Registration Dossier
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EC number: 207-050-4 | CAS number: 428-59-1
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
Key value for chemical safety assessment
Effects on fertility
Link to relevant study records
- Endpoint:
- one-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: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labeling and/or risk assessment.
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Deviations:
- yes
- Remarks:
- (Study included FOB and motor activity endpoints.)
- Qualifier:
- according to guideline
- Guideline:
- other: EPA OPPTS 870.3650
- Deviations:
- yes
- Remarks:
- Study included FOB and motor activity endpoints.
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- other: Crl:CD(SD)
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: Approximately 66 days
- Weight at study initiation: Males: 170.0–235.2 g; Females: 169.1–213.1 g
- Fasting period before study: no data
- Housing:
Pretest/Premating: Individually (except during cohabitation of mating pairs) in stainless steel, wire-mesh cages suspended above cage boards; sexes on separate racks.
Cohabitation: Mating pairs (females placed in males’ cages) on a 1:1 basis in stainless steel, wire-mesh cages suspended above cage boards.
Days 0–19 of gestation: Individually in stainless steel, wire-mesh cages suspended above cage boards; sexes on separate racks.
Day 19 of gestation - Day 4 of lactation: Females assumed pregnant were housed individually in polycarbonate pans with bedding material. Females presumed nonpregnant were housed in the same manner as pregnant females approximately 6 days after the mating pairs were separated.
- Diet (e.g. ad libitum): PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002 (pelleted); ad libitum
- Water (e.g. ad libitum): Tap water from United Water Delaware; ad libitum
- Acclimation period: Pretest Period: 14 days; Quarantine: 7 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18–26°C
- Humidity (%): 30%–70%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12-hours light/12 hours dark - Route of administration:
- inhalation: gas
- Type of inhalation exposure (if applicable):
- whole body
- Vehicle:
- other: filtered and conditioned air
- Details on exposure:
- GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: All exposure chambers were constructed of stainless steel and glass (NYU style) with a nominal internal volume of 350 L. A tangential feed at the chamber inlet promoted uniform chamber distribution of the test atmosphere.
- Method of holding animals in test chamber: During exposure, animals were individually placed in stainless steel wire-mesh modules (one/module) and exposed, whole-body, inside the exposure chamber, except during the mating period when animals were housed as mating pairs in stainless steel wire-mesh modules and exposed in the same manner.
- Source and rate of air: filtered and conditioned air
- Method of conditioning air: filtered and conditioned air
- System of generating vapour: Chamber atmospheres were generated by dilution of the test vapour in air. The test substance was metered into the test chamber inlets using a Brooks model 0145 or 0145E mass flow controller and mixed with filtered and conditioned air, which carried the resulting atmosphere into the exposure chamber. Chamber concentrations of test substance were manually controlled by varying the test substance feed rate to the chamber.
- Temperature, humidity, pressure in air chamber: 19–24°C; 41–82%; pressure not reported
- Air flow rate: 70–114 L/min
- Air change rate: at least 10 air changes per hour
- Treatment of exhaust air: to fume hood
TEST ATMOSPHERE
- Brief description of analytical method used: During each exposure, the atmospheric concentration of the test substance was determined by
gas chromatography at approximately 30-minute intervals in the test chambers. A gas chromatograph equipped with an automated gas sample valve and a flame ionization detector was used.
- Samples taken from breathing zone: yes - Details on mating procedure:
- - M/F ratio per cage: 1:1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: Daily examination of each female for an intravaginal copulation plug or sperm in the vaginal lavage sample.
- Further matings after two unsuccessful attempts: No
- After successful mating each pregnant female was caged (how): Days 0–19 of gestation: Individually in stainless steel, wire-mesh cages suspended above cage boards; sexes on separate racks. On day 19 of gestation for mated females or 6 days after the mating pairs were separated for females without evidence of copulation, females were transferred into polycarbonate pans. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Method of Analysis:
The mean concentrations (± standard error of the mean) of the test substance were 50 ± 0.098 and 250 ± 0.16 ppm in chambers targeted at 50 and 250 ppm, respectively. The mean concentration ± standard error of the mean) for the high concentration chamber was 580 ± 30 ppm. The reason for the relatively high standard error of the mean for the high concentration chamber was because the concentration was targeted to 1000 ppm for the first 6 exposures, and then targeted to 500 ppm for the remainder of the study. Overall daily mean concentrations ranged from 49-52 ppm for the 50 ppm chamber, 250 ppm for the 250 ppm chamber, and 500–1000 ppm for the 1000/500 ppm chamber. The calculated nominal concentrations were generally similar to the actual measured concentrations, with mean nominal concentrations of 58, 264, 564, and 1085 ppm for the 50, 250, 500, and 1000 ppm targeted concentrations, respectively. These data demonstrate that the exposure system and analyses were operating as expected. - Duration of treatment / exposure:
- Exposures for males and females were conducted for the 14 day premating period. Subsequently, males and non-pregnant females were exposed through to the terminal sacrifice. Exposures for females with evidence of mating were conducted during the cohabitation period (up to 2 weeks in duration) and during gestation days 0–19. Gestating P1 females were not exposed after gestation day 19.
Premating period - 14 days exposure
Cohabitation - Up to 14 days exposure
Postcohabitation Exposure:
Males - Up to test days 36–37
Females with no evidence of mating - 19 days (approximate)
Gestation - (GD 0-19)
Lactation - No exposure (LD 0-4) - Frequency of treatment:
- All exposures were conducted for 6 hours/day, 5 days/week during the premating period. Beginning at the mating (cohabitation) period, exposures were conducted for 6 hours/day, 7 days per week. Lactating dams were not exposed.
- Details on study schedule:
- GESTATION:
After being transferred into polycarbonate pans (on day 19 of gestation for mated females, or 6 days after the mating pairs were separated for females without evidence of copulation), female rats were observed at least twice daily for signs of delivery and offspring.
LACTATION:
On lactation days 0 and 4, offspring were individually handled and examined for abnormal behavior and appearance; any dead or abnormal pups were recorded. On Day 0 (Day of deliver = Day 0 of lactation) live and dead pups in each litter were counted by sex and live pups were individually weighed as soon as possible after delivery was completed. On Day 4 (litter sacrifice), pups in each litter were counted by sex and live pups were individually weighed and a gross external examination was performed. If litters died prior to Day 4 of lactation, the female was sacrificed. If the female died prior to Day 4 of lactation, the litter was sacrificed. If individual pups died prior to Day 4 of lactation, the carcass was examined to the extent possible and discarded. F1 pups were not necropsied, organs were not weighed, and there was no microscopic evaluation. - Remarks:
- Doses / Concentrations:
0, 50, 250, 1000/500 ppm (the high level concentration was reduced to 500 ppm on test day 9 [exposure #7])
Basis:
nominal conc. - No. of animals per sex per dose:
- 12 animals/sex/group
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: The test substance is slightly toxic in the rat by the inhalation route with a 2.5-hour ALC of 2000 ppm. The only signs of toxicity observed in rats exposed at 400 ppm in a 2-week inhalation study (4 hr/day; 5 days/wk) were inactivity and slightly deepened respiration; pathological examination revealed no test substance-related changes. No effects on body weight or food consumption were observed nor were there any clinical signs of toxicity in rats exposed at 100 or 400 ppm for 18-weeks (6 hr/day; 5 days/wk). Significant changes in organ/body weight ratios (spleen, kidneys and adrenals in males; liver in females) occurred at 400 ppm. Based on these results, the concentrations selected for the current study are 0, 50, 250, and 1000 ppm. However, due to excessive toxicity, the 1000 ppm concentration was reduced to 500 ppm on test day 9.
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once during pretest (baseline), and weekly thereafter.
BODY WEIGHT: Yes
- Time schedule for examinations:
Quarantine: Twice
Pretest: Weekly
Cohabitation: Weekly
Post-cohabitation: Weekly for males and females without evidence of copulation
Gestation: Days 0, 7, 14, 21G
Lactation: Days 0, 4L
Sacrifice: All animals
FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Premating: Weekly
Cohabitation: None
Post-cohabitation: None for males and females without evidence of copulation
Gestation: Days 0, 7, 14, 21G
Lactation: Days 0, 4L
OTHER:
FOOD EFFICIENCY:
- Calculation of Food Consumption: Feeders were weighed at the beginning and end of the interval. The final weight and amount of spillage (greater than 5 g) were subtracted from initial feeder weight.
- Calculation of Mean Daily Food Efficiency: Calculated from food consumption and body weight data.
HAEMATOLOGY: Yes
- Time schedule for collection of blood: first 5 surviving male and female animals on test day 17
- Anaesthetic used for blood collection: Yes (carbon dioxide)
- Animals fasted: Yes
- How many animals: 5 each sex
- Parameters checked in table [No.1] were examined.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: first 5 surviving male and female animals on test day 17
- Animals fasted: Yes
- How many animals: 5 each sex
- Parameters checked in table [No.2] were examined.
NEUROBEHAVIOURAL EXAMINATION: Yes, (refer to Section 7.9.1, DI.K1.Neuro.R.D-20964 for additional details).
- Time schedule for examinations: Prior to initiation of test substance administration and prior to the end of the premating period
- Dose groups that were examined: all
- Battery of functions tested: Abbreviated Functional Observational Battery (FOB), Motor Activity Evaluation - Litter observations:
- STANDARDISATION OF LITTERS:
- Performed on day 4 postpartum: no
PARAMETERS EXAMINED
On lactation days 0 and 4, offspring were individually handled and examined for abnormal behavior and appearance; any dead or abnormal pups were recorded. On Day 0 (Day of deliver = Day 0 of lactation) live and dead pups in each litter were counted by sex and live pups were individually weighed as soon as possible after delivery was completed. On Day 4 (litter sacrifice), pups in each litter were counted by sex and live pups were individually weighed and a gross external examination was performed. If litters died prior to Day 4 of lactation, the female was sacrificed. If the female died prior to Day 4 of lactation, the litter was sacrificed. If individual pups died prior to Day 4 of lactation, the carcass was examined to the extent possible and discarded. F1 pups were not necropsied, organs were not weighed, and there was no microscopic evaluation.
GROSS EXAMINATION OF DEAD PUPS:
Yes, for external abnormalities; possible cause of death was determined for pups born or found dead - Postmortem examinations (parental animals):
- SACRIFICE SCHEDULE:
Males - After siring litters (approximately 28 days of exposure)
Dams with Litters - Day 4L
Females without evidence of mating - Approximately 26 days after end of cohabitation
F1 litters - PND 4
Gross Pathology - All animals. Uterine implantation sites and ovarian corpora lutea were counted in P1 females.
Organ Weights - All animals
Histopathology - Control and high-dose groups (target organs and gross lesions in intermediate-dose groups)
GROSS NECROPSY: Yes (see table No. 3)
HISTOPATHOLOGY / ORGAN WEIGHTS: The tissues indicated in Table 3 were prepared for microscopic examination and weighed, respectively. - Postmortem examinations (offspring):
- SACRIFICE
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
PARAMETERS EXAMINED
On lactation days 0 and 4, offspring were individually handled and examined for abnormal behavior and appearance; any dead or abnormal pups were recorded.On Day 0 (Day of deliver = Day 0 of lactation) live and dead pups in each litter were counted by sex and live pups were individually weighed as soon as possible after delivery was completed. On Day 4 (litter sacrifice), pups in each litter were counted by sex and live pups were individually weighed and a gross external examination was performed. If litters died prior to Day 4 of lactation, the female was sacrificed. If the female died prior to Day 4 of lactation, the litter was sacrificed. If individual pups died prior to Day 4 of lactation, the carcass was examined to the extent possible and discarded. F1 pups were not necropsied, organs were not weighed, and there was no microscopic evaluation.
GROSS EXAMINATION OF DEAD PUPS:
Yes, for external abnormalities; possible cause of death was determined for pups born or found dead. - Statistics:
- Male and female data were evaluated separately. For litter parameters, the proportion of affected pups per litter or the litter mean were used as the experimental unit for statistical evaluation. The level of significance selected was p < 0.05.
see Table 4 Materials and Methods tables below - Reproductive indices:
- Refer to Table 5 for reproductive indices calculated.
- Offspring viability indices:
- Refer to Table 5 for offspring viability indices calculated.
- Clinical signs:
- effects observed, treatment-related
- 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: oestrous cycle:
- not examined
- Reproductive function: sperm measures:
- not examined
- Reproductive performance:
- no effects observed
- Dose descriptor:
- NOAEC
- Effect level:
- 500 ppm (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: No reproductive effects at the highest concentration tested.
- Remarks on result:
- other: Generation: reproductive (migrated information)
- Dose descriptor:
- NOAEC
- Effect level:
- 50 ppm (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: based on the histopathological and clinical pathology effects, body weight changes, and changes in nutritional parameters observed at 250 ppm and above
- Clinical signs:
- no effects observed
- Mortality / viability:
- no mortality observed
- Body weight and weight changes:
- effects observed, treatment-related
- Sexual maturation:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Histopathological findings:
- not examined
- Dose descriptor:
- NOAEC
- Generation:
- F1
- Effect level:
- 50 ppm (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: pup body weight effects at 250 ppm and above.
- Reproductive effects observed:
- not specified
- Conclusions:
- This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).
The NOAEC for reproduction was 1000/500 ppm based on the absence of effects at the highest concentration tested.
The NOAEC for offspring was 50 ppm based on pup body weight effects at 250 ppm and above. - Executive summary:
A combined repeated dose toxicity study with reproduction/developmental toxicity screening test was conducted with the test substance. Crl:CD(SD) rats (12/sex/concentration) were exposed whole body to 0, 50, 250, or 1000/500 ppm. Due to excessive toxicity, the 1000 ppm concentration was reduced to 500 ppm beginning on exposure 7 (test day 9). Exposures for males and females were conducted for 6 hours per day, 5 days per week from the initiation of the study through the 14 day premating period. Subsequently, males and non-pregnant females were exposed 7 days a week through the terminal sacrifice. Exposures for females with evidence of mating were conducted for 6 hours per day, 7 days per week during the cohabitation period (up to 2 weeks in duration) and during gestation days 0–19. Gestating P1 females were not exposed after gestation day 19. An abbreviated neurobehavioral evaluation consisting of a functional observational battery and motor activity was conducted in P1 rats (12/sex/group) once during pretest and prior to cohabitation. Clinical pathology parameters were measured in P1 rats (5/sex/group) at the end of the premating period (hematology, clinical chemistry) and at terminal sacrifice (coagulation). All P1 rats were given a gross pathological examination and selected tissues were weighed and collected from all adult rats. F1 litter examinations (pup viability, individual pup weights, pup sex, and clinical observations) were performed at birth and on lactation day 4. Uterine implantation sites and ovarian corpora lutea were counted in P1 females.
There were no test substance-related or statistically significant differences in mean number of pregnant animals, number of animals delivering, mating index, fertility index, precoital interval, gestation length, number of corpora lutea, number of implantation sites, or percent of postimplantation loss for any exposure concentration. There were no test substance-related or statistically significant differences in number of litters born, number of pups born or born alive, live born index, viability index, sex ratio, or incidence of clinical observations. Adverse, test substance-related decreases in pup weight occurred in the 250 ppm and above groups. Pup weight for the 250 and 1000/500 ppm groups was significantly lower compared to the control mean on postnatal day 4.
The NOAEC for reproduction was 1000/500 ppm based on the absence of effects at the highest concentration tested. The NOAEC for offspring was 50 ppm based on the pup body weight effects at 250 ppm and above.
Reference
BODY WEIGHT AND FOOD CONSUMPTION: Refer to Section 7.5.2, DI.K1.28Day.InhGas.RD/REPRO/DEV.R.D-20964.KD for details.
HAEMATOLOGY: Refer to Section 7.5.2, DI.K1.28Day.InhGas.RD/REPRO/DEV.R.D-20964.KD for details.
CLINICAL CHEMISTRY: Refer to Section 7.5.2, DI.K1.28Day.InhGas.RD/REPRO/DEV.R.D-20964.KD for details.
HISTOPATHOLOGY / ORGAN WEIGHTS: Refer to Section 7.5.2, DI.K1.28Day.InhGas.RD/REPRO/DEV.R.D-20964.KD for details.
There were no test substance-related or statistically significant differences in mean number of pregnant animals, number of animals delivering, mating index, fertility index, precoital interval, gestation length, number of corpora lutea, number of implantation sites, or percent of postimplantation loss for any exposure concentration. There were no test substance-related or statistically significant differences in number of litters born, number of pups born or born alive, live born index, viability index, sex ratio, or incidence of clinical observations.
BODY WEIGHT (OFFSPRING): Adverse, test substance-related decreases in pup weight occurred in the 250 ppm and above groups. Pup weight for the 250 and 1000/500 ppm groups was significantly lower compared to the control mean on postnatal day 4.
See Table 6 below
Mean Pup Weights |
||||
Group:Concentration (ppm) |
1 0 |
2 50 |
3 250 |
4 1000/500 |
PND 0 |
6.7 0.6(12) |
6.4 0.7(11) |
6.2 0.7(10) |
6.1 0.6(10) |
PND 4 |
11.0 |
9.8 |
9.5 |
9.7 |
Data summarized as: Mean Standard Deviation (n) |
Effect on fertility: via inhalation route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEC
- 3 400 mg/m³
- Study duration:
- subchronic
- Species:
- rat
Additional information
A combined repeated dose toxicity study with reproductive/developmental toxicity screening test was conducted according to OECD Guideline 422 at concentrations of 50, 250, and 1000/500 ppm for 6 hours/day, 5 days/week through premating. They were then exposed 6 hours/day, 7 days/week for the cohabitation (males and females) and gestation (females only). Due to excessive toxicity, the 1000 ppm concentration was reduced to 500 ppm beginning on exposure 7 (test day 9). Test substance-related mortality, clinical signs of toxicity, reductions in body weight, weight gain, food consumption and/or food efficiency were observed in males at ≥250 ppm and in females at 1000/500 ppm. In addition, adverse, test substance-related effects on offspring body weight occurred at ≥250 ppm. There were no test substance-related or statistically significant differences in the reproductive parameters examined. Under the conditions of the study, the No-Observed-Adverse-Effect Concentration (NOAEC) for systemic toxicity was 50 ppm based on the histopathological and clinical pathology effects, body weight changes, and changes in nutritional parameters observed at ≥250 ppm. The NOAEC for reproduction was 1000/500 based on the absence of effects at the highest concentration tested.
Short description of key information:
Inhalation: OECD 422; rats. NOAEC = 1000/500 ppm (6800/3400 mg/m3). No effects were observed in reproductive parameters examined at the highest concentration tested. Reliability = 1.
Justification for selection of Effect on fertility via inhalation route:
OECD guideline; GLP study
Effects on developmental toxicity
Description of key information
Inhalation: OECD 422; rats. NOEC = 50 ppm (340 mg/m3) based on significant reductions in pup weight observed at maternally toxic concentrations of ≥250 ppm. Reliability = 1.
Link to relevant study records
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labeling and/or risk assessment.
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Deviations:
- yes
- Remarks:
- Study included FOB and motor activity endpoints.
- Qualifier:
- according to guideline
- Guideline:
- other: EPA OPPTS 870.3650
- Deviations:
- yes
- Remarks:
- Study included FOB and motor activity endpoints
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- other: Crl:CD(SD)
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: Approximately 66 days
- Weight at study initiation: Males: 170.0–235.2 g; Females: 169.1–213.1 g
- Fasting period before study: no data
- Housing:
Pretest/Premating: Individually (except during cohabitation of mating pairs) in stainless steel, wire-mesh cages suspended above cage boards; sexes on separate racks.
Cohabitation: Mating pairs (females placed in males’ cages) on a 1:1 basis in stainless steel, wire-mesh cages suspended above cage boards.
Days 0–19 of gestation: Individually in stainless steel, wire-mesh cages suspended above cage boards; sexes on separate racks.
Day 19 of gestation - Day 4 of lactation: Females assumed pregnant were housed individually in polycarbonate pans with bedding material. Females presumed nonpregnant were housed in the same manner as pregnant females approximately 6 days after the mating pairs were separated.
- Diet (e.g. ad libitum): PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002 (pelleted); ad libitum
- Water (e.g. ad libitum): Tap water from United Water Delaware; ad libitum
- Acclimation period: Pretest Period: 14 days; Quarantine: 7 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18–26°C
- Humidity (%): 30%–70%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12-hour light/dark - Route of administration:
- inhalation: gas
- Type of inhalation exposure (if applicable):
- whole body
- Vehicle:
- other: filtered and conditioned air
- Details on exposure:
- GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: All exposure chambers were constructed of stainless steel and glass (NYU style) with a nominal internal volume of 350 L. A tangential feed at the chamber inlet promoted uniform chamber distribution of the test atmosphere.
- Method of holding animals in test chamber: During exposure, animals were individually placed in stainless steel wire-mesh modules (one/module) and exposed, whole-body, inside the exposure chamber, except during the mating period when animals were housed as mating pairs in stainless steel wire-mesh modules and exposed in the same manner.
- Source and rate of air: filtered and conditioned air
- Method of conditioning air: filtered and conditioned air
- System of generating vapour: Chamber atmospheres were generated by dilution of the test vapour in air. The test substance was metered into the test chamber inlets using a Brooks model 0145 or 0145E mass flow controller and mixed with filtered and conditioned air, which carried the resulting atmosphere into the exposure chamber. Chamber concentrations of test substance were manually controlled by varying the test substance feed rate to the chamber.
- Temperature, humidity, pressure in air chamber: 19–24°C; 41–82%; pressure not reported
- Air flow rate: 70–114 L/min
- Air change rate: at least 10 air changes per hour
- Treatment of exhaust air: to fume hood
TEST ATMOSPHERE
- Brief description of analytical method used: During each exposure, the atmospheric concentration of the test substance was determined by
gas chromatography at approximately 30-minute intervals in the test chambers. A gas chromatograph equipped with an automated gas sample valve and a flame ionization detector was used.
- Samples taken from breathing zone: yes - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Method of Analysis:
The mean concentrations (± standard error of the mean) of the test substance were 50 ± 0.098 and 250 ± 0.16 ppm in chambers targeted at 50 and 250 ppm, respectively. The mean concentration ± standard error of the mean) for the high concentration chamber was 580 ± 30 ppm. The reason for the relatively high standard error of the mean for the high concentration chamber was because the concentration was targeted to 1000 ppm for the first 6 exposures, and then targeted to 500 ppm for the remainder of the study. Overall daily mean concentrations ranged from 49-52 ppm for the 50 ppm chamber, 250 ppm for the 250 ppm chamber, and 500–1000 ppm for the 1000/500 ppm chamber. The calculated nominal concentrations were generally similar to the actual measured concentrations, with mean nominal concentrations of 58, 264, 564, and 1085 ppm for the 50, 250, 500, and 1000 ppm targeted concentrations, respectively. These data demonstrate that the exposure system and analyses were operating as expected. - Details on mating procedure:
- - M/F ratio per cage: 1:1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: Daily examination of each female for an intravaginal copulation plug or sperm in the vaginal lavage sample.
- Further matings after two unsuccessful attempts: No
- After successful mating each pregnant female was caged (how): Days 0–19 of gestation: Individually in stainless steel, wire-mesh cages suspended above cage boards; sexes on separate racks. On day 19 of gestation for mated females or 6 days after the mating pairs were separated for females without evidence of copulation, females were transferred into polycarbonate pans. - Duration of treatment / exposure:
- Exposures for males and females were conducted for the 14 day premating period. Subsequently, males and non-pregnant females were exposed through to the terminal sacrifice. Exposures for females with evidence of mating were conducted during the cohabitation period (up to 2 weeks in duration) and during gestation days 0–19. Gestating P1 females were not exposed after gestation day 19.
Premating period - 14 days exposure
Cohabitation - Up to 14 days exposure
Postcohabitation Exposure:
Males - Up to test days 36–37
Females with no evidence of mating - 19 days (approximate)
Gestation - (GD 0-19)
Lactation - No exposure (LD 0-4) - Frequency of treatment:
- All exposures were conducted for 6 hours/day, 5 days/week during the premating period. Beginning at the mating (cohabitation) period, exposures were conducted for 6 hours/day, 7 days per week. Lactating dams were not exposed.
- Duration of test:
- GESTATION:
After being transferred into polycarbonate pans (on day 19 of gestation for mated females, or 6 days after the mating pairs were separated for females without evidence of copulation), female rats were observed at least twice daily for signs of delivery and offspring.
LACTATION:
On lactation days 0 and 4, offspring were individually handled and examined for abnormal behavior and appearance; any dead or abnormal pups were recorded. On Day 0 (Day of deliver = Day 0 of lactation) live and dead pups in each litter were counted by sex and live pups were individually weighed as soon as possible after delivery was completed. On Day 4 (litter sacrifice), pups in each litter were counted by sex and live pups were individually weighed and a gross external examination was performed. If litters died prior to Day 4 of lactation, the female was sacrificed. If the female died prior to Day 4 of lactation, the litter was sacrificed. If individual pups died prior to Day 4 of lactation, the carcass was examined to the extent possible and discarded. F1 pups were not necropsied, organs were not weighed, and there was no microscopic evaluation. - Remarks:
- Doses / Concentrations:
0, 50, 250, 1000/500 ppm (the high level concentration was reduced to 500 ppm on test day 9 [exposure #7])
Basis:
nominal conc. - No. of animals per sex per dose:
- 12 females
- Control animals:
- yes, concurrent no treatment
- Details on study design:
- - Dose selection rationale: The test substance is slightly toxic in the rat by the inhalation route with a 2.5-hour ALC of 2000 ppm. The only signs of toxicity observed in rats exposed at 400 ppm in a 2-week inhalation study (4 hr/day; 5 days/wk) were inactivity and slightly deepened respiration; pathological examination revealed no test substance-related changes. No effects on body weight or food consumption were observed nor were there any clinical signs of toxicity in rats exposed at 100 or 400 ppm for 18-weeks (6 hr/day; 5 days/wk). Significant changes in organ/body weight ratios (spleen, kidneys and adrenals in males; liver in females) occurred at 400 ppm. Based on these results, the concentrations selected for the current study are 0, 50, 250, and 1000 ppm. However, due to excessive toxicity, the 1000 ppm concentration was reduced to 500 ppm on test day 9.
- Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once during pretest (baseline), and weekly thereafter.
BODY WEIGHT: Yes
- Time schedule for examinations:
Quarantine: Twice
Pretest: Weekly
Cohabitation: Weekly
Post-cohabitation: Weekly for males and females without evidence of copulation
Gestation: Days 0, 7, 14, 21G
Lactation: Days 0, 4L
Sacrifice: All animals
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Premating: Weekly
Cohabitation: None
Post-cohabitation: None for males and females without evidence of copulation
Gestation: Days 0, 7, 14, 21G
Lactation: Days 0, 4L
POST-MORTEM EXAMINATIONS: Yes
SACRIFICE SCHEDULE:
Males - After siring litters (approximately 28 days of exposure)
Dams with Litters - Day 4L
Females without evidence of mating - Approximately 26 days after end of cohabitation
F1 litters - PND 4
Gross Pathology - All animals. Uterine implantation sites and ovarian corpora lutea were counted in P1 females.
Organ Weights - All animals
Histopathology - Control and high-dose groups (target organs and gross lesions in intermediate-dose groups)
OTHER:
FOOD EFFICIENCY:
- Calculation of Food Consumption:
Feeders were weighed at the beginning and end of the interval. The final weight and amount of spillage (greater than 5 g) were subtracted from initial feeder weight.
- Calculation of Mean Daily Food Efficiency:
From food consumption and body weight data.
HAEMATOLOGY: Yes
- Time schedule for collection of blood: first 5 surviving male and female animals on test day 17
- Anaesthetic used for blood collection: Yes (carbon dioxide)
- Animals fasted: Yes
- How many animals: 5 each sex
- Parameters checked in table [No.1] were examined.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: first 5 surviving male and female animals on test day 17
- Animals fasted: Yes
- How many animals: 5 each sex
- Parameters checked in table [No.2] were examined.
NEUROBEHAVIOURAL EXAMINATION: Yes, (refer to Section 7.9.1, DI.K1.Neuro.R.D-20964 for additional details).
- Time schedule for examinations: Prior to initiation of test substance administration and prior to the end of the premating period
- Dose groups that were examined: all
- Battery of functions tested: Abbreviated Functional Observational Battery (FOB), Motor Activity Evaluation - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No - Fetal examinations:
- - External examinations: Yes: all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
On lactation days 0 and 4, offspring were individually handled and examined for abnormal behavior and appearance; any dead or abnormal pups were recorded. On Day 0 (Day of deliver = Day 0 of lactation) live and dead pups in each litter were counted by sex and live pups were individually weighed as soon as possible after delivery was completed. On Day 4 (litter sacrifice), pups in each litter were counted by sex and live pups were individually weighed and a gross external examination was performed. If litters died prior to Day 4 of lactation, the female was sacrificed. If the female died prior to Day 4 of lactation, the litter was sacrificed. If individual pups died prior to Day 4 of lactation, the carcass was examined to the extent possible and discarded. F1 pups were not necropsied, organs were not weighed, and there was no microscopic evaluation.
Gross Examination of Dead Pups: Yes, for external abnormalities; possible cause of death was determined for pups born or found dead. - Statistics:
- Male and female data were evaluated separately. For litter parameters, the proportion of affected pups per litter or the litter mean were used as the experimental unit for statistical evaluation. The level of significance selected was p < 0.05.
see Table 4 Materials and Methods tables below - Indices:
- Refer to Table 5 for reproductive indices calculated.
- Details on maternal toxic effects:
- Maternal toxic effects:yes
Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY: Refer to Section 7.5.2, DI.K1.28Day.InhGas.RD/REPRO/DEV.R.D-20964.KD for details.
BODY WEIGHT AND FOOD CONSUMPTION: Refer to Section 7.5.2, DI.K1.28Day.InhGas.RD/REPRO/DEV.R.D-20964.KD for details.
HAEMATOLOGY: Refer to Section 7.5.2, DI.K1.28Day.InhGas.RD/REPRO/DEV.R.D-20964.KD for details.
CLINICAL CHEMISTRY: Refer to Section 7.5.2, DI.K1.28Day.InhGas.RD/REPRO/DEV.R.D-20964.KD for details.
HISTOPATHOLOGY / ORGAN WEIGHTS: Refer to Section 7.5.2, DI.K1.28Day.InhGas.RD/REPRO/DEV.R.D-20964.KD for details. - Dose descriptor:
- NOAEC
- Effect level:
- 50 ppm (nominal)
- Based on:
- test mat.
- Basis for effect level:
- other: maternal toxicity
- Dose descriptor:
- NOAEC
- Effect level:
- 50 ppm (nominal)
- Based on:
- test mat.
- Basis for effect level:
- other: developmental toxicity
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:yes
Details on embryotoxic / teratogenic effects:
VIABILITY (OFFSPRING): The failure of 3 P1 adult pairs to produce a litter was not test substance related. One 50 ppm pair and one 250 ppm pair had no gross or microscopic pathology to explain their reproductive failure. The reproductive failure of another 250 ppm pair was apparently due to decreased ovulation by the female as evidenced by decreased corpora lutea.
There were no test substance-related or statistically significant differences in mean number of pregnant animals, number of animals delivering, mating index, fertility index, precoital interval, gestation length, number of corpora lutea, number of implantation sites, or percent of postimplantation loss for any exposure concentration. There were no test substance-related or statistically significant differences in number of litters born, number of pups born or born alive, live born index, viability index, sex ratio, or incidence of clinical observations.
BODY WEIGHT (OFFSPRING): Adverse, test substance-related decreases in pup weight occurred in the 250 ppm and above groups. Pup weight for the 250 and 1000/500 ppm groups was significantly lower compared to the control mean on postnatal day 4.
See Table below. - Abnormalities:
- not specified
- Developmental effects observed:
- not specified
- Conclusions:
- This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).
The NOAEL for reproduction was 1000/500 ppm based on the absence of effects at the highest concentration tested.
The NOAEL for offspring was 50 ppm based on the pup body weight effects at 250 ppm and above.
- Executive summary:
A combined repeated dose toxicity study with reproduction/developmental toxicity screening test was conducted with the test substance. Crl:CD(SD) rats (12/sex/concentration) were exposed whole body to 0, 50, 250, or 1000/500 ppm. Due to excessive toxicity, the 1000 ppm concentration was reduced to 500 ppm beginning on exposure 7 (test day 9). Exposures for males and females were conducted for 6 hours per day, 5 days per week from the initiation of the study through the 14 day premating period. Subsequently, males and non-pregnant females were exposed 7 days a week through the terminal sacrifice. Exposures for females with evidence of mating were conducted for 6 hours per day, 7 days per week during the cohabitation period (up to 2 weeks in duration) and during gestation days 0–19. Gestating P1 females were not exposed after gestation day 19. An abbreviated neurobehavioral evaluation consisting of a functional observational battery and motor activity was conducted in P1 rats (12/sex/group) once during pretest and prior to cohabitation. Clinical pathology parameters were measured in P1 rats (5/sex/group) at the end of the premating period (hematology, clinical chemistry) and at terminal sacrifice (coagulation). All P1 rats were given a gross pathological examination and selected tissues were weighed and collected from all adult rats. F1 litter examinations (pup viability, individual pup weights, pup sex, and clinical observations) were performed at birth and on lactation day 4. Uterine implantation sites and ovarian corpora lutea were counted in P1 females.
There were no test substance-related or statistically significant differences in mean number of pregnant animals, number of animals delivering, mating index, fertility index, precoital interval, gestation length, number of corpora lutea, number of implantation sites, or percent of postimplantation loss for any exposure concentration. There were no test substance-related or statistically significant differences in number of litters born, number of pups born or born alive, live born index, viability index, sex ratio, or incidence of clinical observations. Adverse, test substance-related decreases in pup weight occurred in the 250 ppm and above groups. Pup weight for the 250 and 1000/500 ppm groups was significantly lower compared to the control mean on postnatal day 4.
The NOAEL for reproduction was 1000/500 ppm based on the absence of effects at the highest concentration tested. The NOAEL for offspring was 50 ppm based on the pup body weight effects at 250 ppm and above.
Reference
Mean Pup Weights
|
||||
Group:Concentration (ppm) |
1 0 |
2 50 |
3 250 |
4 1000/500 |
PND 0
|
6.7 0.6(12) |
6.4 0.7(11) |
6.2 0.7(10) |
6.1 0.6(10) |
PND 4
|
11.0
|
9.8
|
9.5
|
9.7
|
Data summarized as: Mean Standard Deviation (n) |
Effect on developmental toxicity: via inhalation route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEC
- 340 mg/m³
- Study duration:
- subchronic
- Species:
- rat
Additional information
A combined repeated dose toxicity study with reproductive/developmental toxicity screening test was conducted according to OECD Guideline 422 at concentrations of 50, 250, and 1000/500 ppm for 6 hours/day, 5 days/week through premating. They were then exposed 6 hours/day, 7 days/week for the cohabitation (males and females) and gestation (females only). Due to excessive toxicity, the 1000 ppm concentration was reduced to 500 ppm beginning on exposure 7 (test day 9). Test substance-related mortality, clinical signs of toxicity, reductions in body weight, weight gain, food consumption and/or food efficiency were observed in males at ≥250 ppm and in females at 1000/500 ppm. In addition, adverse, test substance-related effects on offspring body weight occurred at ≥250 ppm. Pup weights at 250 and 1000/500 ppm were significantly reduced compared to control on postnatal day 4. There were no test substance-related or statistically significant differences in the reproductive parameters examined. Under the conditions of the study, the No-Observed-Adverse-Effect Concentration (NOAEC) for systemic toxicity was 50 ppm based on the histopathological and clinical pathology effects, body weight changes, and changes in nutritional parameters observed at ≥250 ppm. The NOAEC for offspring was 50 ppm based on the pup weight effects observed at maternally toxic concentrations of ≥250 ppm.
Justification for selection of Effect on developmental toxicity: via inhalation route:
OECD guideline, GLP study
Justification for classification or non-classification
The test substance did not adversely affect reproductive function at doses including systemic toxicity. Although some pup weight effects were observed, these effects occurred at concentrations that resulted in maternal toxicity, and were not considered to be primary effects on the developing offspring. Therefore, the substance does not need to be classified for reproductive or developmental toxicity according the EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.
Additional information
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