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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment. Although data provided have a report year after 2009, the study was performed to fulfill needs required by other governmental registrations and/or product stewardship purposes. This study was not performed to specifically fulfill an information requirement under REACH, but since the test data were already available they were provided as part of the REACH submission.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
The exposures for F1 offspring were initiated on PND 22 instead of PND 21.
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
yes
Remarks:
The exposures for F1 offspring were initiated on PND 22 instead of PND 21.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: 99.9769%

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories International, Inc. Raleigh, North Carolina, U.S.A.
- Age at study initiation: (P) approximately 53 days old; (F1) Post Natal (PND) Day 22 at start of treatment
- Weight at study initiation: (P) Males: 215.5-278.8 g; Females: 172.2-213.7 g; (F1) Males: mean wt. 48.1 g; Females: mean wt. 46.4 g
- Fasting period before study: no
- Housing: housed individually in solid-bottom caging
- Diet: PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002, ad libitum, except during exposures
- Water: tap water, ad libitum, except during exposures
- Acclimation period: approximately 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26ºC
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless steel and glass (NYU style)
- Method of holding animals in test chamber: stainless steel, wire-mesh modules
- Source and rate of air: house air
- Method of conditioning air: filtered and conditioned air
- System of generating vapour: Chamber atmospheres were generated by flash evaporation of the liquid test substance in air with a round-bottom evaporation flask. The test substance was metered into the evaporation flasks with a Harvard Apparatus model 22 syringe infusion pump for the 500 ppm exposure chamber or Cole-Palmer Masterflex® console drive pumps (equipped with Fluid Metering Inc. pistons) for other exposure chambers. The Harvard syringe infusion pump and its syringe were kept inside a small refrigerator during use. The Cole-Palmer Masterflex® pumps drew test substance from either a 2- or 5-gallon safety can. The evaporation flasks were placed in Unimantle heaters that were heated up to 175ºC to vaporize the test substance. Heat tapes set at approximately 54-60ºC were used to wrap the glass connection tubes from the evaporation flasks to the chamber inlets for all chambers except for the 500 ppm exposure. Houseline generation air, metered to the evaporation flasks by the Brooks model 5850E mass flow controllers, carried the vaporized test substance into filtered and conditioned air streams leading to the exposure chambers. Chamber concentrations of test substance were controlled by varying the test substance feed rate to the heated flasks. The Harvard syringe infusion pump, Cole-Palmer Masterflex® pumps, Brooks mass flow controllers, and the heat tapes were controlled manually. The Unimantle heaters were controlled and monitored by the Camile Inhalation Toxicology Automated Data System (CITADS).

- Temperature, humidity in air chamber: the temperature ranged from 17-24°C, the relative humidity ranged from 20-82%
- Air flow rate: 240-300 L/min
- Air change rate: 10-13 air changes per hour
- Treatment of exhaust air: exhausted into the exhaust stack

TEST ATMOSPHERE
- Brief description of analytical method used: During each exposure, the atmospheric concentration of the test substance was determined by gas chromatography (GC) approximately 13 times per day in the test chambers.
- Samples taken from breathing zone: yes
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days
- Proof of pregnancy: intravaginal plug and/or sperm referred to as day 0 of gestation
- After successful mating each pregnant female was caged (how): individual cage housing
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of chamber atmosphere were directly injected into a Hewlett Packard model 6890 GC equipped with an automated gas sample valve and a flame ionization detector. All samples were chromatographed isothermally at 80ºC on a 30 m x 0.53 mm Restek RTX 502.2 fused silica glass column.

Duration of treatment / exposure:
6 hours/day, 7 days/week
Parental rats (P1 generation) and the selected F1 males and females were exposed daily for at least 10 weeks prior to cohabitation and up to 2 weeks during the cohabitation period (mating). Exposure during gestation was 20 days (GD 0-19) and during lactation it was 14 days (LD 7-20). Exposure of gestating and lactating females was not conducted during gestation days 20-delivery, and lactation days 0-6 for compliance with animal welfare considerations. F2 offspring were not directly exposed to the test substance.

Males continued to be exposed for 3-4 weeks after the end of the mating period. Females with no evidence of mating or no litter delivered were exposed for at least another 24 days after the end of the mating period. Females with no evidence of mating, but assumed pregnant based on weight gain were exposed for another approximately 6 days following the end of the cohabitation period (or sooner based on GD 14 weight gain).
Frequency of treatment:
6 hours/day, 7 days/week
Details on study schedule:
- F1 parental animals not mated until 10 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 P1 mated animals in the study: approximately 20 weeks
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 500, 1000, 1500, or 2500 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
P1 exposure: 0.0 ± 0.0, 500 ± 1.3, 1000 ± 1.8, 1500 ± 2.1, and 2500 ± 2.5 ppm; F1 exposure: 0.0 ± 0.0, 500 ± 1.2, 1000 ± 1.5, 1500 ± 2.2, and 2500 ± 2.5 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
30/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The proposed exposure concentrations for the current multigeneration reproduction study were 0, 500, 1000, 1500, and 2500 ppm and were based on the decreased body weight, body weight gain, and food consumption that was observed at 1500 ppm and above in the pilot study, as well as the decreased body weight and/or body weight gain in gestating females and fetuses observed at 10000 ppm in the developmental toxicity study.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily; Cage-site examination to detect moribund or dead animals and abnoraml behaviour and/or appearance among animals were conducted once daily at the time of loading the animals into the exposure modules and when the animals were returned to their home cage following exposure, except on days when careful clinical observations were collected. Abnormalities were noted by exception.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once weekly throughout the premating treatment, gestation, and lactation periods. Prior to initiation of the exposure, 3 times during the exposure, and once post-exposure, animals were observed for abnormal behaviour and/or appearance, and the response to a sound stimulus was observed. Observations were performed for the group of rats visible through the exposure chamber window and recorded collectively for the specific exposure concentration since individual animal identification was not visible to the observer.

BODY WEIGHT: Yes
- Time schedule for examinations: All P1 and F1 animals were weighed at least once a week. All animals that were evaluated for developmental landmarks (vaginal patency and preputial separation) wereweighed the day of achievement. P1 and F1 females were weighed on days 0, 7, 14, and 20 of gestation and on days 0, 7, 14, and 21 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: Individual food consumption was determined weekly for all P1 and F1 animals throughout the permating period ending approximately at test day 70. From the food consumption and body weight data, the mean daily food efficiency was calculated. Food consumption was not measured during cohabitation. After mating pairs were separated, food consumption was not measured in males or in females with no evidence of copulation. Individual food consumption of presumed pregnant P1 and F1 females, or females that delivered a litter, was recorded on gestation days 0, 7, 14, and 20 and on lactation days 0, 7, and 14. From the food consumption and body weight data, the mean daily food efficiency was calculated.
Oestrous cyclicity (parental animals):
Vaginal lavage samples were collected daily from all surviving adult P1 and F1 female rats in order to determine the stage of the estrous cycle. Vaginal lavage samples were collected beginning 3 weeks prior to the start of cohabitation and continuing until copulation was confirmed or the cohabitation period had ended. The vaginal lavage sample collected on the day copulation was confirmed was not used for estrous cycle evaluation. Vaginal lavage samples were also collected from all surviving adult P1 and F1 parental female rats at the time of sacrifice. Vaginal lavage samples were examined microscopically for determination of the stage of the estrous cycle (diestrus, estrus, proestrus).
Sperm parameters (parental animals):
Sperm parameters for all P1 and F1 parental males surviving to scheduled sacrifice were evaluated. The right epididymis was removed, and the right cauda epididymis was weighed. Sperm was collected from the right cauda epididymis for evaluation of motility and morphology. The left epididymis and testis were frozen in liquid nitrogen and stored between -65°C and -85°C for sperm and spermatid counts, respectively.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded without pathologic examination. Litters of 8 offspring or fewer were not reduced.

PARAMETERS EXAMINED: At each examination period offspring were individually handled and examined for abnormal behaviour and appearance; any dead or abnormal pups were recorded. Live and dead pups in each litter were couted by sex as soon as possible after delivery was completed. Live pups in each litter were individually weighed (days 0, 4, 7, 14, and 21 postpartum).

GROSS EXAMINATION OF DEAD PUPS: Offspring that were moribund, found dead, or sacrificed because of death of the dam during the lactation period underwent gross pathology evaluation.
Postmortem examinations (parental animals):
SACRIFICE
Adult Males (P1, F1): After siring litters (test days 112-117 for P1 males and test days 119-122 for F1 males)
Pregnant Females (P1, F1): On day of weaning litters (postnatal day 21)
Non-pregnant Females (P1, F1): At least day 25 after the end of cohabitation

GROSS NECROPSY
- All P1 and F1 adult rats received a gross pathological examination, which included examination of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera. The tissues indicated in the tables below were prepared for microscopic examination and weighed, respectively. Paired organs were weighed together. Group mean values, organ weight ratios (% body weight and % brain weight), and combined accessory organs (ASO) values were calculated. Final body weight data were used for the calculation of organ/body weight ratios. The uteri of all cohabited P1 and F1 adult females were examined for the presence and number of implantation sites. A quantitative evaluation of primordial and growing follicles was conducted on 10 lactating F1 females (surviving to scheduled sacrifice) from the control and high-dose (2500 ppm) groups. No treatment-related change was observed; therefore, counting of follicles in the intermediate groups to determine a no-effect level was not required. Six ovarian cross sections were taken from the central area of the ovary using a step section technique. Primordial and growing follicles (up to but not including antral follicles) were enumerated for up to 12 ovarian sections per animal.

HISTOPATHOLOGY / ORGAN WEIGHTS
For P1 and F1 adults surviving to the scheduled sacrifice, all tissues collected at necropsy were processed and evaluated microscopically for 10 rats per sex in the control and high-concentration groups. Subsequent to the initial evaluation of 10 rats/sex in the control and high-concentration groups, suspect target organs were processed and evaluated from the remaining rats at all concentrations (30/sex/concentration). This included the reporductive organs form the P1 and F1 adult females and the femur and teeth from the F1 males. Both the male and female reproductive organs in paired animals that failed to produce a litter (i.e., reproductive failures) were also examined microscopically. In addition, all tissues collected a necropsy from rats that died before the study termination were evaluated microscopically. Most gross lesions in P1 and F1 adults were saved and evaluated microscopically. Selected gross obervations for which a microscopic diagnosis would not be additive were not processed for microscopic evaluation.
Postmortem examinations (offspring):
SACRIFICE: Offspring that were moribund, found dead, or sacrificed because of death of the dam during the lactation period underwent gross pathology evaluation. Pups culled on day 4 postpartum were sacrificed without pathological examination. F1 weanlings/offspring were sacrificed on the day of weaning (postnal day 21, except F1 animals selected for continued evaluation). F2 offspring were sacrificed on postnatal day 21.

GROSS NECROPSY: Three F1 and F2 weanlings/sex/litter (litter size permitting) underwent a gross pathological evaluation. All gross lesions and target organs were preserved for possible future microscopic examination. One weanling/sex/litter was designated fro organ weight and possible histopathological evaluation. These weanlings had selected organs weighed (brain, spleen, and thymus) and/or preserved in formalin for possible future microscopic examination (brain, spleen, thymus, gross lesions, and target organs).

HISTOPATHOLOGY: None of the preserved organs from the F1 and F2 weanlings were subsequently examined microscopically.
Statistics:
Refer to table below for statistics.
Reproductive indices:
Refer to table below for reproductive/offspring viability indices calculated.
Offspring viability indices:
Refer to table below for reproductive/offspring viability indices calculated.

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:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): During exposures, all animals exhibited normal responses to sound stimuli before, during (3 times), and after exposures. There were no early deaths or test substance-related increases in clinical observations in P1 or F1 male rats.

Three P1 female rats were euthanized early during the course of the study. One control animal was euthanized on lactation day 0 due to the loss of her litter. One female at 500 ppm was euthanized on lactation day 0 due to dystocia. One female at 1500 ppm was euthanized on gestation day 8 due to clinical signs which were the result of a uterine stromal sarcoma. These deaths were not considered to be test substance related. There were no test substance-related increases in any clinical observations reported during either the premating, gestation, or lactation phases of the study. The signs observed occurred with low frequency and with no dose response relationship. In addition, the reported observations were generally unremarkable and of a nature commonly seen in rats of this age.

Three F1 female rats were euthanized early during the course of the study. One control animals was euthanized on lactation day 2 due to the loss of her litter. One female at 1000 ppm was euthanized on lactation day 1 due to the loss of her litter. One female at 1500 ppm was euthanized on lactation day 15 due to a fractured nose. There were no adverse clinical signs noted preceding these deaths. These deaths were not considered to be test substance related. There were no test substance-related increases in any clinical observations reported during either the premating, gestation, or lactation phases of the study. The signs observed occurred with low frequency and with no dose response relationship. In addition, the reported observations were generally unremarkable and of a nature commonly seen in rats of this age.

BODY WEIGHT (PARENTAL ANIMALS): In P1 males at 2500 ppm, mean body weights on test days 8 through 85 were statistically significantly decreased, with reductions between 3.4% and 6.6% compared to controls. The effects on body weight were not observed after test day 85, and the mean final body weight at the conclusion of the exposure period (test day 113) was 4.7% lower than controls (not statistically significant). The effects on body weight were accompanied by occasional occurrences of statistically significant reductions in weight gain. Mean body weight gain over the premating period (1-72) was 10.5% lower than controls (statistically significant), and over the entire exposure period (test days 1-113) was 7.5% lower than controls (not statistically significant).

In P1 males at 1500 ppm, there were statistically significant reductions on mean body weights on test days 15, 22, 29, 43, 50, and 57, with reductions between 3.9% and 5.8% compared to controls. The effects on body weight were not observed after test day 57, and the mean final body weight on at the conclusion of the exposure period (test day 113) was 3.8% lower than controls (not statistically significant). The effects on body weight were accompanied by occasional occurrences of statistically significant reductions in weight gain. Mean body weight gain over the premating period (1-72) was 7.2% lower than controls (not statistically significant), and over the entire exposure period (test days 1-113) was 6% lower than controls (not statistically significant).

Since the effects on body weight pareameters in P1 males at 2500 and 1500 ppm were transient effects that were not statistically significant at the end of the exposure period, the reductions in body weight and body weight gain were considered test substance-related non-adverse effects.

In F1 males at 2500 ppm, mean body weights on test days 36 through 113 were statistically significantly decreased, with reductions between 7% and 11%, compared to controls. Mean final body weight at the conclusion of the exposure period (test day 113) was 10.2% lower than controls (statistically significant). The effects on body weight were accompanied by occasional occurrences of statistically significant reductions in weight gain. Mean body weight gain over the premating period (1-71) was 11.9 % lower than controls (statistically significant), and over the entire exposure period (test days 1-113) was 10.9% lower than controls (statistically significant).

In F1 males at 1500 ppm, mean body weights on test days 43 through 113 were statistically significant decreased, with reductions between 5.8% and 8%, compared to controls. Mean final body weight at the conclusion of exposure (test day 113) was 6.9% lower than controls (statistically significant). The effects on body weight were accompanied by occasional occurrences of statistically significant reductions in weight gain. Mean body weight gain over the premating period (1-71) was 8.8% lower than controls (statistically significant), and over the entire exposure period (test days 1-113) was 7.5% lower than controls (statistically significant).

Since the statistically significant effects on body weight parameters in F1 males at 2500 ppm persisted until the end of the exposure period, and due to the magnitude of the effects, the reductions in body weight and body weight gain were considered test substance-related adverse effects. Although the statistically significant effects on body weight parameters in F1 males at 1500 ppm persisted until the end of the exposure period, they were considered test substance-related but non-adverse effects due to the small magnitude of the changes.

There were no test substance-related effects on body weight parameters in P1 or F1 males at 500 or 1000 ppm. Occasional instances of statistically significant increases or decreases in body weight gain did not occur in a dose-related manner and were considered spurious.

In P1 females, there were no statistically significant effects on body weight parameters during premating, gestation, or lactation at any exposure concentration. Occasional instances of statistically significant decreases in body weight gain did not occur in a dose-related manner and were considered spurious.

In F1 females at 2500 ppm, mean body weights on test days 1, 8, 15, and 22 (premating) were statistically significantly decreased, with reductions between 5.9% and 8.2%, compared to controls. Mean final body weight at the conclusion of premating was 4.2% lower than controls (not statistically significant). There were no effects on body weight gain during premating in F1 females, and there were no effects on body weight parameters during gestation or lactation at 2500 ppm. There were no test substance-related effects on body weight parameters in F1 females at ≤1500 ppm. Since the effects on body weight in P1 females at 2500 ppm were transient effects that were not statistically significant at the end of the exposure period, the reductions in body weight was consideered test substance-related non-adverse effects.

FOOD CONSUMPTION (PARENTAL ANIMALS): In P1 males and, there were no test substance-related effects on food consumption or food efficiency at any exposure concentration. Occasional instances of statistically significant increases or decreases in food consumption or food efficiency did not occur in a dose-related manner and were considered spurious.

In F1 males at 2500 ppm, mean daily food consumption was statistically significantly decreased on test day intervals 36-43, 43-50, 57-64, 64-71, and 1-71. Mean daily food consumption over the premating period (test days 1-71) was 7% lower than controls (statistically significant). Mean food efficiency was decreased on test day intervals 29-36, 50-57, 64-71, and 1-71 (5.1% lower than controls). Mean food efficiency over the prematingperiod (test days 1-71) was 5.1% lower than controls (statistically significant). In F1 males at 1500 ppm, mean daily food consumption was statistically significantly decreased on test day intervals 36-43, 57-64, 64-71, and 1-71. Mean daily food consumption over the premating period (test days 1-71) was 5.9% lower than controls (statistically significant). Mean food efficiency was decreased on test day interval 50-57. Since the statistically significant effects on food consumption in F1 males at 2500 ppm persisted until the end of the premating period, due to the magnitude of the effects, and since the reduced food consumptionresulted in adverse effects on body weight and weight gain, the reductions in food consumption were considered test substance-related adverse effects. Although the effect on food consumption at 1500 ppm was statistically significant over the premating period, the effect was considered test substance-related but non-adverse since it did not result in adverse effects on body weight or body weight gain. In F1 males at ≤1000 ppm, there were no test substance-related effects on food consumption or food efficiency. Mean food efficiency over the premating period (test days 1-71) was statistically significantly decreased at 1000 ppm; however, the effect was not observed during any of the individual test day intervals, did not occur in a dose-related manner, and did not impact mean daily food consumption, and was therefore considered spurious.

In F1 females, there were no test substance-related effects on food consumption or food efficiency at any exposure concentration during premating, gestation, or lactation. A statistically significant decrease in food consumption observed at 2500 ppm over interval 8-15 during premating was considered spurious since it was a single occurrence.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS): There were no test substance-related effects on sperm parameters in P1 or F1 males at any exposure concentration. A single occurrence of decreased sperm number (per cauda) in F1 males at 500 was not observed at doses >1000 ppm, was not accompanied by effects on other sperm parameters, and therefore was considered spurious.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS): There were no test substance-related effects on estrous cyclicity in P1 or F1 females at any exposure concentration. The mean cycle length and mean number of cycles were comparable to controls at all exposure concentrations.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): There were no test substance-related effects on reproductive indices or precoital interval in P1 or F1 animals at any exposure concentration. No effects on the following reproductive performance parameters were observed: number of females mated, number of females that littered, mating index, fertility index, gestation index, precoital interval, or gestation length. The number of implantation sites and post-implantation loss were comparable across groups. The decreased post-implantation loss observed in P1 females at 1000 was not observed at doses >1500 ppm, and was a decrease as opposed to an increase in post-implantation loss that would be considered adverse, and therefore was considered spurious.

ORGAN WEIGHTS (PARENTAL ANIMALS): A test substance-related increase in liver and kidney weight parameters was observed in the P1 and F1 adult male rats. In the P1 males, liver and kidney weights were affected at ≥ 1000 ppm and 2500 ppm, respectively; in the F1 males, liver and kidney weights were affected at ≥ 500 ppm and ≥ 1000 ppm, respectively. The increases in relative organ weights were more pronounced in the F1 males primarily due to lower mean terminal body weights in this generation. It is most likely that the liver and kidney weight increases were due to the non adverse induction of metabolic enzymes (hepatocellular and renal tubular, respectively) secondary to xenobiotic exposure. Although liver and kidney histopathology was not conducted in this study, two previous 90-day inhalation studies with the test substance, at maximum exposures of 10,000 pp and 7500 ppm, did not report gross, microscopic or organ weight effects in either the liver or kidneys. There were no test substance-related organ weight effects in the P1 and F1 adult females or the F1 and F2 male and female weanlings

HISTOPATHOLOGY (PARENTAL ANIMALS): In F1 adult males, both the femur and teeth demonstrated microscopic changes associated with the retention of mineral following routine decalcification in tissue processing. In the femur, incomplete decalcification was observed at all exposure levels. The incidence was dose related; and all cases were graded as minimal. In the teeth, increased lamination of the dentin was also observed at all exposure levels and most males were affected. The incidence was not dose related. All cases were graded as minimal or mild and the severity was dose related. The microscopic findings of incomplete decalcification in the femur and increased lamination of the dentin in the teeth in F1 adult males were considered non-adverse test substance-related effects.

Effect levels (P0)

Key result
Dose descriptor:
NOAEC
Remarks:
Reproduction
Effect level:
2 500 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest exposure concentration evaluated
Remarks on result:
other: Generation: P and F1 (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

VIABILITY (OFFSPRING): There were no test substance-related effects observed on the mean number of pups born, live litter size, fetal sex ratio, or postnatal survival in either F1 or F2 offspring at any exposure concentration. The decreased lactation index observed in F2 offspring at 1500 ppm was not observed at 2500 ppm and was not accompanied by effects on other offspring survival indices in either F1 or F2 offspring, and therefore was considered spurious.

CLINICAL SIGNS (OFFSPRING): There were no treatment-related clinical observations noted in F1 or F2 offspring at any exposure concentration.

BODY WEIGHT (OFFSPRING): There were no test substance-related effects on mean pup body weights in F1 or F2 offspring at any exposure concentration. In F2 offspring at 2500 ppm, mean pup body weights were statistically significantly reduced on lactation days 14 and 21, with reductions of 6.1% and 7.2%, respectively, compared to the control group. In F2 offspring at 1500 ppm, mean pup body weights were statistically significantly reduced on lactation day 21, with a reduction of 8.4%, compared to the control group. Due to the small magnitude of the changes, the inherent variability in the pup weights during lactation, the lack of a dose-response relationship, and the lack of a similar effect on body weights in F1 pups, the statistically significant reductions in pup body weights observed in the F2 pups at 1500 and 2500 ppm were considered spurious.

SEXUAL MATURATION (OFFSPRING): There were no test substance-related effects on timing for achievement of vaginal patency or preputial separation in either F1 offspring at any exposure concentration.

ORGAN WEIGHTS (OFFSPRING): In F1 male weanlings, statistically significant decreases (-12.3% to -15.4%) in the 500 and 2500 ppm mean absolute and relative (% brain weight) thymus weights, as compared to control values, were interpreted as spurious since they were not dose related. In F1 female weanlings, a statistically significant increase (8.4%) in the 2500 ppm mean relative (% body weight) brain weight was the result of a moderate decrease (-6.7%) in the mean terminal body weight in this group. The mean absolute brain weight was only decreased 2.6%, relative to the control value. Several other statistically significant differences in mean organ weight values in the F1 female weanlings were considered spurious since they did not demonstrate a dose-related response. These include the absolute brain weight (500 ppm, -4.0%), absolute spleen weight (500 ppm, -16.3%; 2500 ppm, - 17.6%), relative (% brain weight) spleen weight (500 ppm, -13.1%; 2500 ppm, -15.6%), and thymus (500 ppm, -13.0%; 1000 ppm, -12.0%, 2500 ppm, -16.2%). A 14.0% decrease in the mean relative (% brain weight) thymus weight at 2500 ppm was also considered spurious since it was related to the spurious decrease in the mean absolute thymus weight. All individual and mean organ weight differences were considered to be spurious and not related to test substance exposure.

GROSS PATHOLOGY (OFFSPRING): In F1 and F2 weanlings, there were no test substance-related gross observations. All gross observations were consistent with normal background lesions in rats of this age and strain. In F1 and F2 pups, there were no test substance-related gross observations. Observations in pups of “lungs: not expanded” and “stomach: no milk spot” are nonspecific lesions that are commonly seen in pups that are born dead or die shortly after birth.

Effect levels (F1)

Key result
Dose descriptor:
other: NOAEC/Reproduction
Generation:
other: F1
Effect level:
2 500 other: (nominal) ppm (16774 mg/m3)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Highest exposure concentration evaluated
Remarks on result:
other: See remarks
Remarks:
P and F1 (migrated info)

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
This study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
Reproductive Toxicity NOAEC = 2500 ppm (highest exposure concentration)
Executive summary:

A two-generation reproduction study was conducted with the test substance, and involved the production of one set of litters in each generation. In this study, Crl:CD(SD) rats (30/sex/concentration) were exposed via whole-body inhalation for 6 hours per day, 7 days per week to 0, 500, 1000, 1500, or 2500 ppm of test substance vapour in air during the premating, mating, and gestation periods up until gestation day 19. Exposure of lactating dams recommenced beginning on postnatal day 7 and ended on postnatal day 21. Pups were not exposed during lactation. Male and female rats were exposed up until the day before schedule sacrifice.

Following at least 10 weeks of exposure to the test substance (premating), the P1 and F1 generation males and females were co-housed within their respective treatment groups to produce F1 and F2 litters, respectively. Dams were allowed to deliver and rear their offspring until weaning (postnatal day 21). F1 and F2 litters were culled to 4 pups/sex/litter (litter size permitting) on postnatal day 4; all remaining pups were discarded without further evaluation. At weaning, selected F1 offspring (one rat per sex per litter when possible) were randomly selected to serve as parents for the F2 generation. F2 litters were terminated at weaning.

Chamber atmospheres of test substance were generated by flash evaporation of the liquid test substance in air. Vapour concentrations of the test substance were measured by gas chromatography (GC). Temperature, humidity, and airflow were also recorded periodically during each daily exposure. Clinical observations, body weight, and food consumption were determined weekly throughout the study. Litter examinations (live, dead, or missing pups, individual pup weights, clinical observations) were determined at birth, on PND 4, and weekly during the 21-day lactation period. Estrous cycle parameters were evaluated daily for 3 weeks prior to cohabitation and up to the day of presumed mating in P1 and F1 adult rats. The age at either vaginal opening or preputial separation was recorded for the F1 generation. Sperm motility, morphology, concentration in the cauda epididymis, and spermatid concentration in the testis were determined for P1 and F1 adult rats at the terminal sacrifice. Gross postmortem examinations were performed on selected animals, and selected organs were weighed and/or retained for histopathological examination. A quantitative evaluation of ovarian follicles was conducted on 10 lactating F1 females from the control and high-dose (2500 ppm) groups.

The mean concentrations (± standard error of the mean) for exposures of P1 rats were 0.0 ± 0.0, 500 ± 1.3, 1000 ± 1.8, 1500 ± 2.1, and 2500 ± 2.5 ppm in chambers targeted at 0, 500, 1000, 1500, and 2500 ppm, respectively. The mean concentrations for exposures of F1 rats were 0.0 ± 0.0, 500 ± 1.2, 1000 ± 1.5, 1500 ± 2.2, and 2500 ± 2.5 ppm, respectively. Adverse test substance-related systemic toxicity consisted of reductions in body weight and nutritional parameters at 2500 ppm in adult F1 male rats. There were no test substance-related effects on reproductive outcomes at any exposure concentration tested in either the P1 or F1 generations. The data for mating, fertility, precoital interval length, gestation length, and implantation site counts were comparable across all groups tested for each respective generation. Additionally, there were no adverse, treatment-related effects noted on pup survival indices, pup body weights, estrous parameters, sperm parameters, or achievement of puberty at any exposure concentration for any generation. Therefore, the no-observed-adverse-effect-concentration (NOAEC) for reproductive toxicity was 2500 ppm, the highest inhalation exposure concentration evaluated.