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REACH

5-methylhexan-2-one

EC number: 203-737-8 | CAS number: 110-12-3

Life Cycle description
Uses advised against

Toxicological information

Specific investigations: other studies

Currently viewing:

Administrative data

Endpoint:: specific investigations: other studies
Remarks:: Lactational transfer
Type of information:: experimental study
Adequacy of study:: key study
Study period:: 2017
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Justification for type of information:: The study was conducted as part of an OECD 443 extended 1-generation study to demonstrate that the test article or its metabolites can be transferred to the offspring via milk.

Cross-reference

Reason / purpose for cross-reference:: reference to other study

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 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 patterns of the registered substance in coatings, exposure-based waiving is not possible.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

GLP compliance:

yes

Limit test:

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)
Physical Description: Clear, colorless liquid
Storage Conditions: Kept in a controlled temperature area set to maintain 18 °C to 24 °C

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 or test system 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 gas-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 60 ºC) 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 study, 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):

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.

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:

Clinical signs:

no effects observed

Description (incidence and severity):

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

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):

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):

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Reproductive performance:

no effects observed

Description (incidence and severity):

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:

Clinical signs:

no effects observed

Description (incidence and severity):

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

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):

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Sexual maturation:

no effects observed

Description (incidence and severity):

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

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):

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):

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):

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

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

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:

Effect level:

750 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Reproductive effects observed:

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 HC^a Mean (Range)
Parameter	0	380	750	1500	CRL HC^a Mean (Range)
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 HC^a Mean (Range)
Parameter	0	380	750	1500	CRL HC^a Mean (Range)
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
Exposure (ppm):	0	380	750	1500	0	380	750	1500
Adrenal Cortex^a	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
Exposure (ppm):	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
Exposure (ppm):	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
Exposure (ppm):	0	380	750	1500	0	380	750	1500
Adrenal Cortex^a	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
Exposure (ppm):	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. 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.

Data source

Reference

Reference Type:: study report
Title:: Unnamed
Year:: 2019

Materials and methods

Test guideline

Qualifier:: no guideline required

Principles of method if other than guideline:: The principle of the study is to conduct an analysis of lactating animals exposed to the test article to determine if the test article can be found.
GLP compliance:: yes
Type of method:: in vivo
Endpoint addressed:: other: Lactational transfer

Test material

Test material information

Details on test material:: no data

Specific details on test material used for the study:: Identification: Methyl isoamyl ketone (also referred to as MiAK; CAS No. 110 12-3)
Physical Description: Clear, colorless liquid
Storage Conditions: Kept in a controlled temperature area set to maintain 18°C to 24°C
Supplier: Eastman Chemical Company

Test animals

Species:: rat
Strain:: Sprague-Dawley
Sex:: female
Details on test animals or test system and environmental conditions:: Housing
On arrival, the F0 animals were group housed (up to 3 animals of the same sex) until cohabitation. During cohabitation, the 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 (Bed O’ Cobs®) equipped with an automatic watering valve.
Animals were separated during designated procedures/activities. Each cage was clearly labeled with a color-coded cage card indicating study, group, animal number(s), 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. A 12-hour light/12-hour dark cycle was maintained, except when interrupted for designated procedures. Ten or greater air changes per hour with 100 % fresh air (no air recirculation) were maintained in the animal rooms.

Food
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. The feed used on this study needed to have a genistein-equivalent (aglycone) content of less than 300 ppm. PMI Nutrition International, LLC Certified Rodent LabDiet® 5K96 Advanced Protocol® Verified Casein Diet 10 IF consistently analyzed at less than 10.0 ppm total isoflavones (aglycone equivalents of genistein, daidzein, and glycitein).
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.

Water
Municipal tap water after treatment by reverse osmosis was freely available to each animal via an automatic watering system, except during designated procedures.
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.

Animal Enrichment
Animals were socially housed for psychological/environmental enrichment and were provided with environmental enrichment as appropriate to aid in maintaining the animals’ oral health, except during designated procedures.

Veterinary Care
Veterinary care was available throughout the course of the study; however, no examinations or treatments were required.

Administration / exposure

Route of administration:: inhalation: vapour
Vehicle:: air
Details on exposure:: Filtered air or MiAK vapor was administered as 6-hour, daily whole-body inhalation exposures, 7 days per week, at approximately the same time each day, according to the schedule below.
F0 females were exposed to either filtered air or vaporized MiAK concurrently with, and in the same exposure chambers, as animals on a concurrent extended one-generation reproductive toxicity study in Sprague Dawley rats.1 For all females, exposures were conducted for a minimum of 14 days prior to mating and continuing throughout the mating period. Following completion of the mating period, females (with no evidence of mating) continued to be exposed daily until 1 day prior to scheduled euthanasia. Males were not exposed to the test substance, and were only used for breeding purposes.
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 Lactation Day 13, inclusively. During lactation, dams were separated from their litters during the exposure period.
Analytical verification of doses or concentrations:: yes
Duration of treatment / exposure:: 14 days pre-mating, maximum 2 weeks for mating, followed by gestation and to sacrifice at lactation day 13
Frequency of treatment:: daily
Post exposure period:: none

Doses / concentrationsopen all close all

Doses / concentrations 1

Dose / conc.:: 0 ppm

Doses / concentrations 2

Dose / conc.:: 1 500 ppm

Doses / concentrations 3

Dose / conc.:: 750 ppm

Doses / concentrations 4

Dose / conc.:: 380 ppm

No. of animals per sex per dose:: 12
Control animals:: yes, concurrent vehicle
Details on study design:: The study design involved exposing female rats to the test article beginning prior to gestation, throughout pregnancy and into lactation, with an analysis of milk and pup plasma for the test article.

Examinations

Examinations:: 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 (animals visible in exposure chamber only), at the end of exposure, and 1–2 hours postexposure (see Appendix 1 - Study Protocol and Deviations).
During social housing, some observations (e.g., fecal observations) may not have been attributable to an individual animal.

Body Weights
F0 females were weighed individually weekly. 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, and 13.

Food Consumption
Food consumption was quantitatively measured weekly throughout the study period. Once evidence of mating was observed, female food consumption was recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and on Lactation Days 1, 4, 7, 10, and 13.
Positive control:: No

Results and discussion

Details on results:: Whole-body inhalation of MiAK to female Crl:CD(SD) rats for 6 hours daily for at least 14 days prior to mating and continuing throughout the mating, gestation, and lactation periods until Lactation Day 13 resulted in quantifiable levels of MiAK in maternal milk and plasma that increased in a slightly greater than dose-proportional manner from 380 ppm to 750 ppm and roughly increased 2x from 750 ppm to 1500 ppm. For all groups, mean MiAK concentrations in milk were approximately 16-fold higher than in plasma. Even though plasma MiAK concentrations in pups were BLQ (<1000 ng/mL) at all exposure concentrations, the maternal plasma and milk concentration data demonstrate that the offspring were exposed to MiAK during both the gestation (in utero) and neonatal periods (through nursing).

Any other information on results incl. tables

Plasma MIAK Concentrations on Lactation/Postnatal Day 13 (ng/mL)

Target Exposure Concentration (ppm)		Lactation Day 13	Postnatal Day 13
		Dams	Pups
0	Mean	All BLQ < (1000)	All BLQ < (1000)
	S.D.
380	Mean	483	All BLQ < (1000)
	S.D.	881
750	Mean	1540	All BLQ < (1000)
	S.D.	2170
1500	Mean	3100	All BLQ < (1000)
	S.D.	5840

Milk MIAK Concentrations on Lactation Day 13 (ng/mL)

Target Exposure Concentration (ppm)		LD 13
Target Exposure Concentration (ppm)		Females
0	Mean	All BLQ<(1000)
0	S.D.	All BLQ<(1000)
380	Mean	7780
380	S.D.	6610
750	Mean	24,400
750	S.D.	19,200
1500	Mean	51,700
1500	S.D.	55,900

Applicant's summary and conclusion

Conclusions:

Whole-body inhalation of MIAK to female Crl:CD(SD) rats for 6 hours daily for at least 14 days prior to mating and continuing throughout the mating, gestation, and lactation periods until Lactation Day 13 resulted in quantifiable levels of MIAK in maternal milk and plasma that increased in a slightly greater than dose-proportional manner from 380 ppm to 750 ppm and roughly increased 2x from 750 ppm to 1500 ppm. For all groups, mean MIAK concentrations in milk were approximately 16-fold higher than in plasma. Even though plasma MIAK concentrations in pups were BLQ (<1000 ng/mL) at all exposure concentrations, the maternal plasma and milk concentration data demonstrate that the offspring were exposed to MiAK during both the gestation (in utero) and neonatal periods (through nursing).

Executive summary:

The objective of this study was to detect the potential of the test substance, methyl isoamyl ketone (hereafter referred to as MIAK), to be secreted in maternal milk and to assess exposure of the pups via assessment of MIAK in maternal and pup plasma. F₀females were exposed via whole-body inhalation for 6 hours daily for at least 14 days prior to mating and continuing throughout the mating, gestation, and lactation periods until Lactation Day 13, with the exception that exposure was suspended from Gestation Day 21 through Lactation Day 4. A concurrent control group was exposed to humidified, filtered air on a comparable regimen. Overall mean analyzed exposure concentrations were 380, 748, and 1500 ppm for Groups 2, 3, and 4, respectively.

The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, food consumption, reproductive performance, parturition, litter viability and survival, and exposure assessment in milk and plasma.

All F₀females in the control, 380, 750, and 1500 ppm groups survived to the scheduled necropsy. Test substance-related increased incidences of red material around the nose in the 750 and 1500 ppm groups and red material around the mouth in the 1500 ppm group were noted at the end of exposure and 1–2 hours postexposure. In addition, increased incidences of hair loss on various body surfaces were noted for females in the 1500 ppm group at the daily examinations. No other test substance‑related clinical findings were noted at the daily examinations or at the midpoint of exposure, end of exposure, and 1–2 hours postexposure.

Test substance-related lower mean body weight gains, with corresponding effects on mean food consumption, were generally noted for females in the 1500 ppm group throughout the pre‑mating, gestation, and lactation periods, resulting in mean body weights that were lower (up to 5.9%) than the control group. In addition, a lower mean body weight gain was noted for females in the 750 ppm group during Study Days 7–13, without corresponding effects on mean food consumption or mean body weights. No other effects on mean body weights, body weight gains, and food consumption were noted at any exposure concentration.

No test substance-related effects were noted on reproductive performance (female mating, fertility, and conception), gestation length, or the process of parturition at any exposure concentration.

Survival, clinical condition, and growth of the F₁generation was unaffected by test substance exposure at all exposure concentrations.

In conclusion, whole-body inhalation of MIAK to female Crl:CD(SD) rats for 6 hours daily for at least 14 days prior to mating and continuing throughout the mating, gestation, and lactation periods until Lactation Day 13 resulted in quantifiable levels of MIAK in maternal milk and plasma that increased in a slightly greater than dose-proportional manner from 380 ppm to 750 ppm and roughly increased 2x from 750 ppm to 1500 ppm. For all groups, mean MIAK concentrations in milk were approximately 16-fold higher than in plasma. Even though plasma MIAK concentrations in pups were BLQ (<1000 ng/mL) at all exposure concentrations, the maternal plasma and milk concentration data demonstrate that the offspring were exposed to MIAK during both the gestation (in utero) and neonatal periods (through nursing).

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
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