Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

There were no adverse effects on fertility

Link to relevant study records
Reference
Endpoint:
extended one-generation reproductive toxicity - with F2 generation (Cohorts 1A, and 1B with extension)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION

- Pre-existing data:
There are no pre-existing data available that would address the pre-natal developmental toxicity study endpoint (GLP or non-GLP). A rat pre-natal developmental toxicity study is not adequate accordig to the regulation. There are no historical human data that could be used in place of this study.

- (Q)SAR:
There are no accepted QSAR approaches for predicting the outcome of this endpoint. However, there were no Reproductive Toxicity alerts for MiAK.


- In vitro methods
There are no accepted in vitro approaches for predicting the outcome of this endpoint

- Weight of evidence
WoE is not possible as there are not similar substances that could be used for such a comparison.

- Grouping and read-across
The Registrant has not identified any suitable analogues that could be used to provide data for this endpoint. Other similar substances are quite data poor.

- Substance-tailored exposure driven testing
Based on the use patterns of the registered substance in coatings, exposure-based waiving is not possible.
Qualifier:
according to
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
GLP compliance:
yes
Limit test:
no
Justification for study design:
The study design was mandated by ECHA test order. The F2 cohort was not in the original test order but was added after findings in the F1 generation suggested the need to breed the F2 generation for study validation.
Specific details on test material used for the study:
Identification: Methyl isoamyl ketone (also referred to as MiAK; CAS No. 110 12-3)
Batch/Lot No.: TD16040801
Receipt Date: 13 Apr 2017, 27 Jun 2017, 05 Sep 2017, and 19 Dec 2017
Physical Description: Clear, colorless liquid
Purity: 99.33%
Storage Conditions: Kept in a controlled temperature area set to maintain 18°C to 24°C
Supplier: Eastman Chemical Company
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD)
Details on species / strain selection:
The Crl:CD(SD) (Sprague-Dawley) rat has been shown to be an appropriate animal model for this study design
Sex:
male/female
Details on test animals and environmental conditions:
The Crl:CD(SD) rat is recognized as appropriate for reproduction studies. Charles River Ashland has reproductive historical data for the Crl:CD(SD) rat. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
The number of animals selected for this study was based on the OECD Guideline for the Testing of Chemicals, Guideline 443, Extended One-Generation Reproductive Toxicity Study, 28 Jul 2011, which recommends including a sufficient number of mating pairs to yield at least 20 pregnant females per dose group. Given the possibility of nongravid animals, unexpected deaths, total litter losses, or test substance-related moribundity and/or mortality, 24 animals/sex/group was an appropriate number of animals to meet guideline recommendations.

Upon receipt, each animal was identified using a subcutaneously implanted electronic identification chip (BMDS system). Offspring were identified by tattoo markings applied to the digits after parturition and by microchip after weaning.

Pups selected for the F1 generation retained the dam number, followed by a hyphen "-" and the digit tattoo marking (i.e., 9999 01). F2 pups retained the dam number; however, the -01, -02, etc. designation in the F1 dam number was replaced with the corresponding letter of the alphabet (i.e., -01 = A, -02 = B, etc.) on some report tables.

Housing:
On arrival (F0) or following weaning (F1), the animals were group housed (2 to 3 animals of the same sex). During cohabitation, animals were paired for mating in the home cage of the male. Following the breeding period, animals were individually housed. Animals were housed in solid-bottom cages containing appropriate bedding equipped with an automatic watering valve (see Appendix 1 – Study Protocol and Deviations).
Animals were separated during designated procedures/activities. Each cage was clearly labeled with a color-coded cage card indicating study, group, animal, cage number(s), dosage level, and sex. Cages were arranged on the racks in group order.
Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals. The animal facilities at Charles River Ashland are accredited by AAALAC International.

Environmental Conditions:
Target temperatures of 68°F to 78°F (20°C to 26°C) with a relative target humidity of 30% to 70% were maintained (see Appendix 1 – Study Protocol and Deviations). A 12-hour light/12 hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

PMI Nutrition International, LLC Certified Rodent LabDiet® 5K96 Advanced Protocol® Verified Casein Diet 10 IF was provided ad libitum throughout the study, except during designated procedures (see Appendix 1 – Study Protocol and Deviations).
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis are provided by the supplier and are on file at the Testing Facility.
It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study.

Municipal tap water after treatment by reverse osmosis was freely available to each animal via an automatic watering system, except during designated periods. Periodic analysis of the water is performed, and results of these analyses are on file at the Testing Facility. It is considered that there are no known contaminants in the water that could interfere with the outcome of the study.

Animals were socially housed for psychological/environmental enrichment and were provided with environmental enrichment as appropriate to aid in maintaining the animals’ oral health.

Veterinary care was available throughout the course of the study, and animals were examined by the veterinary staff as warranted by clinical signs or other changes. All veterinary examinations and recommended therapeutic treatments, if any, were documented in the study records and reviewed by the Study Director.
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
clean air
Details on exposure:
F0 males were exposed for 6 hours daily for 70 consecutive days prior to mating and continuing through the day prior to euthanasia. F0 females were exposed for 6 hours daily for 70 consecutive days prior to mating and continuing throughout mating, gestation, and lactation, through the day prior to euthanasia.
For females with evidence of mating, exposures during gestation were conducted up to and including Gestation Day 20, at which time exposure was suspended through Lactation Day 4, to avoid confounding effects on parturition and nursing behavior. Exposures resumed on Lactation Day 5 and continued until 1 day prior to scheduled euthanasia. For females with evidence of mating that failed to deliver, exposures were resumed on Postmating Day 25. The offspring selected for the F1 generation began exposure following weaning until the day prior to euthanasia (PND 91 [Cohort 1A] and following the reproductive assessment [Cohort 1B]).
For Cohort 1B females, exposures were suspended during the time of parturition as noted above. All animals were exposed at approximately the same time each day.

The route of administration was inhalation because this is a potential route of exposure for humans and was also the required route of administration according to the ECHA Decision Letter. Historically, this route has been used extensively for studies of this nature.
The exposure levels were determined based on the results of previous studies including an inhalation prenatal developmental toxicity study of MiAK in rats. Bred female Sprague Dawley rats were exposed to MiAK at target exposure concentrations of 0, 380, 750, and 1500 ppm (achieved exposure concentrations of 0, 379, 751, and 1495 ppm) daily from Gestation Day 6 through 19. All animals survived to the scheduled euthanasia. At 1500 ppm, significant clinical findings included hair loss on various body surfaces, reduced reactivity to noise stimulus (near the end of exposure), and clear material around the mouth (at approximately 1 hour postexposure). Lower maternal mean body weight gains and food consumption with correspondingly lower absolute mean body weights were noted at 1500 ppm. Reduced reactivity to noise stimulus was also noted for females in the 750 ppm group. There were no significant maternal findings noted at 380 ppm and there were no maternal macroscopic findings or any effects on fetal survival at any exposure level. However, mean fetal weights at the 1500 ppm exposure concentration were 8.1% lower versus the control group. Fetal weights were unaffected by exposure at the 380 and 750 ppm exposure levels.
Based on these observations, 1500 ppm was selected as the highest exposure level because it had the potential to induce some toxicity. Exposure concentrations of 380 and 750 ppm were selected to characterize the dose-response relationship.
Details on mating procedure:
F0 Breeding Procedure:
After a minimum of 70 days of exposure, 1 female will be cohabitated with 1 male rat of the same treatment group, avoiding sibling mating (Charles River Laboratories will supply non-litter mates), in a solid-bottom cage for mating (home cage of the male). Detection of mating will be confirmed by the appearance of a vaginal copulatory plug or by evidence of sperm in a vaginal lavage. Vaginal lavages will be performed daily during the mating period until evidence of
mating is observed. After confirmation of mating, the female will be returned to an individual solid-bottom cage containing bedding material, and the day will be designated as day 0 of gestation.
A maximum of 14 days will be allowed for mating. If no evidence of copulation is obtained after 14 days, the animals will be separated without further opportunity for mating, and the female will be placed in a solid-bottom cage containing bedding material.

F1 Breeding Phase (Cohort 1B) - F2 Extension:
Due to the lower body weight gains noted for the F1 pups during the neonatal and pre-weaning periods, F1 animals assigned to Cohort 1B will be bred to obtain an F2 generation. All procedures for F1 generation (Cohort 1B) will generally be the same as those for the F0 generation and F1 litters with the following exceptions:
• Daily vaginal lavages will be performed for all Cohort 1B females to determine the stage of estrus beginning on the first PND 102 and continuing until the start of the breeding period, and continuing until evidence of copulation is observed.
• Cohort 1B females will be cohabited (1:1) with a male rat of the same treatment group beginning on the first Postnatal Day 111 in a solid-bottom cage for mating (home cage of the male). Sibling mating will also be avoided for the F1 generation, as parentage is known through birth records maintained in the study data.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyzed exposure concentrations were determined at approximately 45-minute intervals using a gas chromatograph (GC). Samples were collected from the approximate animal-breathing zone of the exposure chamber via 1/8-inch heated stainless steel tubing. Tubing was heated to approximately 60°C using Omega® heat tapes, J-type thermocouples and Model Nos. CN370 or CNi1654 temperature controllers. Under the control of the WINH system, sampling and analyses was performed as follows. The program controls an external multi-position valve (Model E16, Valco Instruments Co., Inc.; Houston, TX) that permits sequential sampling from the exposure room and each exposure chamber. The multi-position valve was heated to approximately 65°C using a disc heater and was controlled using an Omega® temperature controller (Model Nos. CN370 or CNi1654), and J-type thermocouple. Gas sampling injection onto the chromatography column occurs via an internal gas-sampling valve with a sample loop, the chromatograph is displayed and the area under the sample peak is calculated and stored. The WINH system then acquires the stored peak area data and uses an ln-quadratic equation based on the GC calibration curve to calculate the measured concentration in ppm.

Gas Chromatograph Calibration:
The GC was calibrated using ga s-phase standards prepared to contain a known vapor concentration of the test substance in 10-L Tedlar® gas bags (Supelco Analytical; Bellefonte, PA). A known volume of the test substance was injected into a heated glass vaporization bulb (approximately 145°C). Compressed nitrogen (8 L) was delivered through the bulb to dilute and carry the test substance vapor into the bag. A dry test meter (DTM-200A, Elster American Meter Co.; Nebraska City, NE) was used to measure the total volume of nitrogen. The standard bag was then placed in a heated box (approximately 60ºC) to ensure total volatilization of the test substance. The bulb and box was heated using Omega® heat tapes controlled using J-type thermocouples and Model Nos. CN370 or CNi1654 temperature controllers. Prior to being analyzed, the standard was attached at the designated multi-position valve position and opened to sample from the heated box for approximately 2 minutes.


Exposure Atmosphere Homogeneity Assessment:
Homogeneity of the exposure atmosphere within the exposure chambers was evaluated during the method development phase of the study. Four test locations and a reference location were used for sampling. Samples were collected and analyzed on the GC as rapidly as possible alternating from the reference and then to a test location. For each location, the measured concentration was calculated as a percent difference from the reference location. Homogeneity was performed in triplicate for each test substance exposure chamber.

Based on mean differences from the reference location, spatial homogeneity was considered acceptable for all exposure chambers. Temporal stability was evaluated using the concentrations from the reference location. The variability during the homogeneity evaluation was acceptable for all exposure chambers. The generation and exposure trial performed before the start of animal exposure also demonstrated acceptable concentration stability for each exposure chamber.
Duration of treatment / exposure:
Administration of Test Materials:
F0 males were exposed for 6 hours daily for 70 consecutive days prior to mating and continuing through the day prior to euthanasia. F0 females were exposed for 6 hours daily for 70 consecutive days prior to mating and continuing throughout mating, gestation, and lactation, through the day prior to euthanasia. For females with evidence of mating, exposures during gestation were conducted up to and including Gestation Day 20, at which time exposure was suspended through Lactation Day 4, to avoid confounding effects on parturition and nursing behavior. Exposures resumed on Lactation Day 5 and continued until 1 day prior to scheduled euthanasia. For females with evidence of mating that failed to deliver, exposures were resumed on Postmating Day 25. The offspring selected for the F1 generation began exposure following weaning until the day prior to euthanasia (PND 91 [Cohort 1A] and following the reproductive assessment [Cohort 1B]). For Cohort 1B females, exposures were suspended during the time of parturition as noted above. All animals were exposed at approximately the same time each day.
Frequency of treatment:
whole-body inhalation for 6 hours, daily
Details on study schedule:
The testing laboratory for this study uses "F0 and F1" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

The study design was as follows:
1. Filtered Air - Target Exposure Concentration (ppm) 0 ppm - 24 males and 24 females
2. MiAK - Target Exposure Concentration (ppm): 280 - 24 males and 24 females
3. MiAK - Target Exposure Concentration (ppm): 750 - 24 males and 24 females
4. MiAK - Target Exposure Concentration (ppm) 1500 - 24 males and 24 females

Animals in the parental (F0) generation were exposed via whole-body inhalation for 6 hours daily for 70 consecutive days prior to mating and continuing through the day prior to euthanasia. Maternal exposure was suspended from Gestation Day 20 through Lactation Day 4 to prevent confounding effects on parturition and maternal nursing and nesting behavior. The offspring in the F1 generation were potentially exposed in utero during gestation. Direct offspring exposure during the pre-weaning period was demonstrated on a concurrent study4, where quantifiable levels of MiAK were obtained in maternal milk and plasma obtained from dams exposed at the same target exposure concentrations (380 to 1500 ppm). Offspring selected to constitute the F1 generation were exposed beginning at weaning (Postnatal Day 28) and continuing through the day prior to euthanasia. Due to the lower body weight gains noted for the F1 pups during the neonatal and pre-weaning periods (see below), F1 animals assigned to Cohort 1B for follow-up reproductive assessments were bred to obtain an F2 generation. All procedures for F1 animals assigned to Cohort 1B were generally the same as those for the F0 generation/F1 litters, including suspension of exposure for the F1 generation maternal animals from Gestation Day 20 through Lactation Day 4. The offspring in the F2 generation were not directly exposed to test substance atmospheres but were potentially exposed in utero, and through maternal milk during the pre-weaning period.

The following parameters and end points were evaluated in this study:
clinical signs, body weights, body weight gains, food consumption, estrous cycles, reproductive performance, parturition, litter viability and survival, anogenital distance, areolae/nipple anlagen, developmental landmarks, thyroid hormones, clinical pathology, gross necropsy findings, spermatogenic endpoints, organ weights, and histopathologic examinations.

Target exposure concentrations were 380, 750 and 1500 ppm. Overall mean analyzed exposure concentrations were 380, 750, and 1499 ppm for the F0 generation and 379, 750, and 1497 ppm for the F1 generation.
Dose / conc.:
380 ppm (nominal)
Dose / conc.:
750 ppm (nominal)
Dose / conc.:
1 500 ppm (nominal)
No. of animals per sex per dose:
24 per sex per dose
Control animals:
yes
Details on study design:
The study design is according to OECD guideline 443
Parental animals: Observations and examinations:
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, once in the morning and once in the afternoon. Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

Clinical Observations:
Clinical observations were performed once daily throughout the study. During the exposure period, these observations were performed prior to exposure. On exposure days, observations were also recorded at the approximate mid-point of exposure, at the end of exposure, and 1–2 hours postexposure.
During social housing, some observations (e.g., fecal observations) may not have been attributable to an individual animal.


Physical Examinations
The animals were removed from the cage, and a detailed physical examination was performed weekly throughout the study. In addition, detailed physical examinations were conducted on Gestation Days 0, 7, 14, and 20 for all females with evidence of mating and on Lactation Days 1, 7, 14, 21, and 28 (see Appendix 1 – Study Protocol and Deviations).

Body Weights:
Animals were weighed individually weekly throughout the study and prior to the scheduled necropsy (see Appendix 1 – Study Protocol and Deviations). Once evidence of mating was observed, female body weights were recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and on Lactation Days 1, 4, 7, 10, 14, 17, 21, and 28. A fasted weight was recorded on the day of necropsy.

Food Consumption:
Food consumption was quantitatively measured weekly throughout the study period, except during the mating period (see Appendix 1 – Study Protocol and Deviations). Once evidence of mating was observed, female food consumption was recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and Lactation Days 1, 4, 7, 10, 14, 17, 21, and 28.

Food Evaluation:
Food efficiency (body weight gained as a percentage of food consumed) was also calculated and reported for each interval.

Oestrous cyclicity (parental animals):
Vaginal lavages were performed daily and the slides were evaluated microscopically to determine the stage of the estrous cycle of each F0 female for 14 days prior to cohabitation and continuing until evidence of mating was observed or until the end 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 14 days prior to initiation of the mating period and continuing 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 lavages were also performed on the day of necropsy to determine the stage of the estrous cycle.

At the end of the study, the overall pattern of each female was characterized as regularly cycling, irregularly cycling, not cycling, or insufficient data.
Sperm parameters (parental animals):
Immediately upon euthanasia, the reproductive tract of each male was exposed via a ventral mid line incision. The right cauda epididymis was excised and weighed. An incision was made in the distal region of the right cauda epididymis, and it was then placed in Dulbecco's phosphate buffered saline (maintained at approximately 37°C) with 10 mg/mL BSA. After a minimum 10 minute incubation period, a sample of sperm was loaded onto a slide with a 100 µm chamber depth for determination of sperm motility. Because sperm motility can be affected by temperature shock, all pipettes, slides, and diluents were warmed in an incubator, and motility determinations were performed under constant temperature (approximately 37°C). Analysis of a minimum of 200 motile and nonmotile spermatozoa per animal (if possible) in all groups was performed by the analyzer. The motility score (percent) for motile (showing motion only) and progressively motile (showing net forward motion) sperm was reported.

The right epididymis was then placed in modified Davidson’s solution for subsequent microscopic examination. 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 cauda epididymis from all males were weighed, stored frozen, homogenized, and analyzed for determination of homogenization resistant spermatid count and calculation of sperm production rate. An aliquot of each 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 analyzer allowed for the visualization and quantitation of the sperm. A minimum of 200 cells, if possible, or up to 20 fields were counted for each sample.
Litter observations:
Litters were observed for general health/mortality and moribundity twice daily, once in the morning and once in the afternoon. A daily record of litter size was maintained. Animals were not removed from the cage during observation, unless necessary for identification or confirmation of possible findings.

Total litter loss was determined when the last pup in the litter was found dead or euthanized in extremis prior to the scheduled euthanasia. Litters that were euthanized prior to scheduled euthanasia due to reasons unrelated to test substance exposure (e.g., death of the dam) were not considered to be a total litter loss on the data tables and were not included in the pup viability calculations.
Postmortem examinations (parental animals):
All animals were subjected to a complete necropsy examination, which included examination of the external surface, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera. Special attention was paid to the organs of the reproductive system. The numbers of former implantation sites were recorded for females that delivered. The number of unaccounted-for sites was calculated for each female by subtracting the number of pups born from the number of former implantation sites observed. For females that failed to deliver, a pregnancy status was determined, and specific emphasis was placed on anatomic or pathologic findings that may have interfered with pregnancy.
Postmortem examinations (offspring):
All animals were subjected to a complete necropsy examination, which included examination of the external surface, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera. Special attention was paid to the organs of the reproductive system. The numbers of former implantation sites were recorded for females that delivered. The number of unaccounted-for sites was calculated for each female by subtracting the number of pups born from the number of former implantation sites observed. For females that failed to deliver, a pregnancy status was determined, and specific emphasis was placed on anatomic or pathologic findings that may have interfered with pregnancy.
Statistics:
Due to space limits, please see this section of information under "Any Other information on materials and methods incl.tables".
Reproductive indices:
The testing laboratory for this study uses "F0 and F1" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

The day parturition was initiated was designated Lactation Day 0 (Postnatal Day [PND] 0 for pups). During the period of expected parturition, females were observed twice daily for initiation and completion of parturition and for dystocia or other difficulties. All females were allowed to deliver naturally. Beginning on the day parturition was initiated, the numbers of stillborn and live pups were recorded. Individual gestation length was calculated using the date delivery was first observed. The dam and litter remained together until weaning on PND 28.
Offspring viability indices:
Litters were observed for general health/mortality and moribundity twice daily, once in the morning and once in the afternoon. A daily record of litter size was maintained. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

Total litter loss was determined when the last pup in the litter was found dead or euthanized in extremis prior to the scheduled euthanasia. Litters that were euthanized prior to scheduled euthanasia due to reasons unrelated to test substance administration (e.g., death of the dam) were not considered to be a total litter loss on the data tables and were not included in the pup viability calculations.


Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The testing laboratory for this study uses "F0 and F1" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

A test substance-related increased number of F0 males and females with red material around the mouth was noted in the 1500 ppm group at the end of the exposure period and 1–2 hours postexposure. This observation was noted as early as Study Day 0, and continued sporadically through end of the respective exposure periods. To a lesser extent, red material around the eyes and clear material on various body surfaces were also noted at an increased incidence in F0 males and females at the end of the exposure period and 1–2 hours postexposure sporadically throughout the exposure period. There were no other test substance-related observations noted at the daily examinations or postexposure observations. Red material around the nose was noted for F0 males and females across all exposure groups, albeit in a non-exposure responsive manner, and was therefore considered incidental. Other findings noted in the test substance-exposed groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not exposure-related.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
The testing laboratory for this study uses "F0 and F1" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

All F0 parental animals in the test substance-exposed groups survived to the scheduled necropsy.
In the control group, 1 male and 2 females were found dead or euthanized in extremis prior to the scheduled necropsy. Male No. 5563 was euthanized in extremis on Study Day 74 (following the breeding period) due to poor general health; clinical observations of a thin body were noted on the day prior to and on the day of euthanasia, and veterinary observations noted that the animal was quiet, dehydrated with decreased defecation, and had mild piloerection. Microscopic findings in the exocrine pancreas (mild secretory depletion) and gross and microscopic findings in the liver (misshapen median lobe with microscopic correlate of moderate hepatocellular atrophy) both support decreased food consumption. Gross findings were noted in the kidney (adhesions to the liver with microscopic correlate of adhesion, calculi with no microscopic correlate, yellow foci correlating to marked pyelonephritis, and pelvic dilation correlating to marked dilation); urinary bladder (calculi correlating to minimal mineralization, distended and thickened correlating to moderate urothelial hyperplasia, dark red discoloration correlating to minimal congestion); ureter (distended correlating to mild dilation); seminal vesicles (small correlating to marked secretory depletion); thymus (small correlating to marked lymphoid depletion); and stomach (dark red foci correlating to minimal congestion of the glandular portion). Additional significant microscopic findings included minimal or marked acute inflammation in the prostate gland, urinary bladder, and ureter; minimal neutrophilic infiltration in the mesenteric lymph node; moderate myeloid hyperplasia of the sternal bone marrow; and moderate or marked lymphoid depletion of the spleen and axillary lymph node. The myeloid hyperplasia was considered supportive of increased demand for neutrophils due to acute inflammation in the urogenital tract. Lymphoid depletion of the axillary lymph node, spleen, and thymus were considered to be indirect findings related to stress. Based upon review of clinical observations, gross findings, and microscopic observations, the cause of debilitation was urogenital inflammation (affecting the kidneys, urinary bladder, prostate, and seminal vesicles).
Female No. 5686 was found dead on Lactation Day 10. No remarkable clinical observations or noteworthy changes in body weight or food consumption were noted for this female prior to death. In addition, no significant findings were noted during parturition. At necropsy, no gross findings were noted, and significant microscopic observations were limited to mild adrenal cortex vacuolation and marked uterine atrophy. The cause of death was undetermined.

Female No. 5701 was euthanized in extremis on Study Day 56 (prior to the breeding period) due to an accidental right hindlimb injury that occurred following escape from the cage. All other
F0 animals survived to the scheduled necropsies.

Body weight and weight changes:
no effects observed
Description (incidence and severity):
The testing laboratory for this study uses "F0 and F1" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

No test substance related effects on F0 mean body weights and body weight gains were noted in the 380, 750, and 1500 ppm groups. The values in the test substance exposed groups were generally similar to the control group values for the pre-mating period (females) or the entire generation (males). Sporadic, statistically significant mean body weight changes were noted for F0 males and females in the test substance exposed groups during the respective exposure periods; however, these differences were transient, did not occur in a exposure-related manner, and/or were not of sufficient magnitude to affect the overall interval(s) or mean absolute body weights in these groups, and therefore were not considered test substance-related.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
The testing laboratory for this study uses "F0 and F1" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Mean F0 food consumption and food efficiency in the 380, 750, and 1500 ppm group F0 males and females was unaffected by test substance exposure. The values in the test substance exposed groups were generally similar to the control group values for the pre-mating period (females) or the entire generation (males). Slightly higher mean food consumption was noted for F0 males in the 750 and 1500 ppm groups compared to the control group during Study Days 21–56; the differences were generally statistically significant. However, in the absence of corresponding effects on absolute body weights and body weight gains, the higher mean food consumption noted in these groups was considered incidental and not test substance-related. Other statistically significant differences noted in mean food consumption and food efficiency for F0 males and females in the 380, 750, and 1500 ppm groups were slight and/or did not occur in an exposure related manner.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The testing laboratory for this study uses "F0 and F1" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

There were no test substance-related effects on hematology parameters noted for F0 males and females at any exposure level. Statistically significant differences from the control group did not occur in an exposure-related manner, were only observed in 1 sex, and/or were of minimal magnitude; therefore, these differences were not considered test substance-related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The testing laboratory for this study uses "F0 and F1" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Coagulation:
There were no test substance-related effects on coagulation parameters noted for F0 males and females at any exposure level. Statistically significant differences from the control group did not occur in an exposure-related manner, were only observed in 1 sex, and/or were of minimal magnitude; therefore, these differences were not considered test substance-related.

Serum Chemistry:
There were no test substance-related effects on serum chemistry parameters noted for F0 males and females at any exposure level. Mean serum cholesterol levels in F0 males in the 1500 ppm group were elevated (42.7%) compared to the control group; the difference was statistically significant. Mean serum triglycerides in these same animals were lower than the control group and changes in mean serum cholesterol levels in F1 females did not occur in an exposure-related manner, and therefore the changes in serum cholesterol levels in the 1500 ppm group were not considered test substance related. Other statistically significant differences from the control group (calcium and glucose) did not occur in an exposure-related manner, were only observed in 1 sex, and/or were of minimal magnitude; therefore, these differences were not considered test substance related.

Thyroid Hormone Analysis:
There were no test substance-related effects on serum levels of T3 (triiodothyronine), T4 (thyroxine), or TSH (thyroid stimulating hormone) in F0 males and females. Differences from the control group were slight, not statistically significant, and/or did not occur in an exposure related manner. Furthermore, observed changes were not noted in a manner generally considered physiologically relevant (i.e., concentrations of all 3 hormones were reduced compared to the control group, which is not considered physiologically relevant due to the existence of the thyroid feedback loop, where increasing levels of thyroid hormones [T3/T4] generally signal the hypothalamus to reduce TSH secretion).
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
The testing laboratory for this study uses "F0 and F1" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

There were no test substance-related effects on urinalysis parameters noted for F0 males and females at any exposure level. Differences from the control group were slight and not statistically significant, with the following exception. A statistically significantly lower mean urine pH was noted in the 1500 ppm group (pH 6.0) compared to the control group (pH 6.5); however, the difference was only observed in 1 sex and was of minimal magnitude; therefore, this difference was not considered test substance-related.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The testing laboratory for this study uses "F0 and F1" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

Test substance-related microscopic findings involving the adrenal cortex and trachea (males), kidneys, liver, and nasal cavity (males and females) were noted at the F0 generation necropsy.
Test substance-related findings in the kidneys of males and females at ≥ 380 ppm included minimal to moderate chronic progressive nephropathy (variably consisting of basophilic tubules, peri-tubular fibrosis, interstitial mononuclear cells, hyaline casts), and in males additional findings included minimal to mild focal and multifocal granular casts (consisting of dilated tubules containing granular cellular debris located at the junction of the outer and inner stripes of the outer medulla) associated with minimal to moderate hyaline droplet accumulation (consisting of an increased amount of eosinophilic droplets expanding the renal cortex tubular epithelium).
Test substance-related findings in the respiratory tract were noted in the nasal cavity (all levels) of males and females and in the trachea of males only. In Nasal Level I, changes included minimal edema, minimal to mild squamous epithelium hyperplasia, and in one male minimal respiratory epithelium metaplasia. In Nasal Level II, changes included minimal to mild suppurative exudate, minimal to moderate mucous cell hyperplasia, minimal to mild respiratory epithelium hyperplasia, minimal to mild acute inflammation (consisting of neutrophilic inflammatory cells), minimal to mild chronic active inflammation (consisting of mononuclear cells with an active neutrophilic inflammatory cell component), and minimal to mild squamous cell metaplasia; in males only minimal to mild edema and in females only minimal eosinophilic globules. In Nasal Level III, changes included minimal mucous cell hyperplasia; in 1 male minimal olfactory epithelium degeneration (consisting of very few shrunken olfactory epithelial cells with a vacuolated cytoplasm and dark, condensed nucleus), and in females only minimal to mild eosinophilic globules (consisting of accumulation of eosinophilic droplets in olfactory and/or respiratory epithelium). In Nasal Level IV, changes included minimal mucous cell hyperplasia in males only; and minimal to moderate eosinophilic globules, minimal olfactory epithelium degeneration, and minimal acute inflammation in females only. In Nasal Level V, changes included minimal mucous cell hyperplasia; and in females only, minimal to moderate eosinophilic globules. In Nasal Level VI, changes included minimal to mild mucous cell hyperplasia; and in females only, minimal to mild eosinophilic globules. In the trachea of male rats at 380 and 1500 ppm, minimal to mild respiratory epithelium hyperplasia was noted.
Test substance-related minimal to mild hepatocellular hypertrophy was noted in the liver of males and females at ≥ 380 ppm, and was characterized by enlarged hepatocytes, which in females was noticeably centrilobular.
Test substance-related findings were noted in the adrenal gland of males at ≥ 380 ppm and consisted of minimal to moderate cortical vacuolation. When compared to concurrent controls, the incidence and severity of this finding was greater in treated males, and the highest average severity was noted at 750 ppm. Microscopically, this finding was a diffuse change and consisted of distinct cytoplasmic vacuole(s) expanding the epithelial cells of the zona fasciculata. A similar finding was not observed in female rats.
Mineralization of the kidney tubules that was noted at the cortico-medullary junction in the majority of control and test substance-treated females examined was not considered test substance-related.
There were no other test substance-related histologic changes. Remaining histologic changes (to include a malignant thymoma and an adipose tissue leiomyoma in 1 female [No. 5684] at 750 ppm, and mononuclear cell infiltrates in various organs) were considered to be incidental findings, of the nature commonly observed in this strain and age of Sprague Dawley rat, or related to some aspect of experimental manipulation other than exposure to the test substance.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There were no differences compared to the control
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
The testing laboratory for this study uses "F0 and F1" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

No test substance related effects were observed on F0 spermatogenesis endpoints (mean testicular and epididymal sperm numbers and sperm production rate, motility, progressive motility, and morphology) in males at any exposure level. Differences from the control group were slight and were not statistically significant.
Reproductive performance:
no effects observed
Description (incidence and severity):
The testing laboratory for this study uses "F0 and F1" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report.

No test substance related effects on F0 reproductive performance were observed at any exposure level. No statistically significant differences were noted between the control and test substance exposed groups. One mating pair each in the control, 380, and 1500 ppm groups failed to produce a litter.
The mean numbers of days between pairing and coitus in the test substance exposed groups were similar to the control group value. The mean lengths of estrous cycles in these groups were also similar to the control group value. None of these differences were statistically significant.
Data tables are provided in the "attached background materials" section, as fidelity reasons prevent the copying of .PDF tables to IUCLID.
Key result
Dose descriptor:
NOAEC
Effect level:
1 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEC
Effect level:
1 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
The testing laboratory for this study uses "F0 and F1" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report. As the guidance is not specific, the P1 (second parental generation) is assumed to be F1 Cohort 1B, and the F1 section is assumed to be F1 Cohort 1A. Note that the laboratory does not separate cohorts 1A from 1B for reporting purposes until cohort specific data is obtained. So, there is a generic F1 reported until ~ PND 90

Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
The testing laboratory for this study uses "F0 and F1" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report. As the guidance is not specific, the P1 (second parental generation) is assumed to be F1 Cohort 1B, and the F1 section is assumed to be F1 Cohort 1A. Note that the laboratory does not separate cohorts 1A from 1B for reporting purposes until cohort specific data is obtained. So, there is a generic F1 reported until ~ PND 90

No test substance-related effects on survival were noted for F1 males and females at any exposure level.

In the 1500 ppm group, Cohort 1B Female No. 5624-11 was euthanized in extremis on Lactation Day 13 due to an accidental left forelimb injury that occurred following removal of the animal from the inhalation chamber. The moribund condition of this female was not considered test substance-related. In the 750 ppm group, Cohort 1A Male No. 5699-05 was found dead on PND 58. A clinical observation of red material around the nose was noted sporadically for this male during the exposure period, primarily during PND 43–57. There were no gross findings at necropsy. Microscopic observations included minimal hyaline droplet accumulation in the kidney, and the presence of bacterial colonies and minimal to mild mucoid exudate in the lumen of several levels of the nasal cavity. The degree of autolysis in the cecum, duodenum, ileum, and jejunum precluded microscopic evaluation. Based upon review of the clinical observations, the lack of gross findings, and the microscopic changes, the cause of death for this male could not be determined, and thus was considered incidental. All other F1 animals in Cohort 1A and 1B survived to the scheduled necropsies.

A test substance-related increased number of F1 females with red material around the mouth was noted in the 1500 ppm group at the end of the exposure period and 1–2 hours postexposure. This observation was noted as early as PND 44, and continued sporadically through PND 71 for Cohort 1A females or through Lactation Day 23 for Cohort 1B females. This finding was also noted for the F0 generation.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The testing laboratory for this study uses "F0 and F1" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report. As the guidance is not specific, the P1 (second parental generation) is assumed to be F1 Cohort 1B, and the F1 section is assumed to be F1 Cohort 1A. Note that the laboratory does not separate cohorts 1A from 1B for reporting purposes until cohort specific data is obtained. So, there is a generic F1 reported until ~ PND 90

Weekly:
Mean absolute body weight in the 1500 ppm group F1 males were comparable to the control group on PND 28. Lower mean body weight gains were noted in the 1500 ppm group F1 males during PND 28–42 (statistically significant during PND 28¬–35) compared to the control group, which resulted in mean absolute body weights in this group that were 6.9% and 6.3% lower (not statistically significant) during PND 35 and 42, respectively, and considered related to test substance exposure. With the exception of sporadically statistically significantly higher and lower mean body weight gains, mean body weight gains in the 1500 ppm group F1 males were comparable to the control group during the remainder of the exposure period (PND 35–168), as well as when the overall exposure periods (PND 28–90 and 28–168) were evaluated. As a result, mean absolute body weights in this group were comparable to the control group during
PND 49–168.
No test substance related effects on mean body weights and body weight gains were noted for F1 males in the 380 and 750 ppm groups. Mean body weight gains in the 380 ppm group were generally higher than the control group during the exposure period, and when the overall exposure periods (PND 28–90 and 28–168) were evaluated; the difference was statistically significant during PND 28–90. As a result, mean absolute body weights in this group were generally higher than the control group during PND 70-168; the differences were statistically significant during PND 77–84. However, mean absolute body weights and body weight gains in the 750 ppm group were generally comparable to the control group throughout the exposure period, and thus the changes at 380 ppm occurred in a manner that was not exposure responsive.

In the F1 females, mean absolute body weight in the 1500 ppm group was statistically significantly lower (7.8%) than the control group on PND 28, which was consistent with the test substance related effects on body weight noted in this group during the pre-weaning period (see Section 8.3.1.4.). A lower mean body weight gain was noted in the 1500 ppm group F1 females during PND 28–35 compared to the control group, which resulted in absolute mean body weights that continued to be lower (5.3% to 10.0%) during PND 35–49; the differences were generally statistically significant and considered related to test substance exposure. Mean absolute body weights and body weight gains in the 1500 ppm group F1 females were comparable to the control group during the remainder of the exposure period (PND 35–90 [Cohort 1A] or PND 35–105 [Cohort 1B]).
In the 380 and 750 ppm groups, mean body weight gains for F1 females were unaffected by the test substance throughout the exposure period; differences from the control group were slight and not statistically significant. However, mean body weights in these groups were lower (4.2% to 7.8%; statistically significant on PND 28 at 380 ppm only) than the control group during PND 28-35, a result of test substance-related reduced mean body weights and body weight gains noted in this group during the pre-weaning period.


Gestation (Cohort 1B):
Mean F1 maternal body weights and body weight gains were unaffected by test substance exposure during gestation. Differences between the control, 380, 750, and 1500 ppm groups were slight and not statistically significant.

Lactation (Cohort 1B):
Mean F1 maternal body weights and body weight gains were unaffected by test substance exposure during lactation. Differences between the control, 380, 750, and 1500 ppm groups were slight and not statistically significant.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The testing laboratory for this study uses "F0 and F1" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report. As the guidance is not specific, the P1 (second parental generation) is assumed to be F1 Cohort 1B, and the F1 section is assumed to be F1 Cohort 1A. Note that the laboratory does not separate cohorts 1A from 1B for reporting purposes until cohort specific data is obtained. So, there is a generic F1 reported until ~ PND 90

Mean food consumption in the 1500 ppm group F1 females was comparable to the control group; differences were slight and not statistically significant. Mean food efficiency in this group was unaffected by test substance exposure.
No test substance related effects on mean food consumption and food efficiency were noted for F1 females in the 380 and 750 ppm groups. Any statistically significant differences from the control group were transient, did not occur in an exposure-related manner, and/or did not correspond to changes in body weight.
Food efficiency:
no effects observed
Description (incidence and severity):
The testing laboratory for this study uses "F0 and F1" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report. As the guidance is not specific, the P1 (second parental generation) is assumed to be F1 Cohort 1B, and the F1 section is assumed to be F1 Cohort 1A. Note that the laboratory does not separate cohorts 1A from 1B for reporting purposes until cohort specific data is obtained. So, there is a generic F1 reported until ~ PND 90

Weekly
Slightly lower mean food consumption was noted in the 1500 ppm group F1 males during PND 28–42 compared to the control group. The difference during PND 28–35 was statistically significant and the changes corresponded to the lower mean body weight gains noted in this group following weaning and were considered related to test substance exposure. With the exception of statistically significantly higher mean food consumption noted in the 1500 ppm group F1 males during PND 63–70 and 77–84, mean food consumption in this group was comparable to the control group throughout the remainder of the exposure period (PND 42–90). Mean food efficiency in the 1500 ppm group F1 males was unaffected by test substance exposure.
No test substance related effects on mean food consumption and food efficiency were noted for F1 males in the 380 and 750 ppm groups. Higher mean food consumption was noted in the 380 ppm group compared to the control group generally throughout the exposure period (PND 28–90). The differences were occasionally statistically significant, and corresponded to the higher mean body weight gains noted in this group. However, mean food consumption in the 750 ppm group was comparable to the control group throughout the exposure period, and thus the changes at 380 ppm occurred in a manner that was not exposure responsive.
In the F1 females, slightly lower mean food consumption was noted in the 1500 ppm group during PND 28–35 compared to the control group. The difference during was statistically significant and the change corresponded to the lower mean body weight gain noted in this group following weaning and was considered related to test substance exposure. Lower mean food consumption continued to be noted in this group compared to the control group during
PND 35–56; the differences were generally statistically significant (due to individual variance in the data, the value at 1500 ppm during PND 35–42 [15 g/animal/day] was statistically significantly different from the control group value [15 g/animal/day]). Thereafter, mean food consumption in the 1500 ppm group F1 females was comparable to the control group; differences were slight and not statistically significant. Mean food efficiency in this group was unaffected by test substance exposure.
No test substance related effects on mean food consumption and food efficiency were noted for F1 females in the 380 and 750 ppm groups. Any statistically significant differences from the control group were transient, did not occur in an exposure-related manner, and/or did not correspond to changes in body weight.

Gestation (Cohort 1B):
Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, and food efficiency were unaffected by test substance exposure during gestation. Differences between the control, 380, 750, and 1500 ppm groups were slight and not statistically significant, with the following exception: statistically significantly higher mean food efficiency was noted in the 1500 ppm group compared to the control group during Gestation Days 11–14. The difference was transient and a corresponding effect on mean food consumption was not noted at this exposure level; therefore, the difference was not considered test substance-related.

Lactation (Cohort 1B):
Mean F1 maternal food consumption and food efficiency were unaffected by test substance exposure during lactation. Differences between the control, 380, 750, and 1500 ppm groups were slight and not statistically significant, with the following exceptions: statistically significantly lower mean food consumption was noted in the 380 ppm group compared to the control group during Lactation Days 7–10, and consequently when the overall lactation exposure period (Lactation Days 1–28) was evaluated. In the absence of a comparable effect at the higher exposure levels, these differences were not considered test substance-related.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Details on results:
Data tables are provided in the "attached background materials" section, as fidelity reasons prevent the copying of .PDF tables to IUCLID.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The testing laboratory for this study uses "F0 and F1" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report. As the guidance is not specific, the P1 (second parental generation) is assumed to be F1 Cohort 1B, and the F1 section is assumed to be F1 Cohort 1A. Note that the laboratory does not separate cohorts 1A from 1B for reporting purposes until cohort specific data is obtained. So, there is a generic F1 reported until ~ PND 90

There were no test substance-related effects on the number of females cycling or the number of females cycling regularly at any exposure level. Mean estrous cycle lengths in the test substance-exposed groups were similar to the control group
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
The testing laboratory for this study uses "F0 and F1" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report. As the guidance is not specific, the P1 (second parental generation) is assumed to be F1 Cohort 1B, and the F1 section is assumed to be F1 Cohort 1A. Note that the laboratory does not separate cohorts 1A from 1B for reporting purposes until cohort specific data is obtained. So, there is a generic F1 reported until ~ PND 90


No test substance related effects were observed on F1 spermatogenesis endpoints (mean testicular and epididymal sperm numbers and sperm production rate, motility, progressive motility, and morphology) in males at any exposure level. Differences from the control group were slight and were not statistically significant.
Reproductive performance:
no effects observed
Description (incidence and severity):
The testing laboratory for this study uses "F0 and F1" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report. As the guidance is not specific, the P1 (second parental generation) is assumed to be F1 Cohort 1B, and the F1 section is assumed to be F1 Cohort 1A. Note that the laboratory does not separate cohorts 1A from 1B for reporting purposes until cohort specific data is obtained. So, there is a generic F1 reported until ~ PND 90

No test substance related effects on F1 reproductive performance were observed at any exposure level. No statistically significant differences were noted between the control and test substance exposed groups. Males that did not sire a litter numbered 1, 1, 4, and 2 in the control, 380, 750, and 1500 ppm groups, respectively. Females that had evidence of mating but did not deliver numbered 1, 0, 4, and 2 in the same respective groups.
The mean numbers of days between pairing and coitus in the test substance-exposed groups were similar to the control group value. The mean lengths of estrous cycles in these groups were also similar to the control group value. None of these differences were statistically significant.
The testing laboratory for this study uses "F0 and F1" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report. As the guidance is not specific, the P1 (second parental generation) is assumed to be F1 Cohort 1B, and the F1 section is assumed to be F1 Cohort 1A. Note that the laboratory does not separate cohorts 1A from 1B for reporting purposes until cohort specific data is obtained. So, there is a generic F1 reported until ~ PND 90
Key result
Dose descriptor:
NOAEC
Effect level:
1 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Dose descriptor:
NOAEC
Effect level:
1 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
The testing laboratory for this study uses "F0 and F1" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report. As the guidance is not specific, the P1 (second parental generation) is assumed to be F1 Cohort 1B, and the F1 section is assumed to be F1 Cohort 1A. Note that the laboratory does not separate cohorts 1A from 1B for reporting purposes until cohort specific data is obtained. So, there is a generic F1 reported until ~ PND 90

Prior to weaning
The general physical condition (defined as the occurrence and severity of clinical findings) of all F1 pups in this study was unaffected by test substance exposure. Seven (4), 26(6), 5(5), and 29(10) pups (litters) in the control, 380, 750, and 1500 ppm groups, respectively, were found dead or euthanized in extremis. The slightly increased number of unscheduled deaths in the 1500 ppm group did not occur in a exposure-responsive manner, and was primarily due to 2 dams (Nos. 5645 and 5674) in this group, with 11 and 7 pups, respectively, found dead on PND 0 or 4. A slightly increased number of unscheduled deaths was noted in the 380 ppm group compared to the control group; this difference did not occur in an exposure-related manner, and therefore was not considered test substance-related. Four (3), 7(6), 14(11), and 7(6) pups (litters) in the same respective groups were missing and presumed to have been cannibalized. In addition, 8 fetuses from 1 litter in the control group were euthanized due to death of the dam on Lactation Day/PND 10.

After Weaning
No test substance-related effects on survival were noted for F1 males and females at any exposure level.
In the 1500 ppm group, Cohort 1B Female No. 5624-11 was euthanized in extremis on Lactation Day 13 due to an accidental left forelimb injury that occurred following removal of the animal from the inhalation chamber. The moribund condition of this female was not considered test substance-related. In the 750 ppm group, Cohort 1A Male No. 5699-05 was found dead on PND 58. A clinical observation of red material around the nose was noted sporadically for this male during the exposure period, primarily during PND 43–57. There were no gross findings at necropsy. Microscopic observations included minimal hyaline droplet accumulation in the kidney, and the presence of bacterial colonies and minimal to mild mucoid exudate in the lumen of several levels of the nasal cavity. The degree of autolysis in the cecum, duodenum, ileum, and jejunum precluded microscopic evaluation. Based upon review of the clinical observations, the lack of gross findings, and the microscopic changes, the cause of death for this male could not be determined, and thus was considered incidental. All other F1 animals in Cohort 1A and 1B survived to the scheduled necropsies.
A test substance-related increased number of F1 females with red material around the mouth was noted in the 1500 ppm group at the end of the exposure period and 1–2 hours postexposure. This observation was noted as early as PND 44, and continued sporadically through PND 71 for Cohort 1A females or through Lactation Day 23 for Cohort 1B females. This finding was also noted for the F0 generation. There were no other test substance-related observations noted at the daily/detailed physical examinations or postexposure observations. Red material around the nose was noted for F1 males and females across all exposure groups, albeit in a non-exposure responsive manner, and was therefore considered incidental, similar to the F0 generation. Other findings noted in the test substance-exposed groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not exposure-related.


Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The testing laboratory for this study uses "F0 and F1" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report. As the guidance is not specific, the P1 (second parental generation) is assumed to be F1 Cohort 1B, and the F1 section is assumed to be F1 Cohort 1A. Note that the laboratory does not separate cohorts 1A from 1B for reporting purposes until cohort specific data is obtained. So, there is a generic F1 reported until ~ PND 90

No test substance-related effects on survival were noted for F1 males and females at any exposure level.

In the 1500 ppm group, Cohort 1B Female No. 5624-11 was euthanized in extremis on Lactation Day 13 due to an accidental left forelimb injury that occurred following removal of the animal from the inhalation chamber. The moribund condition of this female was not considered test substance-related. In the 750 ppm group, Cohort 1A Male No. 5699-05 was found dead on PND 58. A clinical observation of red material around the nose was noted sporadically for this male during the exposure period, primarily during PND 43–57. There were no gross findings at necropsy. Microscopic observations included minimal hyaline droplet accumulation in the kidney, and the presence of bacterial colonies and minimal to mild mucoid exudate in the lumen of several levels of the nasal cavity. The degree of autolysis in the cecum, duodenum, ileum, and jejunum precluded microscopic evaluation. Based upon review of the clinical observations, the lack of gross findings, and the microscopic changes, the cause of death for this male could not be determined, and thus was considered incidental. All other F1 animals in Cohort 1A and 1B survived to the scheduled necropsies.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The testing laboratory for this study uses "F0 and F1" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report. As the guidance is not specific, the P1 (second parental generation) is assumed to be F1 Cohort 1B, and the F1 section is assumed to be F1 Cohort 1A. Note that the laboratory does not separate cohorts 1A from 1B for reporting purposes until cohort specific data is obtained. So, there is a generic F1 reported until ~ PND 90

Mean absolute body weight in the 1500 ppm group F1 males were comparable to the control group on PND 28. Lower mean body weight gains were noted in the 1500 ppm group F1 males during PND 28–42 (statistically significant during PND 28¬–35) compared to the control group, which resulted in mean absolute body weights in this group that were 6.9% and 6.3% lower (not statistically significant) during PND 35 and 42, respectively, and considered related to test substance exposure. With the exception of sporadically statistically significantly higher and lower mean body weight gains, mean body weight gains in the 1500 ppm group F1 males were comparable to the control group during the remainder of the exposure period (PND 35–168), as well as when the overall exposure periods (PND 28–90 and 28–168) were evaluated. As a result, mean absolute body weights in this group were comparable to the control group during
PND 49–168.
No test substance related effects on mean body weights and body weight gains were noted for
F1 males in the 380 and 750 ppm groups. Mean body weight gains in the 380 ppm group were generally higher than the control group during the exposure period, and when the overall exposure periods (PND 28–90 and 28–168) were evaluated; the difference was statistically significant during PND 28–90. As a result, mean absolute body weights in this group were generally higher than the control group during PND 70-168; the differences were statistically significant during PND 77–84. However, mean absolute body weights and body weight gains in the 750 ppm group were generally comparable to the control group throughout the exposure period, and thus the changes at 380 ppm occurred in a manner that was not exposure responsive.
In the F1 females, mean absolute body weight in the 1500 ppm group was statistically significantly lower (7.8%) than the control group on PND 28, which was consistent with the test substance related effects on body weight noted in this group during the pre-weaning period (see Section 8.3.1.4.). A lower mean body weight gain was noted in the 1500 ppm group F1 females during PND 28–35 compared to the control group, which resulted in absolute mean body weights that continued to be lower (5.3% to 10.0%) during PND 35–49; the differences were generally statistically significant and considered related to test substance exposure. Mean absolute body weights and body weight gains in the 1500 ppm group F1 females were comparable to the control group during the remainder of the exposure period (PND 35–90 [Cohort 1A] or PND 35–105 [Cohort 1B]).
In the 380 and 750 ppm groups, mean body weight gains for F1 females were unaffected by the test substance throughout the exposure period; differences from the control group were slight and not statistically significant. However, mean body weights in these groups were lower (4.2% to 7.8%; statistically significant on PND 28 at 380 ppm only) than the control group during PND 28-35, a result of test substance-related reduced mean body weights and body weight gains noted in this group during the pre-weaning period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The testing laboratory for this study uses "F0 and F1" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report. As the guidance is not specific, the P1 (second parental generation) is assumed to be F1 Cohort 1B, and the F1 section is assumed to be F1 Cohort 1A. Note that the laboratory does not separate cohorts 1A from 1B for reporting purposes until cohort specific data is obtained. So, there is a generic F1 reported until ~ PND 90

Slightly lower mean food consumption was noted in the 1500 ppm group F1 males during PND 28–42 compared to the control group. The difference during PND 28–35 was statistically significant and the changes corresponded to the lower mean body weight gains noted in this group following weaning and were considered related to test substance exposure. With the exception of statistically significantly higher mean food consumption noted in the 1500 ppm group F1 males during PND 63–70 and 77–84, mean food consumption in this group was comparable to the control group throughout the remainder of the exposure period (PND 42–90). Mean food efficiency in the 1500 ppm group F1 males was unaffected by test substance exposure.
No test substance related effects on mean food consumption and food efficiency were noted for F1 males in the 380 and 750 ppm groups. Higher mean food consumption was noted in the 380 ppm group compared to the control group generally throughout the exposure period (PND 28–90). The differences were occasionally statistically significant, and corresponded to the higher mean body weight gains noted in this group. However, mean food consumption in the 750 ppm group was comparable to the control group throughout the exposure period, and thus the changes at 380 ppm occurred in a manner that was not exposure responsive.
In the F1 females, slightly lower mean food consumption was noted in the 1500 ppm group during PND 28–35 compared to the control group. The difference during was statistically significant and the change corresponded to the lower mean body weight gain noted in this group following weaning and was considered related to test substance exposure. Lower mean food consumption continued to be noted in this group compared to the control group during
PND 35–56; the differences were generally statistically significant (due to individual variance in the data, the value at 1500 ppm during PND 35–42 [15 g/animal/day] was statistically significantly different from the control group value [15 g/animal/day]). Thereafter, mean food consumption in the 1500 ppm group F1 females was comparable to the control group; differences were slight and not statistically significant. Mean food efficiency in this group was unaffected by test substance exposure.
No test substance related effects on mean food consumption and food efficiency were noted for F1 females in the 380 and 750 ppm groups. Any statistically significant differences from the control group were transient, did not occur in an exposure-related manner, and/or did not correspond to changes in body weight.
Food efficiency:
effects observed, non-treatment-related
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The testing laboratory for this study uses "F0 and F1" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report. As the guidance is not specific, the P1 (second parental generation) is assumed to be F1 Cohort 1B, and the F1 section is assumed to be F1 Cohort 1A. Note that the laboratory does not separate cohorts 1A from 1B for reporting purposes until cohort specific data is obtained. So, there is a generic F1 reported until ~ PND 90

There were no test substance-related effects on hematology parameters. Statistically significant differences from the control group did not occur in an exposure-related manner, were only observed in a single sex, and/or were of minimal magnitude; therefore, these differences were not considered test substance-related.

Coagulation (Cohort 1A):
There were no test substance-related effects on coagulation parameters. Statistically significant differences from the control group did not occur in an exposure-related manner, were only observed in a single sex, and/or were of minimal magnitude; therefore, these differences were not considered test substance-related.

Serum Chemistry (Cohort 1A):
No test substance-related effects on serum chemistry parameters were noted at any exposure level. Higher (28.0% to 61.2%) mean cholesterol levels were noted for F1 males and females in the 380, 750, and 1500 ppm groups compared to the control group; the differences were statistically significant. Statistically significantly higher mean total protein and globulin levels were noted in the 1500 ppm group F1 males compared to the control group. However, mean albumin was comparable to the control group, mean triglyceride was lower than the control group instead of being correspondingly higher, and comparable changes in mean total protein and globulin levels were not noted in the F1 females. Mean urea nitrogen was also statically significantly elevated in the 1500 ppm group F1 males when compared to concurrent control group; however, there was no corresponding change in the mean creatinine level and a similar change was not noted in the F1 females. Furthermore, other corresponding parameters, including levels of various electrolytes (sodium, chloride, phosphorous, potassium, etc.) were either unaffected or changed in a direction opposite to normal physiological trends. Therefore, the aforementioned differences in the 1500 ppm group F1 males were not considered test substance related. Other statistically significant differences from the control group did not occur in an exposure related manner, lacked histologic correlates, were only observed in 1 sex, were within the Charles River Ashland historical control reference ranges, and/or were of minimal magnitude; therefore, these differences were not considered test substance-related.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
The testing laboratory for this study uses "F0 and F1" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report. As the guidance is not specific, the P1 (second parental generation) is assumed to be F1 Cohort 1B, and the F1 section is assumed to be F1 Cohort 1A. Note that the laboratory does not separate cohorts 1A from 1B for reporting purposes until cohort specific data is obtained. So, there is a generic F1 reported until ~ PND 90

Thyroid Hormone Analysis
PND 4 Culled Pups:
There were no test substance-related effects on serum levels of T3 (triiodothyronine), T4 (thyroxine), or TSH (thyroid stimulating hormone) in pups that were culled on PND 4. Differences from the control group were slight, not statistically significant, and/or did not occur in an exposure related manner. Furthermore, observed changes were not noted in a manner generally considered physiologically relevant (i.e., concentrations of all 3 hormones were reduced compared to the control group, which is not considered physiologically relevant due to the existence of the thyroid feedback loop, where increasing levels of thyroid hormones [T3/T4] generally signal the hypothalamus to reduce TSH secretion).

PND 28 Pups Selected for Hormone Analysis:
There were no test substance-related effects on serum levels of T3 (triiodothyronine), T4 (thyroxine), or TSH (thyroid stimulating hormone) in nonselected pups on PND 28. Differences from the control group were slight, not statistically significant, and/or did not occur in an exposure related manner.

No test substance-related effects on serum chemistry parameters were noted at any exposure level. Higher (28.0% to 61.2%) mean cholesterol levels were noted for F1 males and females in the 380, 750, and 1500 ppm groups compared to the control group; the differences were statistically significant. Statistically significantly higher mean total protein and globulin levels were noted in the 1500 ppm group F1 males compared to the control group. However, mean albumin was comparable to the control group, mean triglyceride was lower than the control group instead of being correspondingly higher, and comparable changes in mean total protein and globulin levels were not noted in the F1 females. Mean urea nitrogen was also statically significantly elevated in the 1500 ppm group F1 males when compared to concurrent control group; however, there was no corresponding change in the mean creatinine level and a similar change was not noted in the F1 females. Furthermore, other corresponding parameters, including levels of various electrolytes (sodium, chloride, phosphorous, potassium, etc.) were either unaffected or changed in a direction opposite to normal physiological trends. Therefore, the aforementioned differences in the 1500 ppm group F1 males were not considered test substance related. Other statistically significant differences from the control group did not occur in an exposure related manner, lacked histologic correlates, were only observed in 1 sex, were within the Charles River Ashland historical control reference ranges, and/or were of minimal magnitude; therefore, these differences were not considered test substance-related.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
The testing laboratory for this study uses "F0 and F1" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report. As the guidance is not specific, the P1 (second parental generation) is assumed to be F1 Cohort 1B, and the F1 section is assumed to be F1 Cohort 1A. Note that the laboratory does not separate cohorts 1A from 1B for reporting purposes until cohort specific data is obtained. So, there is a generic F1 reported until ~ PND 90

There were no test substance-related effects on urinalysis parameters. Differences from the control group were slight, did not occur in an exposure-related manner, and/or were not statistically significant.
Sexual maturation:
no effects observed
Description (incidence and severity):
The testing laboratory for this study uses "F0 and F1" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report. As the guidance is not specific, the P1 (second parental generation) is assumed to be F1 Cohort 1B, and the F1 section is assumed to be F1 Cohort 1A. Note that the laboratory does not separate cohorts 1A from 1B for reporting purposes until cohort specific data is obtained. So, there is a generic F1 reported until ~ PND 90

Mean ages of attainment of balanopreputial separation and mean body weights at the age of attainment were unaffected by test substance exposure. The mean ages of attainment of balanopreputial separation were 44.3, 44.2, and 45.2 days in the 380, 750, and 1500 ppm groups, respectively, when compared to 44.4 days in the control group. Mean body weights at the age of attainment were 210.4 g, 205.1 g, and 205.4 g in the same respective groups compared to 210.3 g in the control group. None of the differences from the control group were statistically significant.

Mean ages of attainment of vaginal patency and mean body weights at the age of attainment were unaffected by test substance exposure. The mean ages of attainment of vaginal patency were 35.4, 35.1, and 35.7 days in the 380, 750, and 1500 ppm groups, respectively, when compared to 34.2 days in the control group. Mean body weights at the age of attainment were 114.4 g, 114.2 g, and 113.1 g in the same respective groups compared to 113.8 g in the control group. None of the differences from the control group were statistically significant.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
The testing laboratory for this study uses "F0 and F1" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report. As the guidance is not specific, the P1 (second parental generation) is assumed to be F1 Cohort 1B, and the F1 section is assumed to be F1 Cohort 1A. Note that the laboratory does not separate cohorts 1A from 1B for reporting purposes until cohort specific data is obtained. So, there is a generic F1 reported until ~ PND 90

No test substance-related effects on anogenital distances (absolute and relative to the cube root of pup body weight) were noted for F1 pups in the 380, 750, and 1500 ppm groups. Differences from the control group were slight and not statistically significant.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
The testing laboratory for this study uses "F0 and F1" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report. As the guidance is not specific, the P1 (second parental generation) is assumed to be F1 Cohort 1B, and the F1 section is assumed to be F1 Cohort 1A. Note that the laboratory does not separate cohorts 1A from 1B for reporting purposes until cohort specific data is obtained. So, there is a generic F1 reported until ~ PND 90

Areolae/nipple anlagen in the F1 male pups was not affected by test substance exposure. There was no retention of nipples noted in any male pup on study on PND 13.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The testing laboratory for this study uses "F0 and F1" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report. As the guidance is not specific, the P1 (second parental generation) is assumed to be F1 Cohort 1B, and the F1 section is assumed to be F1 Cohort 1A. Note that the laboratory does not separate cohorts 1A from 1B for reporting purposes until cohort specific data is obtained. So, there is a generic F1 reported until ~ PND 90

Test substance-related increases in kidney weights were noted in males and females at ≥ 380 ppm, which were statistically significant. Microscopic correlates for these changes in males were hyaline droplets, chronic progressive nephropathy and granular casts; there were no definitive microscopic correlate(s) in females. These kidney weight changes (increase) were similar to the F0 generation animals.
Test substance-related increases in liver weights were noted in males and females at ≥ 380 ppm, reaching statistical significance in males at ≥ 380 ppm and in females at 1500 ppm. Microscopic correlate for this change was hepatocellular hypertrophy in males and females. These liver weight changes (increase) were similar to the F0 generation animals.
Test substance-related increases in thyroids/parathyroids weights were noted in females at 1500 ppm, which were statistically significant. There were no microscopic correlate(s) for this change in the examined thyroid and parathyroid glands, and there was a lack of changes in serum thyroid hormones in the females.
Test substance-related increases in SV/CG/ACC weights were noted in males at ≥ 380 ppm, which was statistically significant at 1500 ppm. There were no microscopic correlate(s) for this change in the examined seminal vesicles and coagulating glands.
There were no other test substance-related effects on organ weights for F1 animals in Cohort 1A. However, some statistically significant differences were observed when the control and test substance-exposed groups were compared. There were, however, no patterns, trends, or correlating data to suggest these values were toxicologically relevant. Thus, other organ weight differences observed were considered incidental and/or related to difference of sexual maturity and unrelated to test substance exposure.
No test substance-related effects on organ weights were noted for F1 males and females in Cohort 1B. However, some statistically significant differences were observed when the control and test substance-exposed groups were compared (decreased left and right testes weight relative to body weight in males at 380 ppm and increased absolute ovary weight and ovary weight relative to body weight in females at 1500 ppm). There were, however, no patterns, trends, or correlating data to suggest these values were toxicologically relevant. Thus, other organ weight differences observed were considered incidental and/or related to difference of sexual maturity and unrelated to test substance exposure.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The testing laboratory for this study uses "F0 and F1" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report. As the guidance is not specific, the P1 (second parental generation) is assumed to be F1 Cohort 1B, and the F1 section is assumed to be F1 Cohort 1A. Note that the laboratory does not separate cohorts 1A from 1B for reporting purposes until cohort specific data is obtained. So, there is a generic F1 reported until ~ PND 90

Unscheduled Deaths:

Seven (4), 26(6), 5(5), and 29(10) pups (litters) in the control, 380, 750, and 1500 ppm groups, respectively, were found dead or euthanized in extremis from PND 0 through the selection of the F1 generation. No internal findings that could be attributed to parental test substance exposure were noted at the necropsies of pups that were found dead or euthanized in extremis. Aside from the absence of milk in the stomach, internal findings were noted for pups in the control, 380, and 1500 ppm groups. In the 1500 ppm group, Pup No. 5618-05 had dark red contents under the skin in the ocular region, Pup Nos. 5645-02 and 5645-06 were noted with the variation of undeveloped renal papilla(e), and Pup No. 5645-10 was noted with renal papilla(e) not fully developed (Woo and Hoar Grade 1) and the developmental variation distended left ureter. In the 380 ppm group, Pup No. 5626-08 had a dark red discolored brain and dark red contents in the cranial cavity (surrounding the brain) and Pup No. 5709-14 had a major blood vessel variation. The aforementioned findings were noted in single animals, occurred infrequently or similarly in the control group, and/or did not occur in an exposure-related manner, and were therefore not considered test substance-related. In the control group, Pup Nos. 5668-01, 5668-02, 5668-03, and 5668-04 had the malformations ectrodactyly and short tail; Pup No. 5668-02 was also noted with the developmental variation undeveloped renal papilla(e) and distended ureters. No other internal findings were noted.

PND 4 Culled Pups:
No internal findings that could be attributed to parental exposure to the test substance were noted at the necropsy of culled pups euthanized on PND 4. Internal findings were noted for pups in the control, 750, and 1500 ppm groups. In the 1500 ppm group, Pup No. 5620-10 had the malformation retroesophageal aortic arch. In the 750 ppm group, Pup No. 5610-07 had the developmental variations white areas in the mesentery and a white discolored mesenteric lymph node, Pup No. 5687-02 had the developmental variation undeveloped renal papilla(e) and distended ureters, Pup No. 5696-03 had the developmental variation hemorrhagic ring around the left iris, and Pup Nos. 5699-10, 5699-11, and 5699-12 had the developmental variation undeveloped renal papilla(e) and/or distended ureter(s); Pup Nos. 5699-10 and 5699-12 also had renal papilla(e) not fully developed (Woo and Hoar Grade 1). The aforementioned findings were noted in single animals, occurred infrequently or similarly in the control group, and/or did not occur in an exposure-related manner, and were therefore not considered test substance-related. In the control group, Pup No. 5633 had the developmental variations white areas in the mesentery and a white discolored mesenteric lymph node(s), Pup No. 5635-06 had the developmental variation hemorrhagic ring around the right iris, and Pup No. 5681-13 had the developmental variation hemorrhagic ring around the left iris. No other internal findings were noted.

PND 28 Nonselected Pups and Pups Euthanized Due to Death of Dam:
No internal findings that could be attributed to parental exposure to the test substance were noted at the necropsy of pups euthanized on PND 28 and pups euthanized due to death of the dam. Internal findings in the test substance-exposed groups were limited to a single pup (No. 5628-01) in the 1500 ppm group with a dilated renal pelvis (right). This finding was noted in single animal, and was therefore not considered test substance-related. In the control group, Pup No. 5691-15 had a diaphragmatic hernia. No other internal findings were noted.

PND 28 Pups Selected for Hormone Analysis:
At the PND 28 necropsy of F1 pups selected for hormone analysis, no internal findings that could be attributed to parental exposure to the test substance were noted at any exposure level. Internal findings were limited to a dilated renal pelvis (right) for Male Pup No. 5644-04 in the 750 ppm group. Because this finding was limited to a single fetus and did not occur in an exposure-related manner, it was not considered test substance-related. No other internal findings were noted.

After Weaning

Test substance-related gross findings, with correlating microscopic findings, were observed in the kidneys and/or liver of F1 (Cohort 1A) males and females. In the kidney of male rats, gross findings included depressed area(s) and mottled. The microscopic correlate for these changes in males was chronic progressive nephropathy. In the liver of one Group 4 female, rough surface correlated microscopically to hepatocellular hypertrophy.
There were no other test-substance related changes. Remaining gross findings were considered to be incidental, of the nature commonly observed in this strain and age of Sprague Dawley rat, and/or were of similar incidence in control and treated animals, and therefore were considered unrelated to test substance exposure. There were no test substance-related macroscopic findings noted for the F1 males and females necropsied on PND 35 or for F1 animals in Cohort 1B.
Histopathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The testing laboratory for this study uses "F0 and F1" for their nomenclature rather than the P0 and P1 (respectively) used in this form. The laboratory nomenclature is retained to be consistent with the study report. As the guidance is not specific, the P1 (second parental generation) is assumed to be F1 Cohort 1B, and the F1 section is assumed to be F1 Cohort 1A. Note that the laboratory does not separate cohorts 1A from 1B for reporting purposes until cohort specific data is obtained. So, there is a generic F1 reported until ~ PND 90

Microscopic findings in the F1 (Cohort 1A) animals that were similar to the F0 animals included minimal to moderate adrenal gland cortex vacuolation in males, although there was no change in F1 male adrenal gland organ weights; minimal to moderate chronic progressive nephropathy in kidney of males and females (non-exposure dependent in the females), minimal to moderate, focal to multifocal granular casts and minimal to moderate hyaline droplet accumulation in kidney of males only; minimal to mild liver hepatocellular hypertrophy; and nasal cavity changes to include minimal to mild mucous cell hyperplasia (Levels II–VI), minimal edema and minimal to mild squamous epithelium hyperplasia (Level I); acute inflammation, chronic active inflammation, and minimal squamous cell metaplasia (Level II). Microscopically, these findings were similar as described in the F0 animals. Lastly, in the trachea, minimal mucous cell hyperplasia was noted in 1 male at 1500 ppm.
Similar to the F0 generation, mineralization of the kidney tubules that was noted at the cortico medullary junction in the majority of control and test substance-treated females examined was not considered test substance-related.24
There were no other test substance-related histologic changes. Remaining histologic changes (to include a malignant lymphoma in 1 female [No. 5669-07] at 1500 ppm) were considered to be incidental findings, of the nature commonly observed in this strain and age of Sprague Dawley rat, or related to some aspect of experimental manipulation other than exposure to the test substance.
Other effects:
not specified
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Data tables are provided in the "attached background materials" section, as fidelity reasons prevent the copying of .PDF tables to IUCLID.
Key result
Dose descriptor:
NOAEC
Effect level:
750 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Clinical signs:
no effects observed
Description (incidence and severity):


PND 0 Litter Data and Postnatal Survival:

The mean number of pups born, live litter size, percentage of males per litter at birth, and postnatal survival between birth and PND 0 (relative to number born), PND 0–1, 1–4 (pre selection), 4 (post-selection)–7, 7–14, 14–21, 21–28, and from birth to PND 4 (pre selection), and PND 4 (post-selection) to PND 28 were unaffected by the test substance at all exposure levels. Differences from the control group were slight, were not statistically significant, and/or did not occur in an exposure related manner.

Observations:

The general physical condition (defined as the occurrence and severity of clinical findings) of all F2 pups in this study was unaffected by test substance exposure. Eight (5), 15(5), 8(4), and 9(6) pups (litters) in the control, 380, 750, and 1500 ppm groups, respectively, were found dead. Five (4), 0(0), 7(5), and 11(8) pups (litters) in the same respective groups were missing and presumed to have been cannibalized. In addition, 8 pups from 1 litter in the 1500 ppm group were euthanized due to death of the dam on Lactation Day/PND 13.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not specified
Description (incidence and severity):
The mean number of pups born, live litter size, percentage of males per litter at birth, and postnatal survival between birth and PND 0 (relative to number born), PND 0–1, 1–4 (pre selection), 4 (post-selection)–7, 7–14, 14–21, 21–28, and from birth to PND 4 (pre selection), and PND 4 (post-selection) to PND 28 were unaffected by the test substance at all exposure levels. Differences from the control group were slight, were not statistically significant, and/or did not occur in an exposure related manner.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):


Mean F2 male and female pup birth weights (PND 1) in the 1500 ppm group was comparable to the control group. Mean pup body weight gains during PND 1–4 and 4–7 and mean absolute pup body weights on PND 4 and 7 in this group were comparable to the control group. However, lower mean pup body weight gains were noted for F2 males and females in the 1500 ppm group during PND 7–14 compared to the control group, which resulted in mean male and female pup body weights in this group that were 8.7% to 4.4%, respectively, lower on PND 14; the difference was statistically significant for males only.
Mean pup body weight gains for males and females in the 1500 ppm group were comparable to the control group during the remainder of the postnatal period (PND 14–28); however, mean absolute body weights for F2 males remained 5.1 % to 6.0% lower (not statistically significant) during PND 21–28. Mean absolute body weight weights for F2 females in the 1500 ppm group were comparable to the control group during the remainder of the postnatal period. In comparison, for the F1 generation, mean male and female pup body weights in the 380, 750, and 1500 ppm groups were 8.3% to 9.4% (males) and 10.9% to 12.6% (females) lower than controls on PND 14 and remained 4.2% to 7.1% (males) and 6.0% to 10.1% (females) lower than controls during the remainder of the preweaning period (PND 21–28).
Mean F2 male and female pup body weights and body weight changes in the 380 and 750 ppm groups were unaffected by parental test substance exposure throughout the postnatal period. Any statistically significant differences noted in these groups compared to the control group were considered transient and had no impact on absolute mean body weights.
Food consumption and compound intake (if feeding study):
not examined
Description (incidence and severity):
The pups were euthanized before weaning, so there was no data to measure.
Food efficiency:
not examined
Description (incidence and severity):
The pups were euthanized before weaning, so there was no data to measure.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
The anogenital distances (absolute and relative to the cube root of pup body weight) in the 380, 750, and 1500 ppm groups were similar to the control group values. Differences from the control group were slight and not statistically significant.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Areolae/nipple anlagen in the F2 male pups was not affected by test substance exposure. There was no retention of nipples noted in any male pup on study on PND 13.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):


There were no test substance-related effects on organ weights. However, some statistically significant differences were observed when the control and test substance-exposed groups were compared (increased thymus weights relative to final body weight in females at 380 and 1500 ppm and relative to brain weight in females at 1500 ppm). There were, however, no patterns, trends, or correlating data to suggest these values were toxicologically relevant. Thus, the organ weight differences observed were considered incidental and/or related to difference of sexual maturity and unrelated to exposure to the test substance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):


Unscheduled Deaths:
Eight (5), 15(5), 8(4), and 9(6) pups (litters) in the control, 380, 750, and 1500 ppm groups, respectively, were found dead from PND 0 to 28. No internal findings that could be attributed to parental test substance exposure were noted at the necropsies of pups that were found dead. Aside from the presence or absence of milk in the stomach, internal findings were limited to pups in the 380 and 750 ppm groups. In the 750 ppm group, Pup No. 5610-12-01 had multiple malformations (an intraventricular septal defect, anophthalmia [bilateral], and a short tail) and developmental variations (7th cervical ribs and malaligned sternebrae). In the 380 ppm group, Pup No. 5651-14-03 had dilated renal pelves. The aforementioned findings were noted in single animals and did not occur in an exposure-related manner, and were therefore not considered test substance-related. No other internal findings were noted.

PND 4 Culled Pups:
No internal findings that could be attributed to parental exposure to the test substance were noted at the necropsy of culled pups euthanized on PND 4. Internal findings were noted in the control, 380, 750, and 1500 ppm groups. One, 2, and 1 pups in the 380, 750, and 1500 ppm groups, respectively, had the developmental variation hemorrhagic ring around the iris (right or left); this finding was also noted in 1 control group pup. In addition, Pup No. 5690-12-06 in the 750 ppm group had renal papillae not fully developed (Woo and Hoar Grade 1) and Pup No. 5639-10-15 in the 380 ppm group had a dark red discolored right eye. The aforementioned findings were noted in single animals, occurred infrequently or similarly in the control group, and/or did not occur in an exposure-related manner, and were therefore not considered test substance-related. In the control group, Pup No. 5705-07-17 had the developmental variation accessory liver lobule, Pup No. 5643-12-04 had renal papillae not fully developed (Woo and Hoar Grade 1), and Pup No. 5635-15-10 had the developmental variation undeveloped renal papilla(e) and distended ureter (right). No other internal findings were noted.

PND 28 Nonselected Pups and Pups Euthanized Due to Death of Dam:

No internal findings that could be attributed to parental exposure to the test substance were noted at the necropsy of pups euthanized on PND 28 and pups euthanized due to death of the dam. Internal findings were limited to a single pup each in the in the test substance-exposed groups. Pup No. 5618-10-03 in the 1500 ppm group, had a small left testis and epididymis, Pup No. 5713-15-11 in the 750 ppm group had a cyst in the left renal cortex, and Pup No. 5676-09-10 in the 380 ppm group had a misshapen spleen. These findings was noted in single animals, and was therefore not considered test substance-related. No other internal findings were noted.

PND 28 Pups Selected for Histopathology:
No test substance-related gross necropsy observations were noted. The gross findings observed were considered to be incidental, of the nature commonly observed in this strain and age of Sprague Dawley rat, and/or were of similar incidence in control and treated animals; therefore, they were considered unrelated to test substance exposure.



Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):


No test-substance related histologic changes were noted in the limited tissues examined. The histologic changes observed were considered to be incidental findings, of the nature commonly observed in this strain and age of Sprague Dawley rat, or related to some aspect of experimental manipulation other than exposure to the test substance.
Other effects:
not specified
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Data tables are provided in the "attached background materials" section, as fidelity reasons prevent the copying of .PDF tables to IUCLID.
Key result
Dose descriptor:
NOAEC
Generation:
F2
Effect level:
750 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Reproductive effects observed:
no

Due to fidelity issues in copy/pasting data tables into IUCLID, aside from the text tables below, all summary tables were edited out of the study report, collated, and are presented in the "attached background material" box as a single .PDF document.


Overall Nominal Exposure Concentrations F0Genereation

Exposure Chamber:

2

3

4

Target Concentration (ppm):

380

750

1500

Nominal Concentration (ppm):

361

688

1516

Standard Deviation:

40.1

23.4

76.2

Number of Exposure Days:

137

137

137

Overall Nominal Exposure Concentrations F1Generation

Exposure Chamber:

2

3

4

Target Concentration (ppm):

380

750

1500

Nominal Concentration (ppm):

330

689

1501

Standard Deviation:

23.9

37.5

79.4

Number of Exposure Days:

151

151

151

 


Overall Mean Exposure Concentrations F0Generation

Exposure Chamber:

1

2

3

4

Target Concentration (ppm):

0

380

750

1500

Mean Concentration (ppm):

0

380

750

1499

Standard Deviation:

0.0

16.0

16.5

26.2

Number of Exposure Days:

137

137

137

137


Overall Mean Exposure Concentrations F1Generation

Exposure Chamber:

1

2

3

4

Target Concentration (ppm):

0

380

750

1500

Mean Concentration (ppm):

0

379

750

1497

Standard Deviation:

0.0

14.9

18.4

40.5

Number of Exposure Days:

151

151

151

151

Results of F0Reproductive Performance

Parameter

Exposure Level (ppm)

CRL HCa

Mean (Range)

0

380

750

1500

Male Mating Index (%)

100.0

100.0

100.0

100.0

97.9 (83.3–100.0)

Female Mating Index (%)

100.0

100.0

100.0

100.0

97.9 (83.3–100.0)

Male Fertility Index (%)

95.7

95.8

100.0

95.8

94.0 (80.0–100.0)

Female Fertility Index (%)

95.7

95.8

100.0

95.8

94.0 (80.0–100.0)

Male Copulation Index (%)

95.7

95.8

100.0

95.8

95.9 (80.0–100.0)

Female Conception Index (%)

95.7

95.8

100.0

95.8

95.9 (80.0–100.0)

Estrous Cycle Length (days)

4.0

4.1

4.1

4.3

4.2 (3.9–5.2)

Pre-Coital Interval (days)

3.0

2.9

2.5

2.2

2.7 (1.8–4.5)

 

a Charles River Ashland historical control data (version 2018.02).


Results of F1Reproductive Performance

Parameter

Exposure Level (ppm)

CRL HCa

Mean (Range)

0

380

750

1500

Male Mating Index (%)

100.0

95.2

100.0

100.0

97.9 (83.3–100.0)

Female Mating Index (%)

100.0

95.2

100.0

100.0

97.9 (83.3–100.0)

Male Fertility Index (%)

95.2

95.2

83.3

90.9

94.0 (80.0–100.0)

Female Fertility Index (%)

95.2

95.2

83.3

90.9

94.0 (80.0–100.0)

Male Copulation Index (%)

95.2

100.0

83.3

90.9

95.9 (80.0–100.0)

Female Conception Index (%)

95.2

100.0

83.3

90.9

95.9 (80.0–100.0)

Estrous Cycle Length (days)

4.1

4.3

4.7

4.0

4.2 (3.9–5.2)

Pre-Coital Interval (days)

2.4

3.0

2.8

2.8

2.7 (1.8–4.5)

 

a Charles River Ashland historical control data (version 2018.02).

Summary of Gross Pathology Findings, F0Generation Necropsy

 

Males

Females

Group

1

2

3

4

1

2

3

4

Exposure (ppm)

0

380

750

1500

0

380

750

1500

No. Animals Examined

23

24

24

24

22

24

24

24

Kidney (No. Examined)

(23)

(24)

(24)

(24)

(22)

(24)

(24)

(24)

   Area(s), depressed

1

1

0

0

1

1

0

0

   Area(s), yellow

1

1

0

0

0

0

0

0

   Rough surface

0

0

1

1

0

0

0

0

Summary of Organ Weight Data, F0Generation Necropsy

 

Males

Females

Group

1

2

3

4

1

2

3

4

Exposure (ppm)

 

0

380

750

1500

0

380

750

1500

No. Animals per Group

23

24

24

24

22

24

24

24

Adrenal glands (No. Weighed)

(23)

(24)

(24)

(24)

(22)

(24)

(24)

(24)

    Absolute value

0.0559

0.0595

0.0628*

0.0637**

0.0678

0.0676

0.0688

0.0774*

    % of body weight

0.010

0.011

0.011

0.011

0.022

0.022

0.022

0.025*

    % of brain weight

2.584

2.775

2.886*

2.932*

3.444

3.416

3.470

3.895*

Kidney (No. Weighed)

(23)

(24)

(24)

(24)

(22)

(24)

(24)

(24)

    Absolute value

3.04

3.49**

3.52**

3.96**

1.79

1.89

1.91

1.99**

    % of body weight

0.554

0.637**

0.630**

0.708**

0.575

0.607

0.611*

0.639**

    % of brain weight

140.329

162.515**

161.700**

182.658**

90.783

95.464

96.850

99.742**

Liver (No. Weighed)

(23)

(24)

(24)

(24)

(22)

(24)

(24)

(24)

    Absolute value

13.51

14.51

16.34**

18.02**

8.07

8.70

8.96*

10.22**

    % of body weight

2.446

2.638*

2.911**

3.224**

2.593

2.784

2.862**

3.303**

    % of brain weight

624.018

675.629

751.222**

831.317**

410.364

438.605

453.804*

515.054**

Based upon statistical analysis of group means, values annotated with an asterisk (*) are significantly different from control group; * = p ≤ 0.05 using Dunnett’s test; ** = p ≤ 0.01 using Dunnett’s test.

Incidence of Selected Histopathologic Findings, F0Generation Necropsy

Exposure (ppm):

Males

Females

0

380

750

1500

0

380

750

1500

Adrenal Cortexa

23

24

24

24

22

0

0

24

Vacuolation

(6)b

(16)

(19)

(19)

(0)

(-)

(-)

(0)

       Minimal

5

13

10

15

0

-

-

0

       Mild

1

1

9

3

0

-

-

0

       Moderate

0

2

0

1

0

-

-

0

Kidneys

23

24

24

24

22

24

24

24

Cast, granular

(0)

(11)

(7)

(11)

(0)

(0)

(0)

(0)

       Minimal

0

7

5

3

0

0

0

0

       Mild

0

4

2

8

0

0

0

0

Droplet, hyaline

(0)

(19)

(18)

(22)

(0)

(0)

(0)

(0)

       Minimal

0

12

13

7

0

0

0

0

       Mild

0

7

5

11

0

0

0

0

       Moderate

0

0

0

4

0

0

0

0

Nephropathy, chronic progressive

(6)

(23)

(20)

(19)

(0)

(3)

(4)

(7)

       Minimal

6

17

14

13

0

3

4

7

       Mild

0

6

6

5

0

0

0

0

       Moderate

0

0

0

1

0

0

0

0

Liver

23

24

24

24

22

24

24

24

Hypertrophy, hepatocellular

(0)

(6)

(3)

(9)

(0)

(7)

(9)

(21)

       Minimal

0

6

3

6

0

7

8

18

       Mild

0

0

0

3

0

0

1

3

Nasal Level I

23

24

24

24

22

24

24

24

Edema

(0)

(0)

(0)

(2)

(0)

(5)

(0)

(0)

       Minimal

0

0

0

2

0

5

0

0

Hyperplasia, squamous epithelium

(0)

(0)

(0)

(3)

(0)

(0)

(2)

(1)

       Minimal

0

0

0

2

0

0

2

0

       Mild

0

0

0

1

0

0

0

1

Metaplasia, respiratory epithelium

(0)

(0)

(0)

(1)

(0)

(0)

(0)

(0)

       Minimal

0

0

0

1

0

0

0

0

Nasal Level II

23

24

24

24

22

24

24

24

Edema

(0)

(1)

(4)

(1)

(0)

(0)

(0)

(0)

       Minimal

0

0

3

0

0

0

0

0

       Mild

0

1

1

1

0

0

0

0

Eosinophilic globules

(0)

(0)

(0)

(0)

(0)

(1)

(6)

(0)

       Minimal

0

0

0

0

0

1

6

0

Exudate, suppurative

(2)

(0)

(3)

(1)

(2)

(0)

(0)

(3)

       Minimal

2

0

2

1

2

0

0

2

       Mild

0

0

1

0

0

0

0

1

Incidence of Selected Histopathologic Findings, F0Generation Necropsy - continued

Exposure (ppm):

Males

Females

0

380

750

1500

0

380

750

1500

Hyperplasia, mucous cell

(0)

(18)

(18)

(14)

(1)

(3)

(4)

(6)

       Minimal

0

1

9

9

1

3

3

3

       Mild

0

15

9

3

0

0

1

2

       Moderate

0

2

0

2

0

0

0

1

Hyperplasia, respiratory epithelium

(1)

(1)

(2)

(5)

(3)

(0)

(0)

(7)

       Minimal

0

1

2

3

3

0

0

6

       Mild

1

0

0

2

0

0

0

1

Inflammation, acute

(0)

(2)

(1)

(0)

(0)

(1)

(0)

(2)

       Minimal

0

1

1

0

0

1

0

2

       Mild

0

1

0

0

0

0

0

0

Inflammation, chronic active

(2)

(5)

(9)

(10)

(4)

(0)

(2)

(7)

       Minimal

1

4

6

6

3

0

2

4

       Mild

1

1

3

4

1

0

0

3

Metaplasia, squamous cell

(2)

(3)

(4)

(5)

(2)

(0)

(0)

(3)

       Minimal

2

3

2

4

2

0

0

2

       Mild

0

0

2

1

0

0

0

1

Nasal Level III

23

24

24

24

22

24

24

24

Degeneration, olfactory epithelium

(0)

(0)

(0)

(1)

(0)

(0)

(0)

(0)

       Minimal

0

0

0

1

0

0

0

0

Eosinophilic globules

(0)

(0)

(0)

(0)

(0)

(1)

(5)

(10)

       Minimal

0

0

0

0

0

0

2

6

       Mild

0

0

0

0

0

1

3

4

Hyperplasia, mucous cell

(0)

(3)

(2)

(1)

(0)

(0)

(0)

(1)

       Minimal

0

3

2

1

0

0

0

1

Nasal Level IV

23

24

24

24

22

24

24

24

Degeneration, olfactory epithelium

(0)

(0)

(0)

(0)

(0)

(0)

(0)

(1)

       Minimal

0

0

0

0

0

0

0

1

Eosinophilic globules

(0)

(0)

(0)

(0)

(0)

(1)

(7)

(9)

       Minimal

0

0

0

0

0

1

6

6

       Mild

0

0

0

0

0

0

1

1

       Moderate

0

0

0

0

0

0

0

2

Hyperplasia, mucous cell

(0)

(3)

(1)

(2)

(0)

(0)

(0)

(0)

       Minimal

0

3

1

2

0

0

0

0

Inflammation, acute

(0)

(0)

(0)

(0)

(0)

(0)

(0)

(2)

       Minimal

0

0

0

0

0

0

0

2

Nasal Level V

23

24

24

24

22

24

24

24

Eosinophilic globules

(0)

(0)

(0)

(0)

(0)

(1)

(6)

(8)

       Minimal

0

0

0

0

0

1

6

7

       Mild

0

0

0

0

0

0

0

0

       Moderate

0

0

0

0

0

0

0

1

Hyperplasia, mucous cell  

(0)

(3)

(2)

(0)

(0)

(0)

(1)

(0)

       Minimal

0

3

2

0

0

0

1

0

Nasal Level VI

23

24

24

24

22

24

24

24

Eosinophilic globules

(0)

(0)

(0)

(0)

(0)

(0)

(5)

(4)

       Minimal

0

0

0

0

0

0

4

4

       Mild

0

0

0

0

0

0

1

0

Hyperplasia, mucous cell

(0)

(2)

(5)

(2)

(0)

(1)

(1)

(0)

       Minimal

0

2

3

2

0

1

1

0

       Mild

0

0

2

0

0

0

0

0

Exposure (ppm):

Males

Females

0

380

750

1500

0

380

750

1500

Trachea

23

24

24

24

22

0

0

24

Hyperplasia, respiratory epithelium

(0)

(2)

(0)

(2)

(0)

(-)

(-)

(0)

      Minimal

0

1

0

0

0

-

-

0

      Mild

0

1

0

2

0

-

-

0

Summary of Thyroid Hormone Data, F1(Cohort 1A)

 

Males

Females

Group

1

2

3

4

1

2

3

4

Exposure (ppm)

0

380

750

1500

0

380

750

1500

No. Animals/Group

10

10

10

10

10

10

10

10

Total T3 (pg/mL)

533

494

544

491

600

518

548

545

Total T4 (pg/mL)

48810

35250

45250

37580

31980

24280*

26130

34020

TSH (ng/mL)

6.1

6.1

6.3

9.2*

3.0

2.8

3.4

4.6

Based upon statistical analysis of group means, values annotated with an asterisk (*) are significantly different from control group; * = p ≤ 0.05 using Dunnett’s test.

Summary of Gross Pathology Findings, F1(Cohort 1A) Generation Necropsy

 

Males

Females

Group

1

2

3

4

1

2

3

4

Exposure (ppm)

0

380

750

1500

0

380

750

1500

No. Animals Examined

21

21

23

22

21

21

24

23

Kidney (No. Examined)

(21)

(21)

(23)

(22)

(21)

(21)

(24)

(23)

   Area(s), depressed

2

0

1

0

0

0

0

0

   Mottled

0

0

1

0

0

0

0

0

Liver (No. Examined)

(21)

(21)

(23)

(22)

(21)

(21)

(24)

(23)

   Rough surface

0

0

0

0

0

0

0

1

Summary of Organ Weight Data, F1(Cohort 1A)

 

Males

Females

Group

1

2

3

4

1

2

3

4

Exposure (ppm)

0

380

750

1500

0

380

750

1500

No. Animals per Group

21

21

23

22

21

21

24

23

Kidney (No. Weighed)

(21)

(21)

(23)

(22)

(21)

(21)

(24)

(23)

    Absolute value

2.61

3.34**

3.18**

3.45**

1.70

1.79

1.80

1.77

    % of body weight

0.640

0.760**

0.750**

0.830**

0.638

0.681*

0.692**

0.704**

    % of brain weight

126.447

161.700**

154.308**

169.535**

88.055

92.826

94.407

95.282

Liver (No. Weighed)

(21)

(21)

(23)

(22)

(21)

(21)

(24)

(23)

    Absolute value

11.62

13.55**

13.93**

15.39**

7.33

7.81

8.18

9.34**

    % of body weight

2.821

3.081**

3.282**

3.695**

2.740

2.968

3.138

3.731**

    % of brain weight

564.547

655.779**

678.219**

755.303**

379.505

405.030

429.576

500.254**

Thyroids/Parathyroids (No. Weighed)

(19)

(21)

(23)

(22)

(21)

(21)

(24)

(23)

    Absolute value

0.0200

0.0187

0.0203

0.0192

0.0140

0.0158

0.0159

0.0172**

    % of body weight

0.005

0.004

0.005

0.005

0.005

0.006

0.006

0.007**

    % of brain weight

0.969

0.905

0.990

0.943

0.726

0.824

0.836

0.921**

SV/CG/ACC Fluid (No. Weighed)

(21)

(21)

(20)

(22)

-

-

-

-

    Absolute value

1.79

1.97

1.89

2.09**

-

-

-

-

    % of body weight

0.441

0.450

0.450

0.504**

-

-

-

-

    % of brain weight

87.150

95.219

92.044

102.500**

-

-

-

-

Based upon statistical analysis of group means, values annotated with an asterisk (*) are significantly different from control group; * = p ≤ 0.05 using Dunnett’s test; ** = p ≤ 0.01 using Dunnett’s test.

Incidence of Selected Histopathologic Findings, F1(Cohort 1A)

Exposure (ppm):

Males

Females

0

380

750

1500

0

380

750

1500

Adrenal Cortexa

21

21

23

22

21

0

0

23

Vacuolation

(2)b

(7)

(12)

(11)

(0)

(-)

(-)

(0)

       Minimal

2

5

10

7

0

-

-

0

       Mild

0

2

2

2

0

-

-

0

       Moderate

0

0

0

2

0

-

-

0

Kidneys

21

21

23

22

21

21

24

23

Cast, granular

(0)

(10)

(9)

(11)

(0)

(0)

(0)

(0)

       Minimal

0

4

6

6

0

0

0

0

       Mild

0

6

3

4

0

0

0

0

       Moderate

0

0

0

1

0

0

0

0

Droplet, hyaline

(0)

(21)

(23)

(21)

(0)

(0)

(0)

(0)

       Minimal

0

10

12

11

0

0

0

0

       Mild

0

8

11

9

0

0

0

0

       Moderate

0

3

0

1

0

0

0

0

Nephropathy, chronic progressive

(9)

(20)

(23)

(22)

(17)

(20)

(22)

(16)

       Minimal

9

5

9

5

14

16

12

13

       Mild

0

6

10

13

3

4

10

3

       Moderate

0

9

4

3

0

0

0

0

       Marked

0

0

0

1

0

0

0

0

Liver

21

21

23

22

21

21

24

23

Hypertrophy, hepatocellular

(0)

(6)

(5)

(11)

(0)

(2)

(6)

(15)

       Minimal

0

6

5

10

0

2

6

11

       Mild

0

0

0

1

0

0

0

4

aNumber of tissues examined from each group.

bNumbers in parentheses represent the number of animals with the finding

Incidence of Selected Histopathologic Findings, F1(Cohort 1A) - continued

Exposure (ppm):

Males

Females

0

380

750

1500

0

380

750

1500

Nasal Level I

21

21

23

22

21

21

24

23

Edema

(0)

(0)

(0)

(0)

(0)

(0)

(2)

(0)

   Minimal

0

0

0

0

0

0

2

0

Hyperplasia, squamous epithelium

(2)

(1)

(0)

(3)

(0)

(0)

(0)

(0)

       Minimal

1

1

0

2

0

0

0

0

       Mild

1

0

0

1

0

0

0

0

Nasal Level II

21

21

23

22

21

21

24

23

Inflammation, acute

(0)

(0)

(0)

(0)

(0)

(0)

(1)

(1)

       Minimal

0

0

0

0

0

0

1

1

Inflammation, chronic active

(0)

(0)

(0)

(0)

(0)

(0)

(0)

(2)

       Minimal

0

0

0

0

0

0

0

1

       Mild

0

0

0

0

0

0

0

1

Hyperplasia, mucous cell

(0)

(4)

(8)

(11)

(3)

(5)

(4)

(6)

       Minimal

0

4

8

10

2

4

3

4

       Mild

0

0

0

1

1

1

1

2

Metaplasia, squamous cell

(0)

(0)

(1)

(1)

(0)

(0)

(1)

(3)

       Minimal

0

0

1

1

0

0

1

3

Nasal Level III

21

21

23

22

21

21

24

23

Hyperplasia, mucous cell

(0)

(0)

(0)

(3)

(0)

(0)

(0)

(2)

       Minimal

0

0

0

3

0

0

0

2

Nasal Level IV

21

21

23

22

21

21

24

23

Hyperplasia, mucous cell

(0)

(1)

(0)

(3)

(0)

(1)

(1)

(1)

       Minimal

0

1

0

3

0

1

1

1

Nasal Level V

21

21

23

22

21

21

24

23

Hyperplasia, mucous cell

(0)

(0)

(0)

(1)

(0)

(0)

(0)

(0)

       Minimal

0

0

0

1

0

0

0

0

Nasal Level VI

21

21

23

22

21

21

24

23

Hyperplasia, mucous cell

(0)

(1)

(1)

(0)

(0)

(2)

(1)

(0)

       Minimal

0

1

1

0

0

2

1

0

Trachea

21

21

23

22

0

0

0

0

Hyperplasia, mucous cell

(0)

(0)

(0)

(1)

(0)

(-)

(-)

(0)

       Minimal

0

0

0

1

0

-

-

0

 

aNumber of tissues examined from each group.

bNumbers in parentheses represent the number of animals with the finding.

Conclusions:
Due to the lack of adverse effects on F0 and F1 males and females throughout the study, an exposure level of 1500 ppm, the highest exposure level evaluated, was considered to be the no observed-adverse-effect level (NOAEL) for F0 and F1 male and female systemic toxicity when methyl isoamyl ketone was administered via whole body inhalation to Crl:CD(SD) rats. Based on the lower mean offspring body weights and body weight gains noted at 1500 ppm during the preweaning period in both the F1 and F2 generations, an exposure level of 750 ppm was considered to be the NOAEC for F1 and F2 neonatal toxicity. There was no evidence of reproductive toxicity at any exposure level based on evaluation of reproductive performance, sperm measurements, and estrous cyclicity in the F0 and F1 generations. Therefore, the NOAEC for F0 and F1 reproductive toxicity was considered to be 1500 ppm.
Executive summary:

The objective of this study was to evaluate the potential adverse effects of MiAK on reproduction in an extended one-generation study.  This included evaluation of life stages not covered by other types of toxicity studies and tested for effects that may occur as a result of pre- and postnatal chemical exposure.  This included determining the effects of the test substance on male and female reproductive processes including gonadal function, estrous cyclicity, mating behavior, conception, gestation, parturition, lactation, and weaning and on survival, growth, and development of the offspring.  A minimum of 1 litter per dam was produced.  Animals were not paired more than once.  As part of this assessment, offspring were also evaluated for effects on the reproductive system, alterations in endocrine function (including thyroid perturbations), and effects on other systemic toxicity parameters.

There were no test substance-related deaths in the F0 and F1 generations at any exposure level.  Test substance-related clinical findings consisted of red material around the mouth and/or eyes and clear material on various body surfaces for males and females at 1500 ppm in the F0 generation and red material around the mouth for F1 females at 1500 ppm.  These findings were generally resolved by the next scheduled observation period and were considered test substance related, but non-adverse.

Lower mean body weight gain was noted for F1 males and females in the 1500 ppm group during the first 10 days of the preweaning exposure period (Postnatal Day 4-14).  Consequently, mean male and female pup body weights in this group were 9.4% and 12.6% lower than controls on PND 14.  Mean body weight gains for males and females were comparable to controls during the remainder of the preweaning period (PND 14–28).  Lower body weight gains and food consumption were also observed for these animals immediately following the initiation of direct inhalation exposures such that mean absolute body weights for F1 males and females were 6.9% and 10.0% lower than controls on PND 35.  Thereafter, mean body weights for the 1500 ppm group were comparable to controls.  Due to the lower body weight gains noted for the F1 pups during the neonatal period, F1 animals assigned to Cohort 1B for follow-up reproductive assessments were bred to obtain an F2 generation.  The effects on mean pup body weight gain noted in the F1 generation at 1500 ppm were replicated in the F2 generation as lower mean pup body weight gains for F2 males and females in the 1500 ppm group during PND 7–14 resulted in mean body weights that were 4.4 to 8.7% lower than the control group on PND 14.  These differences in mean pup body weight gain at the 1500 ppm exposure level, during the preweaning period in both the F1 and F2 generations were considered test substance-related and adverse.

Lower mean body weights during the preweaning period were also noted for F1 pups in the 380 and 750 ppm exposure groups, however, the differences were not dose responsive and were not replicated in the F2 generation.  Thus, any noted differences in mean body weight gains or mean absolute body weights in these groups, in the F1 and F2 generations were considered incidental and unrelated to test substance exposure. With the exception of the aforementioned differences in mean body weight gain during the preweaning period, there were no noted differences in mean body weights, body weight gains, food consumption, and food efficiency for F0 or F1 males and females in the 380, 750 and 1500 ppm exposure groups.  There were no test substance-related effects on F0 and F1 estrous cyclicity, reproductive performance (male and female mating and fertility, male copulation, and female conception indices), precoital interval, mean gestation lengths, the process of parturition, or male spermatogenic parameters.  In addition, F1 and F2 litter viability and survival, anogenital distance and developmental landmarks were unaffected by MiAK exposure.  There were also no test substance related effects on serum levels of T3, T4 or TSH or on clinical pathology parameters (hematology, coagulation, serum chemistry, and urinalysis) for F0 and F1 animals at any exposure level.

 

Microscopically, there were no adverse test substance-related histopathological findings noted for the F0 and F1 generation males and females.  Although chronic progressive nephropathy is a common background finding in the rat kidney, the incidence and severity were increased in test substance-treated rats in both generations when compared to concurrent controls and there was correlation of gross findings in the male kidney to chronic progressive nephropathy.  In the males, the kidney changes of hyaline droplets and granular casts associated with an exacerbation of the spontaneous finding of chronic progressive nephropathy which may be consistent with alpha 2u nephropathy, which is specific to male rats.1  There were no changes in clinical pathology parameters associated with kidney function in the F0 generation.  In the F1 generation, males in the 1500 ppm group were noted with elevated urea nitrogen, however, there were no corresponding changes in creatinine or other kidney-related clinical pathology parameters and no changes in kidney-related clinical pathology parameters in the females, in either generation. Thus, the kidney findings in male rates in both generations were considered nonadverse.  Similarly, findings of chronic progressive nephropathy in female kidneys in both generations were also considered non-adverse based on the magnitude of the change (minimal) and lack of changes in clinical pathology parameters related to kidney function.  

Test substance-related increases in liver, adrenal gland (F0 males only) and kidney weights were noted in both the F0 and F1 generations and correlated with histopathological findings in these organs as noted above.  None of these differences were considered adverse.  There were no other organ weight changes noted in the F0 generation.  Additional organ weight changes noted in animals assigned to F1 generation Cohort 1A included elevated thyroid and parathyroid weights for females and elevated seminal vesicle/coagulating gland/accessory fluid for males in the 1500 ppm group.  These differences were not observed in males and females assigned to Cohort 1B nor in the F2 offspring and were considered nonadverse due to the lack of microscopic correlates, or alterations in serum thyroid hormones.

Microscopic changes in the nasal cavity and trachea were considered minor and nonadverse, and hepatocellular hypertrophy in the liver was considered an adaptive response and nonadverse in both generations.  Adrenal gland cortical vacuolation noted in males only and consisted of a diffuse change where cytoplasmic vacuole(s) were observed to be expanding the epithelial cells of the zona fasciculate.  This finding was not noted in the females and was also considered nonadverse.

In summary, with the exception of lower mean body weight gains and consequently lower mean absolute body weights noted for both F1 and F2 males and females during the preweaning period, there were no adverse test substance-related effects noted in either generation.

Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
3 500 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
The study report does not give values in mg/m3; it gives a NOAEC value in ppm. That value was determined by (MW 114.12)*(750 PPM NOAEL)/24.45 = mg/m3
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The objective of an OECD extended 1 -generation study was to evaluate the potential adverse effects of MiAK on reproduction in an extended one-generation study.  This included evaluation of life stages not covered by other types of toxicity studies and tested for effects that may occur as a result of pre- and postnatal chemical exposure.  This included determining the effects of the test substance on male and female reproductive processes including gonadal function, estrous cyclicity, mating behavior, conception, gestation, parturition, lactation, and weaning and on survival, growth, and development of the offspring.  A minimum of 1 litter per dam was produced.  Animals were not paired more than once.  As part of this assessment, offspring were also evaluated for effects on the reproductive system, alterations in endocrine function (including thyroid perturbations), and effects on other systemic toxicity parameters.

There were no test substance-related deaths in the F0 and F1 generations at any exposure level.  Test substance-related clinical findings consisted of red material around the mouth and/or eyes and clear material on various body surfaces for males and females at 1500 ppm in the F0 generation and red material around the mouth for F1 females at 1500 ppm.  These findings were generally resolved by the next scheduled observation period and were considered test substance related, but non-adverse.

Lower mean body weight gain was noted for F1 males and females in the 1500 ppm group during the first 10 days of the preweaning exposure period (Postnatal Day 4-14).  Consequently, mean male and female pup body weights in this group were 9.4% and 12.6% lower than controls on PND 14.  Mean body weight gains for males and females were comparable to controls during the remainder of the preweaning period (PND 14–28).  Lower body weight gains and food consumption were also observed for these animals immediately following the initiation of direct inhalation exposures such that mean absolute body weights for F1 males and females were 6.9% and 10.0% lower than controls on PND 35.  Thereafter, mean body weights for the 1500 ppm group were comparable to controls.  Due to the lower body weight gains noted for the F1 pups during the neonatal period, F1 animals assigned to Cohort 1B for follow-up reproductive assessments were bred to obtain an F2 generation.  The effects on mean pup body weight gain noted in the F1 generation at 1500 ppm were replicated in the F2 generation as lower mean pup body weight gains for F2 males and females in the 1500 ppm group during PND 7–14 resulted in mean body weights that were 4.4 to 8.7% lower than the control group on PND 14.  These differences in mean pup body weight gain at the 1500 ppm exposure level, during the preweaning period in both the F1 and F2 generations were considered test substance-related and adverse.

Lower mean body weights during the preweaning period were also noted for F1 pups in the 380 and 750 ppm exposure groups, however, the differences were not dose responsive and were not replicated in the F2 generation.  Thus, any noted differences in mean body weight gains or mean absolute body weights in these groups, in the F1 and F2 generations were considered incidental and unrelated to test substance exposure. With the exception of the aforementioned differences in mean body weight gain during the preweaning period, there were no noted differences in mean body weights, body weight gains, food consumption, and food efficiency for F0 or F1 males and females in the 380, 750 and 1500 ppm exposure groups.  There were no test substance-related effects on F0 and F1 estrous cyclicity, reproductive performance (male and female mating and fertility, male copulation, and female conception indices), precoital interval, mean gestation lengths, the process of parturition, or male spermatogenic parameters.  In addition, F1 and F2 litter viability and survival, anogenital distance and developmental landmarks were unaffected by MiAK exposure.  There were also no test substance related effects on serum levels of T3, T4 or TSH or on clinical pathology parameters (hematology, coagulation, serum chemistry, and urinalysis) for F0 and F1 animals at any exposure level.

 

Microscopically, there were no adverse test substance-related histopathological findings noted for the F0 and F1 generation males and females.  Although chronic progressive nephropathy is a common background finding in the rat kidney, the incidence and severity were increased in test substance-treated rats in both generations when compared to concurrent controls and there was correlation of gross findings in the male kidney to chronic progressive nephropathy.  In the males, the kidney changes of hyaline droplets and granular casts associated with an exacerbation of the spontaneous finding of chronic progressive nephropathy which may be consistent with alpha 2u nephropathy, which is specific to male rats.1  There were no changes in clinical pathology parameters associated with kidney function in the F0 generation.  In the F1 generation, males in the 1500 ppm group were noted with elevated urea nitrogen, however, there were no corresponding changes in creatinine or other kidney-related clinical pathology parameters and no changes in kidney-related clinical pathology parameters in the females, in either generation. Thus, the kidney findings in male rates in both generations were considered nonadverse.  Similarly, findings of chronic progressive nephropathy in female kidneys in both generations were also considered non-adverse based on the magnitude of the change (minimal) and lack of changes in clinical pathology parameters related to kidney function.  

Test substance-related increases in liver, adrenal gland (F0 males only) and kidney weights were noted in both the F0 and F1 generations and correlated with histopathological findings in these organs as noted above.  None of these differences were considered adverse.  There were no other organ weight changes noted in the F0 generation.  Additional organ weight changes noted in animals assigned to F1 generation Cohort 1A included elevated thyroid and parathyroid weights for females and elevated seminal vesicle/coagulating gland/accessory fluid for males in the 1500 ppm group.  These differences were not observed in males and females assigned to Cohort 1B nor in the F2 offspring and were considered nonadverse due to the lack of microscopic correlates, or alterations in serum thyroid hormones.

Microscopic changes in the nasal cavity and trachea were considered minor and nonadverse, and hepatocellular hypertrophy in the liver was considered an adaptive response and nonadverse in both generations.  Adrenal gland cortical vacuolation noted in males only and consisted of a diffuse change where cytoplasmic vacuole(s) were observed to be expanding the epithelial cells of the zona fasciculate.  This finding was not noted in the females and was also considered nonadverse.

In summary, with the exception of lower mean body weight gains and consequently lower mean absolute body weights noted for both F1 and F2 males and females during the preweaning period, there were no adverse test substance-related effects noted in either generation.

Effects on developmental toxicity

Description of key information

Developmental toxicity in the form of great vessel malformations was observed in 3 fetuses in a rabbit OECD 414 prenatal developmental toxicity study.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION

Pre-existing data:
There are no pre-existing data available that would address the pre-natal developmental toxicity study endpoint (GLP or non-GLP). A rat pre-natal developmental toxicity study is not adequate according to the regulation. There are no historical human data that could be used in place of this study.

- (Q)SAR:
There are no accepted QSAR approaches for predicting the outcome of this endpoint. However, there were no Reproductive Toxicity alerts for MiAK.

- In vitro methods
There are no accepted in vitro approaches for predicting the outcome of this endpoint

- Weight of evidence
WoE is not possible as there are not similar substances that could be used for such a comparison.

- Grouping and read-across
The Registrant has not identified any suitable analogues that could be used to provide data for this endpoint. Other similar substances are quite data poor.

- Substance-tailored exposure driven testing
Based on the use patternes of the registered substance in coatings, exposure-based waiving is not possible.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
MiAK is clear colorless liquid.The purity of the test substance was 99.33%. The test substance was stored at room temperature (18°C to 24°C), protected from light, and was considered stable under these conditions.
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
Time-mated female New Zealand White [Hra:(NZW)SPF] rabbits were used as the test system on this study. This species and strain of animal is recognized as
appropriate for developmental toxicity studies. The number of animals selected for this study (24 females/group) was based on the US EPA Health
Effects Test Guidelines OPPTS 870.3700, Prenatal Development Toxicity Study, Aug 1998 and the OECD Guidelines for the Testing of Chemicals: Guideline 414, Prenatal Developmental Toxicity Study, Jan 2001, which recommends evaluation of approximately 20 females with implantation sites at necropsy. Given the possibility of nongravid animals, unexpected deaths, or test substance-related moribundity and/or mortality, this was an appropriate number of animals to obtain a sample size of 20 females/group at termination. Time-mated female New Zealand White rabbits were received in 2 shipments; each shipment was received from the same facility and was approximately the same age. One hundred four time-mated female New Zealand White rabbits in total (52/shipment) were received in good health from Covance Research Products, Inc., Denver, PA, on 21 Apr 2017 and 19 May 2017. The time-mated rabbits were received on Gestation Day 1, 2, or 3. The animals were approximately 7 months old upon receipt. Each animal was uniquely identified using a microchip (BMDS system) which was implanted subcutaneously in the dorsoscapular region. The rabbits were observed twice daily for mortality and general changes in appearance and behavior.

All rabbits were housed during the study in an environmentally controlled room. The room temperature and relative humidity controls were set to maintain environmental conditions of 61°F to 71°F (16°C to 22°C) and 30% to 70%, respectively. Room temperature and relative humidity data were monitored continuously and were scheduled for automatic collection on an hourly basis. Actual mean daily temperature ranged from 65.4°F to 68.8°F (18.6°C to 20.4°C)and mean daily relative humidity ranged from 45.3% to 67.7% during the study. Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. The light status (on or off) was recorded once every 15 minutes. Air handling units were set to provide a minimum of 10 fresh air changes per hour.
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
Exposures will be conducted using four 1500-L glass and stainless steel whole-body exposure chambers. One chamber will be dedicated for the filtered air group (0 ppm) and three chambers will be dedicated for each of the three test substance-exposed groups.The chambers will be operated under dynamic conditions and at a slight negative pressure. Exposure system air will be supplied from a breathing quality in-house compressed air source,compressed nitrogen source, and/or from a HEPA- and charcoal-filtered supply air source and will provide a minimum of 12 air changes per hour. The airflow rates for each whole-body
chamber may be monitored by measuring the pressure drop between the ports of an orifice plate using a Dwyer Magnehelic® Indicating Transmitter pressure gauge. Each gauge will be calibrated for conversion from pressure to airflow in standard liters per minute.

Average daily temperature and relative humidity of the exposure atmosphere will be 66 ± 5°F (19 ± 3°C) and 50 ± 20%, respectively. Temperature and relative humidity will be monitored with a temperature and humidity transmitter probe for each chamber. Temperature, relative humidity, chamber ventilation rate, and negative pressure within each exposure chamber will be monitored and recorded using the Inhalation Exposure Data Collection System (WINH). The intended schedule for recording these environmental parameters will be approximately every 45 minutes during each exposure. However, recording these parameters
more frequently or recording at additional intervals is acceptable. In the event of a failure of the automated data collection system (e.g., malfunction of a component or of a hardware connection), manual recording will be used. Oxygen content of the exposure atmospheres will be at least 19% and will be documented during method development, and at least once during the exposure phase. The daily exposure duration will be 6 hours. Exposures will be initiated when the generation of test atmospheres is started and will be terminated after 6 hours when the generation is stopped. The test substance chambers will not be opened until at least the T99 period has elapsed. The continued animal exposure during this chamber clearance period is considered to be the reciprocal equivalent of the “under exposure” experienced during the chamber equilibration (concentration build up) period. Therefore, the daily exposure duration will not be adjusted for the calculated T99 for the exposure systems.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyzed exposure concentrations were determined at approximately 45 minute intervals using a gas chromatograph (GC). Samples were collected from the approximate animal-breathing zone of the exposure chamber via 1/8-inch heated stainless steel tubing. Tubing was heated to approximately 60°C using heat tapes,
J-type thermocouples, and temperature controllers. Under the control of the WINH system, sampling and analyses was performed as follows. The program controls an external multi-position valve that permits sequential sampling from the exposure room and each exposure chamber. The multi-position valve was heated to approximately 65°C using a disc heater and was controlled using a temperature controller and J-type thermocouple. Gas sampling injection onto the chromatography column occurs via an internal gas-sampling valve with a sample loop, the chromatograph is displayed and the area under the sample peak was calculated and stored. The WINH system then acquires the stored peak area data and uses an ln-quadratic equation based on the GC calibration curve to calculate the measured concentration in ppm.
Details on mating procedure:
Time-mated rabbits will be received by Charles River in 2 cohorts, each cohort will be received from the same facility and be approximately the same age. The animals will arrive on Gestation Day 1, 2 or 3
Duration of treatment / exposure:
gestation day 7 through 28
Frequency of treatment:
Daily
Duration of test:
6 hours per day
Dose / conc.:
500 ppm
Dose / conc.:
1 250 ppm
Dose / conc.:
2 500 ppm
No. of animals per sex per dose:
24 per group
Control animals:
yes
Details on study design:
This study was conducted in 2 phases (Phase I and Phase II) due to the limitations in the total number of animals that can be placed in the stainless steel whole-body exposure chambers at one time.For each phase, methyl isoamyl ketone (MiAK) was administered via whole-body inhalation exposure for 6 hours per day to 3 groups of 12 time-mated female New Zealand White [Hra:(NZW)SPF] rabbits from Gestation Days 7–28. Target exposure concentrations were 500,
1250, and 2500 ppm. Overall mean analyzed exposure concentrations were 508, 1257, and 2493 ppm for Phase I and 499, 1262, and 2497 ppm for Phase II for the 500, 1250, and 2500 ppm groups, respectively. Each phase also contained a concurrent control group, composed of 12 time-mated females, that was exposed to humidified, filtered air on a comparable regimen. The females for both phases were approximately 7 months of age at the initiation of exposure. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. On Gestation Day 29, a laparohysterectomy was performed on each female. The uteri, placentae, and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.
Maternal examinations:
All rabbits were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Individual clinical observations were recorded daily from the day of receipt through Gestation Day 29 (prior to exposure during the treatment period) . Animals were also observed for signs of toxicity at approximately the midpoint of exposure, the end of the exposure period, and 1–2 hours following exposure.
Ovaries and uterine content:
Gravid uterine weight was collected and net body weight (the Gestation Day 29 body weight exclusive of the weight of the uterus and contents) and net body weight change (the Gestation Day 0–29 body weight change exclusive of the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy.
Fetal examinations:
External, visceral, and skeletal findings were recorded .viable fetus with Crown-rump measurements, degrees of autolysis and gross
examinations, if possible, were recorded for late resorptions.
Statistics:
All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-exposed group to the control group.
Historical control data:
yes
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Maternal toxicity was apparent in the 2500 ppm group as evidenced by adverse clinical observations (general irritation-related findings of partial closure of the eyes and/or clear material around the mouth and/or nose) noted at the midpoint of exposure, end of the daily exposure, and/or postexposure observations.
Dermal irritation (if dermal study):
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A test substance-related lower mean body weight gain was noted in at 2500 ppm group during entire exposure period (Gestation Days 7–29)
Description (incidence and severity):
Mean food consumption in the 2500 ppm group during Gestation Days 7–10, 10–13, and 13–20, and when the entire exposure period (Gestation Days 7–29) wre lower in 2500 ppm group when compared to the control.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Mean food consumption, evaluated as g/animal/day and g/kg/day, was significantly (p < 0.01) lower in the 2500 ppm group during Gestation Days 7–10, 10–13, and 13–20, and when the entire exposure period (Gestation Days 7–29) was evaluated when compared to the control group, which corresponded to the lower mean body weight gains or body weight losses. The differences were considered adverse.
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
At the scheduled necropsy on Gestation Day 29, no test substance-related internal findings were noted at exposure levels of 500, 1250, and 2500 ppm.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Intrauterine growth and survival were unaffected by test substance exposure at exposure levels of 500, 1250, and 2500 ppm. Parameters evaluated included postimplantation loss, number and percentage of viable fetuses, mean fetal body weights and fetal sex ratios. Mean number of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups. Differences from the control group were slight and not statistically significant.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Details on maternal toxic effects:
Data tables are provided in the "attached background materials" section, as fidelity reasons prevent the copying of .PDF tables to IUCLID.
Key result
Dose descriptor:
NOAEL
Effect level:
1 250 ppm
Based on:
test mat.
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
The heart and great vessel associated visceral malformations retroesophageal aortic arch, Tetralogy of Fallot, interventricular septal defect, and bulbous aorta in the 2500 ppm group were considered test substance-related and adverse.
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEC
Effect level:
1 250 ppm
Based on:
test mat.
Basis for effect level:
visceral malformations
Abnormalities:
effects observed, treatment-related
Localisation:
visceral/soft tissue: cardiovascular
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
2 500 ppm
Treatment related:
yes
Relation to maternal toxicity:
developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Overall Mean Nominal Exposure Concentration Phase I

Exposure Chamber:

2

3

4

Target Concentration (ppm):

500

1250

2500

Nominal Concentration (ppm)

544

1351

2716

Standard Deviation:

34.4

54.6

142.2

N:

24

24

24


Overall Mean Nominal Exposure Concentration Phase II

Exposure Chamber:

2

3

4

Target Concentration (ppm):

500

1250

2500

Nominal Concentration (ppm)

480

1211

2764

Standard Deviation:

8.5

71.4

64.8

N:

24

24

24


Overall Mean Exposure Concentration Phase I

Exposure Chamber:

1

2

3

4

Target Concentration (ppm):

0

500

1250

2500

Mean Concentration (ppm)

0

508

1257

2493

Standard Deviation:

0.0

20.5

33.0

64.4

N:

24

24

24

24


Overall Mean Exposure Concentration Phase II

Exposure Chamber:

1

2

3

4

Target Concentration (ppm):

0

500

1250

2500

Nominal Concentration (ppm)

0

499

1262

2497

Standard Deviation:

0.0

7.1

26.1

29.2

N:

24

24

24

24

Conclusions:
Based on the adverse clinical findings of eye, nasal, and oral irritation, and adverse effects on maternal food consumption with correspondingly lower mean body weight gain during Gestation Days 7 through 20 in the 2500 ppm group, the no-observed-adverse-effect level (NOAEL) for maternal toxicity was 1250 ppm when methyl isoamyl ketone was administered by whole-body inhalation exposure to time-mated New Zealand White rabbits. Based on heart and great vessel visceral malformations at the 2500 ppm exposure level, 1250 ppm was considered to be the NOAEL for embryo/fetal developmental toxicity.
Executive summary:

Methyl isoamyl ketone (MiAK) was administered via whole-body inhalation exposure for 6 hours per day to 3 groups of 12 time-mated female New Zealand White [Hra:(NZW) SPF] rabbits from Gestation Days 7–28. Target exposure concentrations were 500, 1250, and 2500 ppm. Overall mean analyzed exposure concentrations were 508, 1257, and 2493 ppm for Phase I and 499, 1262, and 2497 ppm for Phase II for the 500, 1250, and 2500 ppm groups, respectively. All females survived to the scheduled necropsy. Test substance-related increased incidences of decreased defecation were noted for 12 of 24 females in the 2500 ppm group at the daily examinations. At the midpoint of exposure, end of the daily exposure, and/or postexposure observations, test substance-related, adverse general irritation-related findings of partial closure of the eyes and/or clear material around the mouth and/or nose were noted for females in the 2500 ppm group. Clear material around the nose was also noted sporadically for 7 of 24 females in the 1250 ppm group at the midpoint of exposure; however, this finding was transient and not observed at the postdosing observation, and therefore was not considered adverse. No other test substance-related clinical observations were noted at any exposure level. Test substance-related lower mean body weight gains or body weight losses, with correspondingly reduced mean food consumption, were noted in the 2500 ppm group during Gestation Days 7–10, 10–13, and 13–20, resulting in a lower mean body weight gain when the entire exposure period (Gestation Days 7–29) was evaluated compared to the control group. These decreases in body weight gain generally correlated with an approximate 30% decrease in food consumption during this time period and were considered adverse. Mean gravid uterine weights, mean net body weights and net body weight changes in the 500, 1250, and 2500 ppm groups were unaffected by test substance exposure. No test substance-related maternal macroscopic findings were noted at the scheduled necropsy at any exposure level. Intrauterine growth and survival were unaffected by maternal test substance exposure. Heart and great vessel visceral malformations were noted in 3 fetuses from different litters within the 2500 ppm group, including retroesophageal aortic arch, Tetralogy of Fallot, an interventricular septal defect, and bulbous aorta, were considered test substance-related and adverse. There were no fetal skeletal malformations noted in the 2500 ppm group and fetal morphology was unaffected by test substance exposure in the 500 and 1250 ppm groups. Based on the adverse clinical findings of eye, nasal, and oral irritation, and adverse effects on maternal food consumption with correspondingly lower mean body weight gain during Gestation Days 7 through 20 in the 2500 ppm group, the no-observed-adverse-effect level (NOAEL) for maternal toxicity was 1250 ppm when methyl isoamyl ketone was administered by whole-body inhalation exposure to time-mated New Zealand White rabbits. Based on heart and great vessel visceral malformations at the 2500 ppm exposure level, 1250 ppm was the NOAEL for embryo/fetal developmental toxicity.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted according to OECD guidelines in a GLP-compliant facility
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
Sexually mature, virgin female Sprague Dawley [Crl:CD(SD)] rats were used as the test system on this study. One hundred twenty-five sexually mature, virgin female Crl:CD(SD) rats were received in good health from Charles River Laboratories, Inc., Raleigh, NC, on 15 November 2011. The animals were approximately 79 days old upon receipt. Each female was examined by a qualified biologist on the day of receipt. The day following receipt, all animals were weighed and clinical observations were recorded. Each rat was uniquely identified by a Monel® metal ear tag displaying the animal number and housed for 14 days for acclimation purposes. During the acclimation period, the rats were observed twice daily for mortality and changes in general appearance and behavior.

Upon arrival and until pairing, all rats were individually housed in clean, stainless steel wire-mesh cages suspended above cage-board. The cage-board was changed at least 3 times per week. The rats were paired for mating in the home cage of the male. Following positive evidence of mating, the females were returned to individual suspended wire-mesh cages; nesting material was not required as the females were euthanized prior to the date of expected parturition. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996).


All rats were housed throughout the acclimation period and during the study in an environmentally controlled room. The room temperature and humidity controls were set to maintain environmental conditions of 71°F ± 5°F (22°C ± 3°C) and 50% ± 20%, respectively. Room temperature and relative humidity data were monitored continuously and were scheduled for automatic collection on an hourly basis. These data are summarized in Appendix D. Actual mean daily temperature ranged from 69.7°F to 71.4°F (20.9°C to 21.9°C) and mean daily relative humidity ranged from 31.5% to 41.6% during the study. Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. The light status (on or off) was recorded once every 15 minutes. Air handling units were set to provide a minimum of 10 fresh air changes per hour.


Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:


Exposures were conducted in four 1000-L stainless steel and glass whole-body inhalation exposure chambers. One chamber was dedicated for each group. All chambers were operated under dynamic conditions with at least 12 to 15 air changes per hour and at a slight negative pressure. Chamber supply air was provided from a HEPA-and charcoal-filtered, temperature and humidity-controlled source. Treatment of exhaust air consisted of charcoal and HEPA filtration. Animals were individually housed in stainless steel wire-mesh caging suspended over cage boards during the exposures. Food and water were withheld during the exposure period. Animals were housed in an animal colony room during non-exposure hours.

Temperature, relative humidity, ventilation rate, and negative pressure within each chamber were continually monitored and recorded at approximately 45-minute intervals during the 6-hour exposure period. The mean temperature and mean relative humidity were to be between 18°C to 26°C and 30% to 70%, respectively. Oxygen content was measured during the method development phase and was at least 19% for all chambers.

Vapors of MiAK were generated using a glass-bead column-type vaporization system. The column for each test substance chamber was filled with various sized glass beads and heated to approximately 160°C using an Omega® heat tape controlled by an Omega® PID temperature controller and process monitor and J-type thermocouple. An FMI Lab Pump was used to meter liquid test substance from the bulk test substance container to the top of the bead column while a pressure regulator and rotameter-type flowmeter were used to meter nitrogen to the bottom of the column. Vaporization occurred as the test substance flowed through the column. The resulting vapor was piped into the chamber inlet where the concentration was reduced to the desired level by mixing with the chamber ventilation air.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyzed exposure concentrations were determined at approximately 45-minute intervals using a gas chromatograph (GC) under the control of the WINH data acquisition software application. Samples were collected from the approximate animal-breathing zone of the inhalation exposure chamber via heated stainless steel sampling lines. Test atmosphere samples were collected automatically using a heated external multi-position valve. For each sample, the chromatograph was displayed and the area under the sample peak was calculated and stored. The concentration in parts per million (ppm) was calculated using a ln-quadratic equation based on the GC calibration curve. Spatial homogeneity of exposure concentrations within each test substance exposure chamber was evaluated during the method development phase of the study by comparison of analyzed concentrations at 4 test locations to concentrations at a reference location. Spatial homogeneity was found to be adequate for each chamber. The variability during the homogeneity was acceptable for each test substance chamber.
Details on mating procedure:


At the conclusion of the acclimation period, all available females were weighed and examined in detail for physical abnormalities. At the discretion of the Study Director, each animal judged to be in good health and meeting acceptable body weight requirements was placed in a suspended wire-mesh cage with a resident male from the same strain and source for breeding. Resident males were untreated, sexually mature rats utilized exclusively for breeding. These rats were maintained under similar laboratory conditions as the females. A breeding record containing the male and female identification numbers and the dates of cohabitation was maintained. The selected females were approximately 13 weeks old when paired for breeding.

Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage and verified by a second biologist. Each mating pair was examined daily. The day on which evidence of mating was identified was termed gestation day 0 and the animals were separated.

The experimental design consisted of 3 test substance-treated groups and 1 control group, composed of 25 rats per group. The bred females were assigned to groups using a WTDMS™ computer program which randomized the animals based on stratification of the gestation day 0 body weights in a block design. Animals not assigned to study were transferred to the WIL Research stock colony or euthanized by carbon dioxide inhalation and discarded. Body weight values ranged from 228 g to 305 g on gestation day 0.

Duration of treatment / exposure:
14 days
Frequency of treatment:
daily
Duration of test:
6 hours per day
No. of animals per sex per dose:
25 per group
Control animals:
yes, concurrent vehicle
Maternal examinations:
Laparohysterectomies and macroscopic examinations were performed blind to treatmentgroup. All females were euthanized on gestation day 20 by carbon dioxide inhalation. The thoracic, abdominal, and pelvic cavities were opened by a ventral mid-line incision, and the contents were examined. In all instances, the postmortem findings were correlated with the antemortem comments, and any abnormalities were recorded.
Ovaries and uterine content:
The uterus and ovaries were then exposed and excised. The number of corpora lutea on each ovary was recorded. The trimmed uterus was weighed and opened, and the number and location of all fetuses, early and late resorptions, and the total number of implantation sites were recorded. The placentae were also examined. The individual uterine distribution of implantation sites was documented using the following procedure. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn.

Maternal tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as indicated by the gross findings. Representative sections of corresponding organs from a sufficient number of control animals were retained for comparison. In addition, the liver from each female was weighed and preserved in 10% neutral-buffered formalin for possible future histopathologic examination. The carcass of each female was then discarded.

Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss.

Fetal examinations:



Fetal examinations were performed blind to treatment group. Each viable fetus was examined externally, individually sexed, weighed, euthanized by a subcutaneous injection of sodium pentobarbital in the scapular region (if necessary), and tagged for identification. Fetal tags contained the WIL Research study number, the female number, and the fetus number. The detailed external examination of each fetus included, but was not limited to, an examination of the eyes, palate, and external orifices, and each finding was recorded. Crown-rump measurements and degrees of autolysis were recorded for late resorptions, a gross external examination was performed (if possible), and the tissues were discarded.

Each viable fetus was subjected to a visceral examination using a modification of the Stuckhardt and Poppe fresh dissection technique to include the heart and major blood vessels. The sex of each fetus was confirmed by internal examination. Fetal kidneys were examined and graded for renal papillae development. Heads from approximately one-half of the fetuses in each litter were placed in Bouin's fixative for subsequent soft-tissue examination by the Wilson sectioning technique. The heads from the remaining one-half of the fetuses were examined by a midcoronal slice. All carcasses were eviscerated and fixed in 100% ethyl alcohol.

Following fixation in alcohol, each fetus was macerated in potassium hydroxide and stained with Alizarin Red S and Alcian Blue. Fetuses were then examined for skeletal malformations and developmental variations.

External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence, representing slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life). The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison and calculating the number of affected fetuses in a litter on a proportional basis.

Statistics:


All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group. Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Data obtained from nongravid animals were excluded from statistical analyses. Where applicable, the litter was used as the experimental unit.

Mean maternal body weights (absolute and net), body weight changes (absolute and net), and food consumption, gravid uterine weights, liver weights, numbers of corpora lutea, implantation sites, and viable fetuses, and fetal body weights (separately by sex and combined) were subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-treated groups to the control group. Mean litter proportions (percent per litter) of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and fetal sex distribution), total fetal malformations and developmental variations (external, visceral, skeletal, and combined) and each particular external, visceral, and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test (Kruskal and Wallis, 1952) to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test (Dunn, 1964) was used to compare the test substance-treated groups to the control group.

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Decreased reation to loud stimuli (startle reflex)
Dermal irritation (if dermal study):
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:


A test substance-related, significant (p<0.01) lack of body weight gain was noted in the1500 ppm group following the initiation of exposure (gestation days 6-9), compared to a mean body weight gain in the control group. Mean body weight gain in this group was similar to the control group during gestation days 9-12. However, significantly (p<0.01) lower mean body weight gains were noted at 1500 ppm for the remainder of the exposure period (gestation days 12-15 and 15-20) and when the overall exposure period (gestation days 6-20) was evaluated, relative to the control group. As a result, mean body weights in this group were significantly (p<0.05 or p<0.01) lower (up to 9.0%) than the control group during gestation days 15-20. In addition, mean net body weight and net body weight gain in the 1500 ppm group were significantly (p<0.01) lower than the control group. The lower mean body weight gains noted during the later portion of the gestational period were in part due to a slightly lower (not statistically significant) mean gravid uterine weight that was secondary to the reduced fetal weights noted in this group. However, mean net body weight and net body weight gain were also lower indicating maternal toxicity was also noted in this group.


Mean food consumption, evaluated as g/animal/day and g/kg/day, in the 1500 ppm group was significantly (p<0.01) lower than the control group throughout the exposure period (gestation days 6-20). These lower food consumption values generally corresponded with body weight effects in this group and were considered test substance-related. In the 750 ppm group, significantly (p<0.05 or p<0.01) lower mean food consumption was noted during gestation days 6-7, 8-9, 19-20, 6-9, 9-12, and/or 6-20. However, these lower food consumption values were slight and occurred in the absence of body weight effects in this group. Therefore, these lower values were considered to be treatment- related, but not adverse.



Key result
Dose descriptor:
NOAEC
Effect level:
379 ppm (analytical)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOAEC
Effect level:
751 ppm (analytical)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:


External malformations were noted in 0(0), 1(1), 0(0), and 2(2) fetuses (litters) in the control, 380, 750, and 1500 ppm groups, respectively. In the 1500 ppm group, fetus no. 33764-13 was noted with exencephaly without open eyelids (bilateral; skeletally, the frontal and parietal bones, bilateral, and the interparietal and supraoccipital bones were absent), omphalocele (several loops of the intestine protruded through an opening in the umbilicus, remnants of a membranous sac), cleft lip (upper left; skeletal confirmation revealed small left premaxilla bone, small and misshapen left maxilla bone, and left nasal bone smaller than normal), and anophthalmia (skeletally, the orbits were smaller than normal, bilateral) and fetus no. 33815-08 was noted with localized fetal edema (thorax) and anophthalmia (right). These external malformations occurred infrequently, affecting only 2 litters, and the mean litter proportions of these findings were within the range of values in the WIL historical control data. Furthermore, the fetuses in which these malformations were noted had fetal weights of 1.7 g and 1.8 g, respectively; malformations are not uncommon in fetuses with severely reduced weights. Therefore, these external malformations were not considered test substance-related. In addition, fetus no. 33791-02 in the 380 ppm group was noted with polydactyly (extra digit, left hindpaw; skeletally, distal phalanx ossified); however, this finding did not occur in an exposure-related manner and was not considered test substance-related. There were no external developmental variations noted at any dosage level.

Visceral Malformations and Variations

There were no visceral malformations noted at any dosage level. Visceral developmental variations were limited to 2(2), 1(1), 4(4), and 1(1) fetuses (litters) in the control, 380, 750, and 1500 ppm groups, respectively and consisted of major blood vessel variation, pale spleen, and distended ureter(s). These visceral variations were not observed in an exposure-related manner, were noted at similar frequencies in the concurrent control group, and/or the mean litter proportions of these findings were within the ranges of values in the WIL historical control data. Therefore, no relationship to the test substance was evident.

Renal papilla(e) that were not fully developed (Woo and Hoar Grade 1) was observed in 1 and 3 fetuses in the control and 750 ppm groups, respectively. This finding was not classified as either a malformation or developmental variation, was not included on the summary tables, and was not considered to be test substance-related because it occurred infrequently and in a manner that was not exposure-related.

Skeletal Malformations and Variations

No test substance-related skeletal developmental malformations were noted at any exposure level. Skeletal malformations were noted in 1 fetus in each of the 750 and 1500 ppm groups. Fetus no. 33815-08 in the 1500 ppm group was noted with costal cartilage anomaly (right costal cartilage nos. 1 and 2 fused and associated with sternum in normal no. 2 position; no right costal cartilage associated with sternum in normal no. 1 position), rib anomaly (right rib nos. 3 and 4 fused, medial through distal; right costal cartilage nos. 3 and 4 associated with sternum in normal no. 3 and 4 positions, respectively), and vertebral anomaly without associated rib anomaly (right cervical arch no. 6 absent; left cervical arch nos. 5 and 6 fused; right cervical arch nos. 1 through 3 small and misshapen). Fetus no. 33815-08 also had localized fetal edema and anophthalmia (see Section 6.8.1.). In addition, fetus no. 33811-15 in the 750 ppm group was noted with costal cartilage anomaly (left costal cartilage no. 1 fused to costal cartilage arising from 7th cervical rib and associated with sternum in normal no. 1 position). These findings were limited to single fetuses, occurred in the presence of an unusually low fetal body weight, and/or the mean litter proportions were within the range of values in the WIL historical control data. Therefore, these findings were not considered test substance-related.

Skeletal developmental variations noted in the treated groups consisted of 14th rudimentary rib(s), cervical centrum no. 1 ossified, 7th cervical rib(s), sternebra(e) nos. 5 and/or 6 unossified, hyoid unossified, sternebra(e) malaligned (slight or moderate), 27 presacral vertebrae, sternebra(e) nos. 1, 2, 3, and/or 4 unossified, reduced ossification of the vertebral arches, reduced ossification of the 13th rib(s), and pubis unossified. These findings occurred infrequently, were noted similarly in the concurrent control group, and/or were noted in a manner that was not exposure-related. Furthermore, when evaluated on a mean litter proportion basis, the values were within the range of values in the WIL historical control data and differences from the control group were not statistically significant. Therefore, these skeletal developmental variations were not considered to be test substance-related.

Summery of External, Visceral and Skeletal Examinations.

The numbers of fetuses (litters) available for morphological evaluation were 346(24),

350(24), 386(25), and 348(24) in the control, 380, 750, and 1500 ppm groups, respectively. Malformations were observed in 0(0), 1(1), 1(1), and 2(2) fetuses (litters) in these same respective exposure groups and were considered spontaneous in origin. External and skeletal malformations in the 1500 ppm group were limited to 1 or 2 fetuses with severely reduced fetal weights. The findings noted in the 380 and 750 ppm groups
did not occur in an exposure-related manner and were limited to single fetuses. In
addition, the mean litter proportions of all malformations and developmental variations
were not statistically different from the concurrent control group and/or were within the
range of values in the WIL historical control data. Therefore, no fetal malformations or
developmental variations were attributed to test substance exposure
Key result
Dose descriptor:
NOAEC
Effect level:
750 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
changes in litter size and weights
Key result
Abnormalities:
no effects observed
Developmental effects observed:
not specified
Conclusions:
In this study, clinical findings, lower mean body weights and mean body weight gains,and corresponding reduced mean food consumption were noted for females in the 1500 ppm group. In addition, reduced reactivity to a noise stimulus was noted for females at 750 and 1500 ppm. Therefore, the no-observed-adverse-effect level (NOAEL) for maternal toxicity was considered to be 380 ppm. Based on reduced fetal weights in the 1500 ppm group, 750 ppm was considered to be the NOAEL for embryo/fetal development when MiAK was administered by full body inhalation to bred Crl:CD(SD) rats.
Executive summary:

Methyl isoamyl ketone (MiAK), was administered via whole body inhalation exposure to 3 groups of 25 bred female Crl:CD(SD) rats for 6 hours daily from gestation days 6 through 19. Target exposure levels were 380, 750, and 1500 ppm, corresponding to mean measured exposure concentrations of 379, 751, and 1495 ppm, respectively. A concurrent control group composed of 25 bred females was exposed to filtered air on a comparable regimen. The females were approximately 14 weeks of age at the initiation of exposure. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. On gestation day 20, a laparohysterectomy was performed on each female and the liver was weighed. The liver and any tissues with gross lesions were preserved for possible future histopathologic examination. The uteri, placentae, and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations. All females survived to the scheduled euthanasia. In the 1500 ppm group, test substance-related hair loss on the hindlimbs, rump, and abdominal, thoracic, and/or urogenital areas was noted at the daily examinations and/or at the scheduled necropsy and clear material around the mouth was noted approximately 1 hour following exposure. In addition, test substance-related reduced reactivity to a noise stimulus near the end of exposure was noted for females at 750 and 1500 ppm compared to the control group. In the 1500 ppm group, test substance-related lower mean body weight gains and food consumption were noted generally throughout the exposure period (gestation days 6-20). In addition, mean net body weight and net body weight gain at 1500 ppm were lower than the control group. Test substance-related lower mean food consumption was noted in the 750 ppm group sporadically throughout the exposure period; these reductions did not correspond with effects on mean body weight and were not considered adverse. Mean body weights, body weight gains, and food consumption at 380 ppm and mean net body weights and net body weight gains at 380 and 750 ppm were unaffected by test substance exposure. No test substance-related macroscopic findings were noted at 380 and 750 ppm and no effects on mean liver weights were noted at 380, 750, and 1500 ppm. Test substance-related lower mean fetal weights were noted in the 1500 ppm group when compared with the control group. As a result, mean gravid uterine weight in this group was lower than the control group. Intrauterine growth and mean gravid uterine weights at 380 and 750 ppm and intrauterine survival at 380, 750, and 1500 ppm were unaffected by test substance administration. No test substance-related effects on fetal morphology were noted at any dosage level.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
5 834.36 mg/m³
Study duration:
subchronic
Species:
rabbit
Quality of whole database:
The study report does not give values in mg/m3; it gives a NOAEC value in ppm. That value was determined by (MW 114.12)*(1250 PPM NOAEC)/24.45 = mg/m3
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Methyl isoamyl ketone (MiAK) was administered via whole-body inhalation exposure for 6 hours per day to 3 groups of 12 time-mated female New Zealand White [Hra:(NZW) SPF] rabbits from Gestation Days 7–28. Target exposure concentrations were 500, 1250, and 2500 ppm. Overall mean analyzed exposure concentrations were 508, 1257, and 2493 ppm for Phase I and 499, 1262, and 2497 ppm for Phase II for the 500, 1250, and 2500 ppm groups, respectively. All females survived to the scheduled necropsy. Test substance-related increased incidences of decreased defecation were noted for 12 of 24 females in the 2500 ppm group at the daily examinations. At the midpoint of exposure, end of the daily exposure, and/or postexposure observations, test substance-related, adverse general irritation-related findings of partial closure of the eyes and/or clear material around the mouth and/or nose were noted for females in the 2500 ppm group. Clear material around the nose was also noted sporadically for 7 of 24 females in the 1250 ppm group at the midpoint of exposure; however, this finding was transient and not observed at the postdosing observation, and therefore was not considered adverse. No other test substance-related clinical observations were noted at any exposure level. Test substance-related lower mean body weight gains or body weight losses, with correspondingly reduced mean food consumption, were noted in the 2500 ppm group during Gestation Days 7–10, 10–13, and 13–20, resulting in a lower mean body weight gain when the entire exposure period (Gestation Days 7–29) was evaluated compared to the control group. These decreases in body weight gain generally correlated with an approximate 30% decrease in food consumption during this time period and were considered adverse. Mean gravid uterine weights, mean net body weights and net body weight changes in the 500, 1250, and 2500 ppm groups were unaffected by test substance exposure. No test substance-related maternal macroscopic findings were noted at the scheduled necropsy at any exposure level. Intrauterine growth and survival were unaffected by maternal test substance exposure. Heart and great vessel visceral malformations were noted in 3 fetuses from different litters within the 2500 ppm group, including retroesophageal aortic arch, Tetralogy of Fallot, an interventricular septal defect, and bulbous aorta, were considered test substance-related and adverse. There were no fetal skeletal malformations noted in the 2500 ppm group and fetal morphology was unaffected by test substance exposure in the 500 and 1250 ppm groups. Based on the adverse clinical findings of eye, nasal, and oral irritation, and adverse effects on maternal food consumption with correspondingly lower mean body weight gain during Gestation Days 7 through 20 in the 2500 ppm group, the no-observed-adverse-effect level (NOAEL) for maternal toxicity was 1250 ppm when methyl isoamyl ketone was administered by whole-body inhalation exposure to time-mated New Zealand White rabbits. Based on heart and great vessel visceral malformations at the 2500 ppm exposure level, 1250 ppm was the NOAEL for embryo/fetal developmental toxicity.

Justification for classification or non-classification

In a rabbit pre-natal developmental study by inhalation, there were 3 instances of rare great vessel malformations observed in the high dose (2 malformations) and mid dose (1 malformation). The LR feels that this is sufficient for category 2 classification (Repro Cat. 2).