups. Mean relative cauda epididymal (left and right) weights in the 750 ppm group were reduced (statistically significant at p<0.01) compared to the control group values. Additionally, the mean absolute prostate weights in the 250, 500 and 750 ppm groups and seminal vesicle weights in the 500 and 750 ppm groups were reduced compared to the control group values. The differences in the prostate weights and the 750 ppm group seminal vesicle weight were statistically significant (p<0.05 or p<0.01). The reduced epididymal weights in the 750 ppm group correlated with the reduced epididymal sperm numbers observed in this same group. Although there were no macroscopic or microscopic observations that correlated with the changes in prostate and seminal vesicle weights, the reductions were considered to be test article-related because of the reductions in fertility and/or litter size observed in the 250, 500 and 750 ppm groups.
Mean absolute adrenal and pituitary weights in the 750 ppm group males were decreased compared to the control group values. The differences were statistically significant (p<0.01). Similar reductions were not observed in the treated females. There were no changes in the relative weights of these organs, and there were no macroscopic or microscopic changes that correlated with the weight reductions; therefore, the decrease in adrenal weight was considered to be incidental and unrelated to exposure to the test article. Because of the infertility observed in the F0 animals in 750 ppm group and since similar reductions in pituitary weights were observed in the 500 ppm group F1 males, the reduced pituitary weights were considered to be test article-related.
Mean relative liver weights were increased in the 500 and 750 ppm group males and the 750 ppm group females. The differences from the control group values were statistically significant (p<0.01) for the 750 ppm group animals. These increases correlated to the microscopic findings of vacuolation and increased glycogen.
Statistically significant (p<0.05 or p<0.01) increases in organ weights were observed in the 500 ppm group males (relative liver weight), in the 750 ppm group males (relative kidney, spleen and left testis weights) and in the 750 ppm group females (relative liver and kidney weights). These changes were considered secondary to the decreases in body weights in these animals, and were not direct test article effects.
There were no test article-related effects on organ weights in the 100 ppm group males and females.

GROSS PATHOLOGY (PARENTAL ANIMALS)

Female no. 32821 in the 500 ppm group was euthanised in extremis on study day 119 due to an ocular abnormality. At the time of necropsy, the left eye was enlarged and reddened. This female was also nongravid.
At the scheduled necropsy, possible test article-related macroscopic changes were observed in the testis and epididymis of the 500 and 750 ppm group males. Low incidences of small left and/or right epididymides and small and/or soft testes were observed in one or two of these males. All other macroscopic observations were considered spontaneous, incidental and unrelated to test article exposure.
Evaluation of the calculated differences between the number of pups born and the number of implantation sites counted (unaccounted sites) in the 750 ppm group females was precluded by 100% infertility in this group. In the 500 ppm group, the mean number of unaccounted sites was comparable to the control group value, but the mean number of implantation sites was reduced (statistically significant at p<0.01) in comparison to the control group value. A statistically significant (p<0.01) increase in the mean number of unaccounted sites was observed in the 250 ppm group (1.3 unaccounted sites per dam) when compared to the concurrent control group value (0.3 unaccounted sites/dam). However, no exposure-relationship was evident; therefore, this increase was not considered to be an effect of the test article. The mean number of implantation sites was decreased (not statistically significant) in the 250 ppm group. The mean numbers of unaccounted sites and implantation sites in the 100 ppm group females were similar to the control group values; none of the differences were statistically significant.

HISTOPATHOLOGY (PARENTAL ANIMALS)

One female in the 500 ppm group was euthanised in extremis due to an ocular abnormality. No microscopic findings related to the moribundity of the animal were observed.
Test article-related microscopic changes were observed in the liver (250, 500 and 750 ppm group males and females), kidney (500 and 750 ppm group males and females), spleen (250, 500 and 750 ppm group males and females) and ovary (500 and 750 ppm group females).
In the liver, an exposure-related increase in the incidence and severity of minimal to mild centrilobular hepatocellular vacuolation, consisting of discrete, nonstaining cytoplasmic vacuoles, was observed in the 250, 500 and 750 ppm group males and the 500 and 750 ppm group females. The differences from the control group values were statistically significant (p<0.05). The vacuoles were morphologically compatible with lipid. Increased glycogen, microscopically appearing as cytoplasmic clearing of hepatocytes, was also increased in incidence and severity in the 250, 500 and 750 ppm group males and females; the differences from the control group values were statistically significant (p<0.05) in the 500 ppm group females and in both sexes in the 750 ppm group. The increased glycogen was observed throughout the lobule (panlobular distribution). Although test article-related, both of these changes (increased glycogen and centrilobular vacuolation) are considered to be reversible changes that are not biologically adverse.
In the kidney, test article-related increases in the incidences of minimal to mild pelvic mineralisation and transitional epithelial hyperplasia were observed in the 500 and 750 ppm group males and females; the differences from the control group value were generally statistically significant (p<0.05) for the 500 and 750 ppm group females. A slight increase in pelvic mineralisation was also observed in the 250 ppm group females. Pelvic mineralisation was characterised by the presence of small areas of mineral along the transitional epithelium lining the renal pelvis. This change was sometimes associated with hyperplasia of the transitional epithelium, which is considered secondary to irritation from the mineral. No large calculi were observed. There were no associated changes in the renal cortex andlor medulla.
A slight increase in the severity of brown pigment in the spleen was observed in the 250, 500 and 750 ppm group males and females. The differences from the control group values were not statistically significant. Spleens from control group animals had primarily mild amounts of pigment, while spleens of the 250, 500 and 750 ppm group animals had primarily mild to moderate amounts of pigment. The increases in severity were not exposure-related; however, because the consistency of this finding between sexes and between generations, it was considered to be test article-related. The pigment was stained positively for iron with Perl's iron stain, and morphologically consistent with hemoglobin. However, hematology was not conducted on these animals. Because of the minimal nature of the change, this finding was not considered to be toxicologically significant.
Test article-related changes in the ovaries of the females consisted of decreased corpora lutea (500 and 750 ppm groups) and increases in follicular cysts and interstitial cell hyperplasia (750 ppm group only). The differences from the control group values were statistically significant (p<0.05) in the 750 ppm group. Decreased corpora lutea was diagnosed when there were either no corpora lutea present (a grade of "severe" implied that the animals did not ovulate recently, perhaps during the last four to six cycles) or that there were fewer recent corpora lutea present than would be expected for a regularly cycling animal (a grade of "mild" implied that ovulation did not occur consistently within the last few estrous cycles). It should be noted that some of the animals did have new corpora lutea from the most recent cycle, but did not show a good continuum of recent to very old degenerating corpora lutea, as would be expected for a regularly cycling animal. Decreased corpora lutea was considered to be a test article-related effect in the 500 and 750 ppm groups, and although it correlates with the decreased number of litters in these groups, it does not fully account for the absence of litters in the 750 ppm group. Enumeration of the numbers of corpora lutea in five sections per ovary revealed a statistically significant decreased (p<0.01) in the number of corpora lutea in the 750 ppm group when compared to the control group value, and confirmed the microscopic findings. Corpora lutea counts in the 500 ppm group females were also decreased compared to those in the control group, although the differences were not statistically significant.
Decreased numbers of corpora lutea were often observed in animals that had follicular and/or luteinised follicular cysts. Both types of cysts were large, fluid-filled, and frequently multiple and bilateral. Follicular cysts were lined by degenerating granulosa and theca cells, and a degenerating oocyte was sometimes observed within the cyst. Luteinised follicular cysts were lined by a variable number of layers of degenerating or viable luteal cells. A slight increase in the incidence of both types of cysts was observed in the 750 ppm group. However, cysts were also observed in the control group females, and in females in the 750 ppm group that had no microscopic evidence of decreased corpora lutea.
Interstitial cell hyperplasia of the ovary was also observed with increased incidence in the 750 ppm group females. The difference from the control group value was not statistically significant. Since interstitial tissue forms from the thecal cells from the atretic follicles (those that grow but are not ovulated and subsequently degenerate), this change was considered to be a reflection of the interruption of the estrous cycle in the affected animals. All of these changes (decreased corpora lutea, increased follicular and luteinised follicular cysts and interstitial cell hyperplasia) are changes that have been described in aging animals. Enumeration of the number of primordial follicles in five sections per ovary did not reveal any statistically significant changes in the numbers of primordial oocytes between the 750 ppm and control groups.
(continued in any other information on results)

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
100 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: body weight gain and food consumption/food efficiency
Dose descriptor:
NOAEL
Effect level:
100 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Developmental effects
Remarks on result:
other: Generation: F1 and F2 (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

F1 LITTER DATA

VIABILITY (OFFSPRING)

Evaluation of pup viability in the 750 ppm group was precluded by F0 infertility. The mean number of pups born (12.5 and 8.5 pups per dam) and live litter size on PND 0 (12.3 and 8.3 pups per dam) in the 250 and 500 ppm groups, respectively, were reduced compared to the control group values (15.0 pups born per dam and 14.4 viable pups per dam on PND 0). The differences were statistically significant (p<0.01) in the 500 ppm group. The percentages of males per litter at birth in these groups were similar to the control group value. The number of pups born, percentage of males per litter at birth and live litter size in the 100 ppm group were unaffected by test article exposure.
Pup survival throughout the pre-weaning period was not affected by parental exposure in the 100, 250 and 500 ppm groups. The only statistically significant difference from the control group was a slight reduction (p<0.05) in viability in the 500 ppm group during PND 14-21.
This reduction was not of sufficient magnitude to be considered toxicologically significant.

CLINICAL SIGNS (OFFSPRING)

Pups that were found dead or euthanised in extremes during the postnatal period numbered 16, 20, 8 and 5 in the control, 100, 250 and 500 ppm groups, respectively. In the same respective groups, 0, 1, I and 1 pups were missing and presumed to have been cannibalised. The general physical condition of the F1 pups during the postnatal period was similar in all groups, including the control group.

BODY WEIGHT (OFFSPRING)

Mean F1 pup body weight gains in the 500 ppm group males and females were slightly increased (not statistically significant) before selection (PND 1-4). Mean pup body weight gains in this group were reduced in comparison to the control group values after culling on PND 4 (PND 4-7, 7-14, 14-21 and 21-28); the differences were statistically significant (p<0.01) for the males during PND 4-7 and 7-14 and for both sexes during PND 21-28 (first week of F1 exposure). Mean pup body weights in the 500 ppm group males and females were increased (generally statistically significant at p<0.05) on PND 1 and 4 (before selection). The body weight increases in these pups prior to culling were attributed to the reduced litter sizes in this group (decreased intralitter competition) and were not considered to be direct effects of test article exposure. Following culling on PND 4, mean pup body weights in the 500 ppm group males were similar to the control group value on PND 7, 14 and 21 and reduced relative to the control group values on PND 28; the PND 28 difference (13.7%) was statistically significant (p<0.01). Mean pup body weights in the 500 ppm group females following culling were similar to the control group values on PND 7, 14 and 21, but were 9.7% lower than the control group value on PND 28 (not statistically significant).
Mean pup body weight gains in the 250 ppm group males and females were similar to the control values during PND 1-4, but were slightly reduced during PND 4-7, 7-14, 14-21 and 21-28; only the PND 21-28 difference for the males was statistically significant (p<0.01). Mean pup body weights in the 250 ppm group males were similar to the control group values on PND 1, 4, 7, 14 and 21, but were slightly reduced on PND 28; the PND 28 difference (8.9%) was statistically significant (p<0.01). Mean pup body weights in the 250 ppm group females were similar to the control group values at all intervals; no statistically significant differences from the control group were observed.
Mean pup body weight gains and pup body weights in the 100 ppm group males and females were similar to the control group values throughout the postnatal period. The only statistically significant difference (p<0.05) was a slight reduction in body weight gain in the 100 ppm group males during PND 21-28. However, the reduction in mean body weight gain was slight and not of sufficient magnitude to affect absolute body weight. Therefore, this reduction was not considered to be test article-related.

SEXUAL MATURATION (OFFSPRING)

A delay in the day of acquisition of balanopreputial separation was observed in the 500 ppm group F1 males. The mean ages at balanopreputial separation were 44.2, 45.9, 45.7 and 48.3 days in the control, 100, 250 and 500 ppm groups, respectively. The difference from the control group was statistically significant (p<0.01) for the 500 ppm group. All males in the control and exposure groups had balanopreputial separation by PND 58. Mean body weight on the day of acquisition in the 500 ppm group was similar to the control group value. Therefore, the delay in acquisition of balanopreputial separation was attributed to the reduced body weights of the males in the 500 ppm group.
The mean day of acquisition of balanopreputial separation and mean body weight on the day of acquisition in the 100 and 250 ppm group F1 males were not affected by exposure to the test article. Differences from the control group values were slight and were not statistically significant.
Vaginal patency was not affected in any of the F1 female pups. The mean ages at acquisition of vaginal patency were 36.0, 34.1, 36.1 and 37.8 days in the control, 100, 250 and 500 ppm groups, respectively. All female pups had vaginal opening by PND 45.
Mean body weights in the exposure group females on the day of acquisition of vaginal patency were similar to the control group values. No statistically significant differences were noted.

ORGAN WEIGHTS (OFFSPRING)

Mean absolute brain weights in the 100 and 250 ppm group males and females were reduced compared to the control group values. With the exception of the value for the 250 ppm group females, the differences were statistically significant (p<0.05 or p<0.01). The reductions in the females did not occur in an exposure-related manner. Mean brain weights relative to final body weight in these males and females were similar to the control group values. Mean absolute and relative spleen and thymus weights in the 100 and 250 ppm group males and females were similar to the control group values; none of the differences were statistically significant.

GROSS PATHOLOGY (OFFSPRING)

At the necropsy of F1 weanlings selected for organ weights on PND 21, no exposure-related internal findings were observed. The only internal findings noted were a dilated renal pelvis in one pup each in the control and 100 ppm groups and a small spleen in one pup in the 100 ppm group. All other pups were internally normal.

F2 LITTER DATA


VIABILITY (OFFSPRING)

The mean number of pups born (8.6 pups per dam) and live litter size on PND 0 (8.6 pups per dam) in the 500 ppm group were reduced compared to the control group values (14.9 pups per dam and 14.5 pups on PND 0, respectively). The differences were statistically significant (p<0.01). The percentage of males per litter in this group was not statistically significantly different from the control group value. The mean number of pups born (13.1 pups per dam) and live litter size on day 0 (12.5 pups per dam) in the 250 ppm group were reduced compared to the control group values; the differences were not statistically significant. The mean numbers of pups born and live litter size in the 100 ppm group and the percentage of males per litter in the 100 and 250 ppm groups were similar in the control group.
Pup survival throughout the postnatal period was not affected by parental exposure in the 100, 250 and 500 ppm groups; none of the differences from the control group were statistically significant.

CLINICAL SIGNS (OFFSPRING)

F2 pups that were found dead or euthanised in extremis during the postnatal period numbered 11, 5, 20 and 2 in the control, 100, 250 and 500 ppm groups, respectively. In the same respective exposure groups, 1, 2, 2 and 0 pups were missing and presumed cannibalised. The general physical condition of the F2 pups during the postnatal period was unaffected by parental exposure to the test article. Clinical findings noted in the 100, 250 and 500 ppm groups, including smallness of size, edema, malrotation of the forelimbs and uneven hair growth, were not observed in an exposure-related manner or were noted for only one or two pups per group, and were considered to be incidental.

BODY WEIGHT (OFFSPRING)

Mean F2 pup body weight gains in the 500 ppm group males and females were similar to the control group values before selection (PND 1-4). After culling on PND 4 (PND 4-7, 7-14 and 14-21), mean pup body weight gains in this group were reduced in comparison to the control group values; the differences were statistically significant (p<0.01). Mean pup body weights in the 500 ppm group males and females were similar to the control group values on PND 1, 4 (before selection) and 7. On PND 14 and 21, mean pup body weights in these animals were reduced and statistically significant (p<0.01) relative to the control group values.
Mean pup body weights and body weight gains in the 100 and 250 ppm group males and females were similar to those in the control group. Differences were slight and not statistically significant.

SEXUAL MATURATION (OFFSPRING)

Not examined.

ORGAN WEIGHTS (OFFSPRING)

Mean spleen weights (absolute and relative to final body weight) in the 500 ppm group F2 male and female pups were reduced compared to the control group values. The differences were statistically significant (p<0.01). These decreases were considered to be test article-related.
Mean absolute brain weights in the 500 ppm group males and females and the mean absolute thymus weight in the 500 ppm group males were reduced compared to those in the control group. The differences were statistically significant (p<0.05 or p<0.01). However, the relative brain weights in the 500 ppm group males and females and relative thymus weight in the males were similar to those in the control group.
Mean absolute and relative organ weights in the 100 and 250 ppm group F2 pups were similar to those in the control group. No statistically significant differences were observed.

GROSS PATHOLOGY (OFFSPRING)

No internal findings that were attributed to parental exposure to the test article were noted at the necropsies of pups that were found dead or euthanised in extremis. With the exception of the presence or absence of the milk in the stomach, remarkable findings were limited to the following. One control group pup had mandibular micrognathia, aglossia and a hemorrhagic ring around the iris. Skeletally, mandibular micrognathia consisted of small mandibular bones that were fused medially and tympanic rings that were moderately reduced in ossification. Another pup in the control group had renal papillae that were not fully developed. One pup in the 250 ppm group had hemorrhagic rings around the irides. No other internal findings were observed.
At the necropsy of F2 pups not selected for organ weights on PND 21, no macroscopic finds that could be attributed to F1 parental exposure were observed. Two pups in the 100 ppm group had a dilated renal pelvis; one of these pups also had a distended ureter. One pup in the control group had white areas on the lungs. No other internal findings were observed.
At the PND 21 necropsy of the F2 weanling pups selected for organ weights, no macroscopic findings that could be attributed to F1 parental exposure were observed. One male and one female in the control group and one male in the 250 ppm group had an enlarged spleen. One male in the 250 ppm group had an accessory spleen. No other internal findings were observed.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Reproductive effects

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

(continued from details on results (parental animals))

A slight increase (not statistically significant) in the incidence of seminiferous tubule degeneration was observed in the testis of the 750 ppm group males. This change correlated with the macroscopic observation of small and/or soft testes. Affected tubules had layers of germ cells missing. The most severely affected tubules were lined only with Sertoli cells. Although the incidence was 1/25 in the control group and 6/25 in the 750 ppm group, it should be noted that four of the affected males had only one or several tubules affected (graded as "minimal"). Additionally, there were no changes in the testis weight to correlate with the degeneration. Seminiferous tubule degeneration was not increased in the other exposure groups. Therefore, the seminiferous tubule degeneration in the 750 ppm group males was not considered to be an adverse effect of test article exposure. Small epididymides, observed sporadically at necropsy in the 500 and/or 750 ppm groups, were not confirmed microscopically.

Multiple sections of brain were examined microscopically in the F0 control and 750 ppm groups (males and females) in an effort to determine the cause of the decreased brain weights observed. The areas of the brain specifically examined for these two groups included the cerebral cortex, hippocampus, basal ganglia, cerebral peduncles, pons, tectum, central gray matter, thalamus, hypothalamus, cerebellum and nucleus gracilis. No test article-related morphologic changes were observed in any of the areas.

All other microscopic observations were considered to be spontaneous, incidental and unrelated to test article exposure.

GESTATION LENGTH AND PARTURITION

There were no gravid females in the 750 ppm group. Mean gestation lengths in the 100, 250 and 500 ppm groups were unaffected by test article exposure. The mean lengths of gestation were 21.9, 22.0 and 22.2 days in the same respective exposure groups compared to 22.0 days in the concurrent control group and 21.8 days in the WIL historical control data. Female no. 32884 in the 100 ppm group had difficulty delivering one pup that was presented in a breech position on gestation day 24. No signs of dystocia were observed in any other exposure group. Therefore, this single incident of difficult parturition in the low exposure group was not attributed to the test article.

F1 GENERATION

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)

F1 male no. 32800-04 in the 500 ppm group was euthanised in extremis during the second week of exposure (PND 33, study week 18). Prior to euthanasia, this animal was lethargic and had shallow respiration and pale eyes. Male no. 32923-04 in the control group was euthanised in extremis on study day 154 due to possible mechanical trauma (nose and upper incisors deviated to the right). Prior to euthanasia, this animal had red material around the eyes and nose and an unkempt appearance. All other F1 males and females survived to the scheduled necropsy.

There were no exposure-related clinical observations in the F1treated males and females at the weekly examinations or one hour following completion of exposure. Clinical findings were observed infrequently, with similar incidence in the control group and/or did not occur in an exposure-related manner.

BODY WEIGHT (PARENTAL ANIMALS)

WEEKLY - Mean body weight gains in the 500 ppm group Fl males were reduced compared to the control group values during study weeks 19-20 and 20-21. The difference was statistically significant (p<0.01). During the remainder of the F1 generation (study weeks 21-22 through 36-37), mean body weight gains in these males were similar to the control group values. Cumulative body weight gains (from study week 20) in the 500 ppm group males (study weeks 20-22 through 20-37) were similar to the control group values. Mean body weights in the 500 ppm group males were 9.3%-18.5% lower than the control group values throughout the generation (study weeks 19-37). The differences were statistically significant (p<0.01).

In the 500 ppm group F1 females, mean body weight gains and cumulative body weight gains were similar to the control group values throughout the pre-mating period (study weeks 19-28); none of the differences were statistically significant. Mean body weights in these females were 6.7%-11.8% lower than those in the control group during weeks 19-28. The differences were statistically significant (p<0.05 or p<0.01). The reductions in F1 female body weights in the 500 ppm group were attributed to the reduced mean body weight gain observed during the first week of F1 exposure (PND 21-28). Following weaning (study week 37), mean body weight in the 500 ppm group females was comparable to that in the control group.

Mean body weights, body weight gains and cumulative body weight gains in the 100 and 250 ppm group males and females were generally similar to the control group values, with the following exceptions. Mean body weights in the 100 ppm group males were reduced and usually statistically significant (p<0.05) when compared to the control group values during study weeks 25-37. These reductions did not occur in an exposure-related manner; therefore, these changes were not attributed to test article exposure.

GESTATION - Mean maternal body weight gain in the 500 ppm group females was similar to that in the control group during gestation days 0-4 and 4-7. Mean body weight gains in these females were reduced (generally statistically significant at p<0.05 or p<0.01) throughout the remainder of gestation (days 7-11, 11-14 and 14-20) and when the entire gestation period (days 0-20) was evaluated. Mean body weights in the 500 ppm group females were similar to the control group values during gestation days 0, 4 and 7, but were slightly reduced throughout the remainder of gestation (days 11, 14 and 20) when compared to the control group values. The differences were statistically significant (p<0.05 or p<0.01) on gestation days 14 and 20. Mean body weight in these females was lower than the control group value at the initiation of breeding, and therefore was reduced (4.3% lower than the control group value) on gestation day 0. The reductions in body weights and body weight gains late in gestation were attributed to the reduced litter size in this group.

Mean body weight gains in the 250 ppm group females were similar to the control group values during gestation days 0-4, 4-7, 7-11 and 11-14, but were reduced during gestation days 14-20 and when the overall gestation period (days 0-20) was evaluated; the differences were not statistically significant. Mean gestation body weight in the 250 ppm group females were similar to the control group values throughout gestation.

Mean gestation body weights and body weight gains in the 100 ppm group females were not affected by exposure to the test article. The only statistically significant difference from the control group was a transient reduction (p<0.05) in mean body weight gain in the 100 ppm group during gestation days 4-7. A similar reduction was not observed in the next highest exposure group; therefore, the decrease was not attributed to the test article.

LACTATION - There were no test article-related effects on lactation body weights or body weight changes in the 100, 250 and 500 ppm groups. The only statistically significant differences (p<0.05) were reduced mean body weights on lactation days 4, 7 and 14 in the 500 ppm group. The reductions in mean body weight in the 500 ppm group were slight; therefore, the reductions were not considered to be test article-related.

FOOD CONSUMPTION (PARENTAL ANIMALS)

WEEKLY - Food consumption (g/animal/day) in the 500 ppm group Fl males was reduced during weeks 19-20. The difference from the control group values was statistically significant (p<0.05). No other adverse effects on food consumption, g/animal/day and g/kg/day, or food efficiency were observed in the 100, 250 and 500 ppm group F1 males and females during the pre-mating (males and females) or post-mating (males only) periods. A statistically significant reduction in g/animal/day food consumption was noted for the 100 ppm group males during weeks 31-32. However, the reduction was not observed in an exposure-related manner; therefore, no relationship to treatment was apparent. Statistically significant (p<0.05 or p<0.01) increases in g/kg/day food consumption were noted in the 250 ppm group males (weeks 25-26 and 26-27) and the 500 ppm group males and females (weeks 20-21 through 27-28 for both sexes and weeks 31-32, 32-33 and 33-34 for the males). These increased g/kg/day food consumption values were not considered to be direct effects of test article exposure, but were attributed to the reduced body weights noted in these groups. No other statistically significant differences from the control group values were noted.

GESTATION - Food consumption in the 250 ppm group females was similar to that in the control group during gestation, with the following exception. During gestation days 4-7, g/kg/day food consumption was increased and statistically significant (p<0.05) compared to the control group value. No exposure-related increases were observed in the 500 ppm group; therefore, no relationship to treatment was evident. Food efficiency in the 250 ppm group females was similar to that in the control group during gestation days 0-4 and 4-7. During gestation days 7-11, 11-14 and 14-20, food efficiency in these females was slightly reduced compared to the control group values. The differences were not statistically significant. However, when the entire gestation period (days 0-20) was evaluated, food efficiency in these females was reduced. The difference from the control group value was statistically significant (p<0.05).

Food consumption (g/animal/day and g/kg/day) and food efficiency in the 100 ppm group females were unaffected by treatment throughout gestation. Food efficiency was slightly reduced in these females during gestation days 4-7 and 11-14 compared to the control group values. The difference was statistically significant (p<0.05) during gestation days 4-7. However, no exposure-related reductions in food efficiency were noted at the high exposure levels during gestation days 4-7, and consistent, sustained reductions were not observed in the 100 ppm group females. Therefore, the slight reductions in food efficiency in these females were not considered to be exposure-related.

LACTATION - Food consumption, evaluated as g/animal/day and g/kg/day, in the 500 ppm group females was reduced throughout the entire lactation period (lactation days 1-4, 4-7, 7-14, 14-21 and 1-21). The majority of the differences from the control group values were statistically significant (p<0.05 or p<0.01). Food efficiency in these females was similar to the control group values throughout lactation; differences were slight and not statistically significant. The reduction in food consumption during lactation was attributed to the reduced mean litter size in this group that caused reduced maternal lactational demand.

Food consumption and food efficiency in the 100 and 250 ppm group females throughout lactation were not affected by exposure to the test article. Differences from the control group were slight and not statistically significant.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)

The mean lengths of the estrous cycle were 4.5, 4.5, 4.9 and 5.1 days in the control, 100, 250 and 500 ppm groups, respectively. Although all the values were within the historical control range (4.1-5.1 days), estrous cycle lengths could not be determined for three and four animals in the 250 and 500 ppm groups, respectively, compared to one each in the control and 100 ppm groups because no complete estrous cycles occurred in these females. Therefore, the increases in the 250 and 500 ppm group values were attributed to the test article.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)

Test article-related reductions were observed in sperm motility (74.4%) and the percentage of morphologically normal sperm (95.3%) in the 500 ppm group F1 males compared to the control group values (88.9% and 99.5%, respectively). The differences were statistically significant (p<0.01). Abnormal sperm morphology consisted of normally shaped head separated from the flagellum, normal flagellum with absent head and misshapen head (microcephalic) with normal flagellum. The mean testicular and epididymal sperm numbers and the sperm production rate in the 500 ppm group males were similar to those in the control group.

Sperm motility (84,8%) in the 250 ppm group males was slightly reduced compared to the control group value (88.9%). The difference was statistically significant (p<0.05). However, the 250 ppm group value was slightly higher than the mean value in the WIL historical control data (83.2%). Therefore, this reduction was not attributed to exposure to the test article. The mean testicular and epididymal sperm numbers, sperm production rate and the percentage of morphologically normal sperm in the 250 ppm group males were similar to those in the control group.

Spermatogenic endpoints in the 100 ppm group males were not affected by test article exposure. The only statistically significant difference from the control group was a reduction (p<0.01) in the percentage of morphologically normal sperm. However, a similar reduction was not observed at the next highest exposure level. Therefore, this reduction was not attributed to the test article.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)

Male fertility indices were 87.5%, 68.0%, 64.0% and 70.8% in the control, 100, 250 and 500 ppm groups, respectively. Female fertility indices were 88.0%, 68.0%; 64.0% and 72.0% in the same respective groups. The differences from the control group values were not statistically significant. However, in the WIL historical control data only one of 33 data sets had a value less than the fertility indices observed in the 100, 250 and 500 ppm groups. The mean values for male and female fertility in the WIL historical control data are 89.3% and 90.4%, respectively. Although the reduction in fertility indices did not occur in an exposure-related manner, because of the reduced fertility (52%) in the F0 500 ppm group, only 11 litters of the F0 generation were represented in the F1 500 ppm group. Therefore, the fertility index in the F1 500 ppm group may have been biased because the F0 animals most sensitive to the effects of the test article were not represented in the F1 generation.

Male mating indices were 100.0%, 84.0%, 80.0% and 100.0% in the control, 100, 250 and 500 ppm groups, respectively. Female mating indices were 100.0, 84.0%, 80.0% and 100.0% in the same respective groups. The differences from the control group values were not statistically significant. Males that did not sire a litter numbered 3, 8, 9 and 7 in the control, 100, 250 and 500 ppm groups, respectively. Females that had evidence of mating, but did not deliver, numbered 3, 4, 4 and 8 in the same respective groups. All of these females were nongravid, with one exception. Female no. 32825-02 in the 500 ppm group had evidence of mating, did not deliver and was gravid (two former implantation sites).

No effects of exposure were observed on the mean number of days between pairing and coitus in the exposure groups. Differences from the control group value were slight and were not statistically significant.

ORGAN WEIGHTS (PARENTAL ANIMALS)

Organ weight changes relative to the control group were observed in the brain (100, 250 and 500 ppm group males and 500 ppm group females), liver (500 ppm group males and females), left and right epididymis (500 ppm group males), thymus (250 and 500 ppm group males) and pituitary gland (500 ppm group males).

Mean absolute brain weights were reduced (statistically significant at p<0.01) in the 100, 250 and 500 ppm group males and in the 500 ppm group females. The mean brain weights relative to final body weights were similar to the control group values in these animals. The decreases in the 100 and 250 ppm group males did not occur in an exposure-related manner. There were no corresponding macroscopic or microscopic changes in the brain.

Mean liver weights (absolute and relative) were increased in the 500 ppm group males and females. The difference from the control group value for relative liver weight was statistically significant (p<0.01) for the males. These increases, although slight, were considered to be test article-related since the liver weight increased despite decreasing body weight. Microscopically, increased glycogen and centrilobular hepatocellular vacuolation correlated with the weight increase.

Mean absolute left and right epididymal weights (total and cauda) in the 500 ppm group males were reduced compared to the control group values; the majority of the values were statistically significant (p<0.05 or p<0.01) when compared to the control group values. A similar decrease in weight was observed in these organs in the F0 males. Although there were no correlating macroscopic or microscopic changes, these reductions in epididymal weights were considered to be test article-related because of the consistency between generations and the effects observed on fertility in the Fl generation.

Mean absolute pituitary weight in the 500 ppm group males was decreased compared to the control group value; the difference was statistically significant (p<0.01). There was no change in relative weight of this organ, and there were no macroscopic or microscopic changes that correlated with the weight reductions. However, because similar reductions in pituitary weights were observed in the 750 ppm group Fo males, the reduced pituitary weights were considered to be test article-related.

Thymus gland weights (absolute and relative) were increased in the 250 and 500 ppm group males; the differences were often statistically significant (p<0.05 or p<0.01). No correlating microscopic changes were observed; therefore, the biological significance of the increased thymus weights in the 250 and 500 ppm group males is unknown.

Other statistically significant changes in organ weights (mean absolute seminal vesicle/coagulating gland and mean absolute kidney) were not considered test article-related effects because the changes were not exposure-related and the mean relative weights were unaffected.

GROSS PATHOLOGY (PARENTAL ANIMALS)

F1 male no. 32800-04 in the 500 ppm group was euthanised in extremis during the second week of exposure (PND 33). At necropsy, this animal had a dark red area on the lung and dark red areas in the stomach. Male no. 32923-04 in the control group was euthanized in extremis on study day 154 due to possible mechanical trauma. This animal had a distended and gas-filled cecum, dark red area on the lungs, small seminal vesicles and malaligned upper incisors. No internal evidence of mechanical injury was observed. All other animals survived to the scheduled necropsy.

No exposure-related internal findings were observed in males or females in the treated groups at the scheduled necropsy. Findings were observed infrequently, were noted in single animals and/or did not occur in an exposure-related manner.

The calculated difference between the number of pups born and the number of implantation sites counted (unaccounted sites) at the scheduled necropsy was increased (not statistically significant) in the 500 ppm group females (1.2 unaccounted sites per dam) when compared to the concurrent control group value (0.5 unaccounted sites per dam). The 500 ppm group value was above the range of the WIL reproductive historical control data (0.8 ± 0.17 unaccounted sites per dam). This increase was attributed to the test article. The mean number of implantation sites in the 500 ppm group females (9.8 sites per dam) and the 250 ppm group females (13.5 sites per dam) were reduced compared to the control group value (15.5 sites per dam). The difference was statistically significant (p<0.01) for the 500 ppm group. The mean numbers of unaccounted sites in the 100 and 250 ppm group females and the mean number of implantation sites in the 100 ppm group females were similar to those in the control group.

HISTOPATHOLOGY (PARENTAL ANIMALS)

One male in each of the control and 500 ppm group were euthanised in extremis. The cause of moribundity for these two animals could not be determined from the microscopic examination.

Test article-related effects were observed microscopically in the liver (100, 250 and 500 ppm group females and 250 and 500 ppm group males), spleen and ovary (500 ppm group females) and the kidney (500 ppm group males and 250 and 500 ppm group females).

In the liver, increased incidences of minimal to mild centrilobular hepatocellular vacuolation were observed in the 250 and 500 ppm group males and females when compared to that in the control group; with the exception of the 250 ppm group females, the differences were statistically significant (p<0.05). The vacuolation was much more prominent in the males than in the females, and the incidence was exposure-related in the affected groups. The amount of glycogen (incidence andlor degree) observed was increased all treated group males and females. When compared to the control group value, the differences were statistically significant (p<0.05) for the value of the 500 ppm group males and the 100, 250 and 500 ppm group females. Glycogen, although present in many control group animals, was increased in degree in the affected animals (primarily graded as "minimal" to "mild" in the control groups and "mild" to "moderate" in the treated groups), although the increased degree was not exposure-related. Both of these changes were also observed in the treated males and females in the F0 generation, and both were considered to be reversible and not biologically adverse.

In the spleen, an increase in the severity of brown pigment was observed in the 500 ppm group females. The difference from the control group value was not statistically significant. This lesion was also observed in the F0 750 ppm group males and females. The pigment was morphologically compatible with hemosiderin, and was demonstrated to contain iron by Perl's iron stain applied to spleens from selected F0 animals. The slight increase in pigment, although test article-related, was not considered to be toxicologically significant, as the increase was slight, there were no correlating macroscopic changes or clinical signs referable to anemia (such as pale tissues), and there were no correlating weight changes in the spleen.

In the ovary, test article-related findings were limited to the 500 ppm group and consisted of an increased incidence of cysts (combined follicular and luteinized follicular) and an increased incidence of interstitial cell hyperplasia. Both of these changes were observed as test article-related effects in the F0 750 ppm group females. Decreased numbers of corpora lutea were observed in 3, 3, 7 and 4 animals in the control, 100, 250 and 500 ppm group F1 females, respectively. Although this change was considered to be test article-related in the F0 females in the 750 ppm group and possibly in the 500 ppm group, a test article-related effect was not demonstrated in the F1 500 ppm group because of the lack of dose response and the minimal nature of the increase. Additionally, the qualitative change was not supported by quantitative evaluation of the number of corpora lutea in the control and 500 ppm groups. However, in this study, enumeration of the corpora lutea per ovary may have been an insensitive method for detecting subtle morphologic changes in the type of corpora lutea present. Enumeration of the number of primordial follicles in five sections per ovary resulted in a statistically significant (p<0.05) increase in the 500 ppm group. This increase was not considered to be an adverse effect.

In the kidney, a very slight increase in pelvic mineralisation, sometimes associated with transitional epithelial hyperplasia, was observed in the 250 ppm group females and the 500 ppm group males and females. The differences from the control group values were not statistically significant. The mineral was considered to be an effect of exposure, since a slight increase was also observed in the 500 and 750 ppm group F0 animals. Transitional epithelial hyperplasia was sometimes observed in the mineralised areas, but was also observed in animals without mineral, associated with inflammation or other changes. The hyperplasia was considered to be a secondary effect of the mineral, and not a direct effect of the test article. Minimal mononuclear cell infiltration was observed in the kidney of some animals in all groups, including the control group. Although the difference was statistically significant (p<0.05) in the 500 ppm group males, this change is a common incidental finding in rats of this strain and age, and the increase was considered to be incidental.

All other microscopic observations were considered to be spontaneous, incidental and unrelated to exposure to the test article.

GESTATION LENGTH AND PARTURITION

The mean lengths of gestation in the F1 exposure groups were similar to those in the control group. The mean gestation lengths were 21.8, 21.9 and 22.2 days in the 100, 250 and 500 ppm groups, respectively, compared to 22.0 days in the concurrent control group and 21.8 days in the WIL historical control data. The differences from the concurrent control group were not statistically significant. During the period of delivery, female no. 32818-11 in the 250 ppm group was observed to be lethargic and having difficulty with parturition; this female delivered pups over three days (PND 0-2). No signs of dystocia were observed in the 100 and 500 ppm groups; therefore, the single occurrence of difficult delivery in the 250 ppm group was not attributed to test article exposure.

Applicant's summary and conclusion

Conclusions:
In conclusion, whole-body inhalation exposure of Crl:CD(SD)IGS BR rats and the resulting offspring to vapor concentrations of 1-Bromopropane (100, 250, 500 and 750 ppm [F0 only]) for 6 hours per day, 7 days per week for a minimum of 70 days prior to mating and continuing until euthanasia produced no post-exposure clinical observations different from the control group animals exposed to filtered air. There were no mortalities related to exposure in the F0 generation. One F1 male in the 500 ppm group was euthanised in extremis during the second week of exposure. Body weight data (parental and pup) were reduced in the 500 (F0, F1 and F2) and 750 ppm (F0) groups. No macroscopic or microscopic pathology in brain tissue was observed. Complete lack of offspring was observed at exposures to 750 ppm, and a statistically decreased number of offspring compared to the control group was observed at exposures to 500 ppm (both generations). Non-statistically significant reductions in the number of offspring were observed in 250 ppm group for both generations. Fertility indices were statistically significantly reduced for the F0 500 ppm group. Reductions (not statistically significant) were observed in the 100, 250 and 500 ppm F1 groups. Extended estrous cycle lengths and an increase in the number of animals for which estrous cycle length could not be determined were observed in the 250 (F1), 500 (F0 and F1) and 750 (F0) ppm groups. The number of days between pairing and coitus were increased in the 500 and 750 ppm F0 groups. Reductions in organ weights were observed in the ovaries (500 and 750 ppm group F0 females), epididymides in the 250 (F0), 500 (F0 and F1) and 750 (F0) ppm group males, prostate (250, 500 and 750 ppm group F0 males), seminal vesicles (500 and 750 ppm group F0 males), pituitary in the 500 (F1) and 750 (F0) ppm group males and spleen in the F2 male and female weanlings. Organ weight differences from the control group values were also observed in the thymus (increase), liver (increase) and brain (decrease) in some groups. Generally, absolute values were different in treated animals compared to the control group values, but not when weights were expressed relative to body weights. Absolute brain weights were in the expected range for age, sex and strain, and no clinical or microscopic changes were correlated with the weight differences. No pathology associated with the thymus weight increases, and increased lipid vacuolation and glycogen content of the liver was considered reversible, and not of biological significance. Spermatogenic endpoints were adversely affected in the 500 (F0 and F1) and 750 (F0) ppm group males. Microscopic findings were observed in the ovaries in the 500 (F0 and F1) and 750 (F0) ppm group females.
Executive summary:

This study was designed to determine the potential adverse effects of 1-Bromopropane on reproductive capabilities in two generations of Crl:CD(SD)IGS BR rats. One litter was produced in each generation. This report encompasses growth, mating, parturition and lactational phases of the F0 and F1 generations and the subsequent growth, development and survival of the offspring from these generations. For clarification of the terminology used to differentiate the generations in the report text, the offspring of the F0 generation were defined as the F1 litters; pups from the F1 litters were selected to constitute the F1 generation. The offspring of the F1 generation were defined as the F2 litters. The study was conducted in accordance with EPA OPPTS 870.3800 and to GLP standard.

In conclusion, whole-body inhalation exposure of Crl:CD(SD)IGS BR rats and the resulting offspring to vapor concentrations of 1-Bromopropane (100, 250, 500 and 750 ppm [F0 only]) for 6 hours per day, 7 days per week for a minimum of 70 days prior to mating and continuing until euthanasia produced no post-exposure clinical observations different from the control group animals exposed to filtered air. There were no mortalities related to exposure in the F0 generation. One F1 male in the 500 ppm group was euthanised in extreris during the second week of exposure. Body weight data (parental and pup) were reduced in the 500 (F0, F1 and F2) and 750 ppm (F0) groups. No macroscopic or microscopic pathology in brain tissue was observed. Complete lack of offspring was observed at exposures to 750 ppm, and a statistically decreased number of offspring compared to the control group was observed at exposures to 500 ppm (both generations). Non-statistically significant reductions in the number of offspring were observed in 250 ppm group for both generations. Fertility indices were statistically significantly reduced for the F0 500 ppm group. Reductions (not statistically significant) were observed in the 100, 250 and 500 ppm F1 groups. Extended estrous cycle lengths and an increase in the number of animals for which estrous cycle length could not be determined were observed in the 250 (F1), 500 (F0 and F1) and 750 (F0) ppm groups. The number of days between pairing and coitus were increased in the 500 and 750 ppm F0 groups. Reductions in organ weights were observed in the ovaries (500 and 750 ppm group F0 females), epididymides in the 250 (F0), 500 (F0 and F1) and 750 (F0) ppm group males, prostate (250, 500 and 750 ppm group F0 males), seminal vesicles (500 and 750 ppm group F0 males), pituitary in the 500 (F1) and 750 (F0) ppm group males and spleen in the F2 male and female weanlings. Organ weight differences from the control group values were also observed in the thymus (increase), liver (increase) and brain (decrease) in some groups. Generally, absolute values were different in treated animals compared to the control group values, but not when weights were expressed relative to body weights. Absolute brain weights were in the expected range for age, sex and strain, and no clinical or microscopic changes were correlated with the weight differences. No pathology associated with the thymus weight increases, and increased lipid vacuolation and glycogen content of the liver was considered reversible, and not of biological significance. Spermatogenic endpoints were adversely affected in the 500 (F0 and F1) and 750 (F0) ppm group males. Microscopic findings were observed in the ovaries in the 500 (F0 and F1) and 750 (F0) ppm group females.