(2E)-1,1,1,4,4,4-hexafluoro-2-butene

Toxicity to reproduction

Administrative data

Endpoint:

two-generation reproductive toxicity

Remarks:

Inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 Mar 2017 - 23 Aug 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The developmental toxicity study was performed to comply with product stewardship as well as other global regulatory requirements.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

Not specified

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Version / remarks:

Not specified

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-​Butene, 1,​1,​1,​4,​4,​4-​hexafluoro-​, (2E)​-

Cas Number:

66711-86-2

IUPAC Name:

2-​Butene, 1,​1,​1,​4,​4,​4-​hexafluoro-​, (2E)​-

Test material form:

gas

Remarks:

colorless

Specific details on test material used for the study:

Storage conditions: controlled temperature area set to maintain 18°C to 24°C

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

Details on species / strain selection:

The Crl:WI(Han) rat is recognized as appropriate for reproduction studies. The testing laboratory, Charles River Ashland, has reproductive historical data for the Crl:WI(Han) rat. Previous general toxicity studies were conducted in this strain of rat. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC (males) or Kingston, NY (females), USA
- Age at study initiation: 6 wks (P0), PND 28 (P1)
- Weight at study initiation: 182-249g (males, P0), females: 147-206g (females, P0); 68-110g (males, P1), females: 40-102g (females, P1)
- Fasting period before study: No
- Housing: On arrival (P0) or following selection (P1), animals were group housed (2 to 3 animals of the same sex). 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 equipped with an automatic watering valve, except during inhalation exposures. Animals were separated during designated procedures/activities.
- Diet: PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 (ad libitum)
- Water: Municipal tap water after treatment by reverse osmosis (ad libitum)
- Acclimation period: 14 days

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.Periodic analysis of the water is performed, and results of these analyses are on file at the Testing Facility.

ENVIRONMENTAL CONDITIONS (target)
- Temperature (°C): 20-26
- Humidity (%): 30-70
- Air changes (per hr): 10 or more
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 28 Mar 2017 To: 23 Aug 2018

Administration / exposure

Route of administration:

inhalation: gas

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure atmospheres generated by releasing the neat test substance in gas form from the original cylinder and mixing it with sufficient humidified air to achieve the desired target concentrations
- Temperature, humidity, pressure in air chamber: The mean temperature and relative humidity were to be between 19°C and 25°C and 30% and 70%, respectively; Chamber at a slight negative pressure; Oxygen content of the exposure atmospheres estimated at 20.9% for all groups (measured during the method development phase)
- Air flow rate: The chambers were operated under dynamic conditions, and with a minimum of 12 air changes per hour

TEST ATMOSPHERE
- Brief description of analytical method used: Analyzed exposure concentrations were determined at approximately 45-minute intervals using a gas chromatograph (GC).
- Samples taken from breathing zone: yes, samples were collected from the approximate animal-breathing zone of the exposure chamber

Details on mating procedure:

After a minimum of 70 days of exposure, the animals were paired on a 1:1 basis within each group, avoiding sibling mating. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage. Vaginal lavages were performed daily during the mating period until evidence of mating was observed. If evidence of mating was not apparent after 14 days or 3 estrous periods, the animals were separated, with no further opportunity for mating. Each generation was mated once to produce 1 litter/dam (the F1 and F2 litters). Animals cohabited over a 12-hour dark cycle were considered to have been paired for 1 day.

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. Homogeneity of exposure concentrations 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. Temporal stability was evaluated using the concentrations from the reference location.

Duration of treatment / exposure:

6 hours (at approximately the same time each day) for a minimum of 70 consecutive days prior to mating and continuing throughout mating, gestation, and lactation, through the day prior to euthanasia. Exposure was suspended from Gestation Day 21 through Lactation Day 4.

Frequency of treatment:

Once daily

Details on study schedule:

- P0 and P1 females were exposed via whole body inhalation for 6 hours daily for a minimum of 70 consecutive days prior to mating and continuing throughout mating, gestation, and lactation, through the day prior to euthanasia. Exposure was suspended from gestation day 21 through lactation day 4. The offspring selected to become the P1 parental generation began exposure following weaning (PND 28).

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5 000 ppm (nominal)

Remarks:

Analytical concentrations: 4994 ppm (P0) and 5000 ppm (P1)

Doses / concentrations 2

Dose / conc.:

7 500 ppm (nominal)

Remarks:

Analytical concentrations: 7490 ppm (P0) and 7455 ppm (P1)

Doses / concentrations 3

Dose / conc.:

12 500 ppm (nominal)

Remarks:

Analytical concentrations: 12,420 ppm (P0) and 12,433 ppm (P1)

No. of animals per sex per dose:

30

Control animals:

yes

Details on study design:

- Dose selection rationale:
The exposure levels were based on the results of previous repeated dose inhalation toxicity studies in rats up to 90-days as well as a prenatal developmental toxicity study in rats. In these studies, animals were exposed to the test substance for 6 hours per day for either 5 or 7 days per week. There was limited male mortality at 15000 ppm; however, all females survived throughout the studies. There were some transient clinical signs of tremors, transient body weight and food consumption reductions, reduced fetal and placental weights, and fetal retardation in bone ossification noted at 15000 ppm. Therefore, 12500 ppm was the highest exposure concentration in the current study. The route of administration was whole body inhalation exposure because the intended use of the test substance indicates that this would be a likely route of human exposure.

Positive control:

No

Examinations

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily prior to exposure. Observations were also recorded at the approximate midpoint of exposure and approximately 1 hour following the completion of the 6-hour exposure. Detailed physical examination was performed weekly prior to exposure. Throughout the study, animals were observed for general health/mortality and moribundity twice daily, once in the morning and once in the afternoon

BODY WEIGHT: Yes
- Time schedule for examinations: weekly throughout the study and prior to the scheduled necropsy. 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, 11, 14, 17, 21, 24, and 28. Terminal body weights were not collected from animals found dead or euthanized moribund.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- 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 Lactation Days 1, 4, 7, 11, 14, 17, 21, 24, and 28. Food efficiency (body weight gained as a percentage of food consumed) was calculated weekly.

Oestrous cyclicity (parental animals):

For all females (P0), vaginal lavages were performed daily for 21 days prior to the initiation of the mating period and continuing until evidence of mating was observed or until the end of the mating period. The slides were microscopically examined to determine the stage of the estrous cycle. 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] for 21 consecutive days before cohabitation and until the detection of evidence of mating). Estrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in a subsequent cycle. The cycle during which evidence of mating was observed for a given animal was not included in the individual mean estrous cycle length calculation. Vaginal lavages were also performed on the day of necropsy to determine the stage of the estrous cycle.

Sperm parameters (parental animals):

Immediately upon euthanasia, sperm samples were collected from the distal region of the right cauda epididymis for determination of sperm motility (P0 animals). 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:
Percent Motile (or Progressively Motile) Sperm = (Number of Motile (or Progressively Motile) Sperm/Total Number of Sperm Counted) x 100.
Sperm morphology was evaluated by light microscopy. 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. A minimum of 200 cells, if possible, or up to 20 fields were counted for each sample. The sperm production rate was calculated as follows:
Sperm Production Rate = No. of Sperm Per Gram of Tissue/ 6.1 Days (the rate of turnover of the germinal epithelium).

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Eight pups from each litter of equal sex distribution (if possible) will be randomly selected. For litters consisting of fewer than eight pups, adjustments for litter size will not be performed. Following selection, the nonselected PND 4 pups were euthanized by an intraperitoneal injection of sodium pentobarbital and discarded.

PARAMETERS EXAMINED (F1 and F2 Litters)
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., drowning, mechanical injury, inadvertent removal from room, early group termination, or 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 observations were performed on PND 1, 4, 7, 14, 21, 24, and 28. Pups were individually sexed on PND 0, 4, 14, 21, and 28. Pups were weighed individually on PND 1, 4, 7, 11, 14, 17, 21, 24, and 28.

Each male pup was observed for balanopreputial separation beginning on PND 35. The age at which balanopreputial separation was first observed was recorded for each pup. Examination of the males continued daily until balanopreputial separation was present. Body weights were recorded at the age of attainment of this landmark.

Each female pup was observed for vaginal perforation beginning on PND 25. The age at which vaginal patency was first observed was recorded for each pup. Examination of the females continued daily until vaginal patency was present. Body weights were recorded at the age of attainment of this landmark.

GROSS EXAMINATION OF DEAD PUPS:
Necropsy was conducted for animals that died on study, and specified tissues were retained. Intact offspring that were found dead during PND 0–4 were necropsied using a fresh dissection technique, which included examination of the heart and major vessels. Pups with external abnormalities that would warrant further skeletal examination were eviscerated and stained for subsequent skeletal evaluation. Findings were recorded as developmental variations or malformations, as appropriate. A gross necropsy was performed on any pup found dead after PND 4 and prior to weaning. Cannibalized pups were discarded without necropsy.

Postmortem examinations (parental animals):

All animals were subjected to a complete necropsy examination (P0 and P1), which included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal, and pelvic cavities, including viscera. 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. Numbers of corpora lutea were also recorded for females necropsied during gestation through Lactation Day 4.
Main organs were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals found dead or euthanized in poor condition or in extremis. Paired organs were weighed together. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated.
Pathological evaluation was performed by a board-certified veterinary pathologist. Tissues for microscopic examination as specified in the guidance were evaluated from all animals in the control and high-dose groups and from all animals found dead and euthanized in extremis. Gross lesions were examined from all animals (including low and mid-dose groups). The prostate gland from all F1 males was evaluated microscopically. In addition, reproductive organs of all animals suspected of reduced fertility were subjected to histopathological examination. The prostate (target organ) was examined from all F1 males in the low- and mid-dose groups.

Postmortem examinations (offspring):

Pups (F1 and F2) were euthanised on post natal day 21, all animals were subjected to a complete necropsy examination, with emphasis on developmental morphology and organs of the reproductive system. Brain, spleen and thymus were weighed at necropsy from 1 pup/sex/litter at the scheduled euthanasia. Gross lesions were collected and preserved in 10% neutral buffered formalin for possible future histopathological examination.

Statistics:

Each mean was presented with the standard deviation (S.D.) and the number of animals or cages (N) used to calculate the mean. Where applicable, the litter was used as the experimental unit. All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex. Parental mating, fertility, copulation, and conception indices were analyzed using the Chi-square test with Yates’ correction factor. Parental and offspring body weights and body weight changes, parental food consumption and food efficiency data, estrous cycle lengths, precoital intervals, gestation lengths, former implantation sites, live litter sizes, unaccounted-for sites, numbers of pups born, age and weight at attainment of developmental landmarks, absolute and relative organ weights, sperm production rates, epididymal and testicular sperm numbers, and ovarian primordial follicle counts were subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunnett's test was used to compare the test substance-treated groups to the control group. Mean litter proportions of postnatal survival and pup sexes at birth (percentage of males per litter), percentages of motile and progressively motile sperm, and percentages of sperm with normal morphology were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the nonparametric ANOVA revealed significant (p < 0.05) intergroup variance, Dunn’s test was used to compare the test substance-treated groups to the control group.

Reproductive indices:

Male (female) mating index (%) = (No. Of males (females) with evidence of mating (or confirmed pregnancy/ Total no. Of males (females) used for mating) x 100;
Male fertility index (%) = (No. Of males siring a litter/ Total no. Of males used for mating) x 100;
Male copulation index (%) = (No. Of males siring a litter/ No. Of males with evidence of mating (or females confirmed pregnant) x 100;
Female fertility index (%) = (No. Of females with confirmed pregnancy/ Total no. Of females used for mating) x 100;
Female conception index (%) = (No. Of females with confirmed pregnancy/ No. Of females with evidence of mating (or confirmed pregnancy)) x 100

Offspring viability indices:

Litter parameters were defined as follows:
Mean Live Litter Size = Total No. Viable Pups on PND 0 / No. Litters with Viable Pups on PND 0;
Postnatal Survival Between Birth and PND 0 or PND 4 (Pre-Selection) (% Per Litter) = (Sum of (Viable Pups Per Litter on PND 0 or PND 4 [Pre-Selection]/No. of Pups Born/Litter/ No. of Litters Per Group) x 100;
Postnatal Survival for All Other Intervals (% Per Litter) = (Sum of (Viable Pups Per Litter at End of Interval N/Viable Pups Per Litter at Start of Interval N)/ No. of Litters/Group) x 100
Where N = PND 0–1, 1–4 (Pre-Selection), 4 (Post-Selection)–7, 7–14, 14–21, 21–24, 24–28, or 4 (Post-Selection)–28

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the 12500-ppm group, test substance-related clinical observations of tremors were noted for males and females and clonic convulsions were noted for females at the midpoint of exposure. These findings were considered adverse. In addition, increased incidences of red material around the nose and red and clear material around the mouth were noted for F0 males and females in all test substance-exposed groups compared to the control group primarily at approximately 1 hour postexposure, generally in a dose-related manner. No other test substance-related clinical observations were noted during the F0 generation at the detailed physical examinations, daily examinations, or at the midpoint of exposure and 1 hour following exposure. Findings noted in the test substance-treated groups, including hair loss on various body surfaces, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.

Mortality:

mortality observed, treatment-related

Description (incidence):

One 12500ppm group male was euthanized in extremis on Study Day 121 due to clinical findings of head tilt, hypoactivity, unkempt appearance, red material around the nose and mouth, and yellow material on the urogenital area at approximately 1 hour postexposure on the day of euthanasia. In addition, one 12500-ppm group male was found dead on Study Day 126; clinical findings for this male included pale extremities, shallow respiration, and red material around the nose, forelimbs, and facial area at approximately 1 hour postexposure on the day of death. No gross findings were noted in the first male that died, and no histologic lesions indicating the cause of death were noted. Gross findings of a few white pinpoint areas in the left lung lobe were noted in the second male that died, but these were not correlated to a microscopic finding, and no cause of death was determined. Both of these animals were in the highest exposure group (12500ppm), so a test substance-related effect cannot be excluded. All other F0 parental animals in the control, 5000, 7500, and 12500ppm groups survived to the scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A test substance-related, statistically significantly lower mean body weight gain was noted for males in the 12500ppm group following the first week of exposure (Study Days 0–7). As a result, mean body weights in this group were statistically significantly lower (up to 6.4%) than the control group during Study Days 7–49. However, mean body weights and body weight gains in this group were similar to the control group throughout the remainder of the generation and when the entire premating period (Study Days 0–69) and entire generation (Study Days 0–132) were evaluated. Therefore, the initial effect on mean body weight gain was not considered adverse.
Mean body weight gains for females in the 12500ppm group were sporadically higher than the control group during the premating period (Study Days 0–69); differences from the control group were statistically significant during Study Days 35–42 and 42–49 and when the entire premating period was evaluated. As a result, mean body weights in this group were statistically significantly higher (up to 6.6%) than the control group during Study Days 42–69, and remained statistically significantly higher than the control group following the lactation period (Study Day 132). This correlated to a slight increase in mean food consumption, so a potential relationship to treatment cannot be dismissed; however, the magnitude of change was minimal and not considered adverse. No test substance-related effects on mean body weights or body weight gains were noted for males and females in the 5000 and 7500 ppm groups. The values in the test substance-treated groups were generally similar to the control group values for the pre-mating period (females) or the entire generation (males). Statistically significant differences from the control group were transient, discordant in direction of change, and/or did not occur in a dose-related manner.
Mean maternal body weights and body weight gains were unaffected by test substance exposure during gestation. Slightly higher mean body weights were noted in the 12500ppm group throughout gestation; the differences were statistically significant, on Gestation Days 17 and 20. These changes were attributed to the effects on body weights and body weight gains during the pre-mating period, as mean body weight gains and food consumption were similar to the control group throughout gestation. Statistically significantly lower mean body weight gains were noted for females in the 5000 and 7500 ppm group during Gestation Days 0–4; however, this did notoccur in a dose-related manner and were not considered test substance-related. Other differences between the control, 5000, 7500, and 12500 ppm groups were slight and not statistically significant.
Mean maternal body weights and body weight gains were unaffected by test substance exposure during lactation. Mean body weights in the 12500ppm group were 3.9% higher than the control group on Lactation Day 1. Mean body weights in this group remained higher (3.7% to 6.7%)
than the control group throughout the remainder of lactation, but were due the effects on body weights and body weight gains during the pre-mating period; differences from the control group were generally statistically significant. Other differences between the control, 5000, 7500, and 12500 ppm groups were slight and not statistically significant.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Statistically significantly lower mean food consumption and food efficiency were noted for males in the 12500ppm group following the first week of exposure (Days 0–7), corresponding to the lower mean body weight gain noted for these males during this period. This was considered test substance-related; however, this effect was transient, as mean food consumption and food efficiency were generally similar to the control group throughout the remainder of the premating and postmating periods. Therefore, this was not considered adverse. No other test substance-related effects were noted on food consumption or food efficiency for males at any exposure concentration. Statistically significantly higher mean food efficiency was noted for males in the 7500 and 12500 ppm groups during Days 63–69; however, this was transient and did not occur in a dose-related manner. During Days 126–132, statistically significantly higher mean food consumption was noted for males in the 5000 and 12500 ppm groups and statistically significantly higher mean food efficiency was noted for males in the 7500-ppm group; however, these differences were transient and were not considered test substance-related.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Mean food consumption and food efficiency for females in the 12500-ppm group were generally slightly higher than the control group during the pre-mating period (Study Days 0–69); differences from the control group were statistically significant during Study Days 28–35 and 42–49 (food consumption). These results corresponded to sporadically higher mean body weight gains for females in this group; therefore, a potential relationship to treatment cannot be dismissed, however, the magnitude of change was minimal and not considered adverse. No other test substance-related effects were noted on food consumption or food efficiency at any dosage concentration. Statistically significant differences from the control group included higher mean food consumption in the 5000 and 7500 ppm groups during Study Days 28–35 and higher mean food efficiency in the 7500-ppm group during Study Days 0–7; however, these results were transient and did not result in effects on mean body weight gain.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test substance-related histologic changes. Remaining histologic changes were considered to be incidental findings. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations. There were no microscopic findings in females that would be expected to result in reduced fertility. There were histologic changes that may have contributed to reduced fertility in some males; mild to moderate neutrophilic infiltrates in the prostate were noted in two 5000 ppm group males that had reduced fertility matings. There were no test substance related effects on the incidence or severity of this microscopic finding.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The mean lengths of estrous cycles in the exposed groups were similar to the control group value.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

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 concentration. Differences from the control group were slight and were not statistically significant.

Reproductive performance:

no effects observed

Description (incidence and severity):

F0 male and female reproductive parameters are presented in the table included below. 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, 2, 3, and 1 males in the control, 5000, 7500, and 12500 ppm groups, respectively, did not sire a litter. One, 2, 3, and 1 females in these same respective groups were determined to be nongravid. The mean numbers of days between pairing and coitus in the test substance-treated 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.

Details on results (P0)

No test substance-related effects were noted on mean gestation lengths or the process of parturition at any dosage level. Mean F0 gestation lengths in the test substance-treated groups were similar to the control group value. Differences were slight and were not statistically significant. The mean gestation lengths in the 5000, 7500, and 12500 ppm groups were 21.7, 21.5, and 21.8 days, respectively, compared to mean gestation lengths of 21.9 days in the concurrent control group and 21.7 days in the Charles River Ashland historical control data (Version 2.3). No signs of dystocia were noted at any exposure level.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 7 500 ppm (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical signs

mortality

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the 12500-ppm group, test substance-related clinical observations of tremors were noted for males and females at the midpoint of exposure. These findings were considered adverse. Tremors was also noted for one control on PND 76; however, this was believed to have been entered in error. In addition, increased incidences of red material around the nose and/or mouth were noted for F1 males in all test substance-exposed group and females in the 12500-ppm group compared to the control group, primarily at approximately 1 hour postexposure. No other test substance-related clinical observations were noted during the generation at the detailed physical examinations, daily examinations, or at the midpoint of exposure and 1 hour following exposure. Findings noted in the test substance-treated groups, including hair loss on various body surfaces, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

In the control group, three females were euthanized in extremis. Deaths were related to presence of adenocarcinoma (Mammary gland in salivary gland region) (one female, euthanized PND 147), or to uterine inflammation with bacteria (one other female, euthanized PND 130) and for one female cause of death could not be determined (euthanized PND 28). At 5000 ppm, one female was found dead on PND 39 with fractured calvarium/ brain hemorrhage. At high dose, one male was found dead on PND 125 and two other males were euthanized (on PND 98 and 135, respectively), cause of death could not be determined. All other F1 parental animals in the control, 5000, 7500, and 12500 ppm groups survived to the scheduled necropsy. Because there were equal number of unscheduled deaths in the 0 ppm and 12,500 ppm groups, and there was no clear cause of mortality found in the high dose animals, the deaths/moribundities were not attributed to the test substance.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A test substance-related lower mean body weight gain was noted for males and females in the 12500-ppm following the initiation of exposure (PND 28–35); the differences from the control group were statistically significant. Mean body weight gains for these animals were generally similar to or higher than the control group throughout the remainder of the generation, and there were no effects on mean body weights in this group; therefore, this initial effect on body weight gain was not considered adverse. Statistically significantly higher mean body weight gains were noted for males in the 12500 ppm group during PND 84–91 and 119–126 and for females in the 12500-ppm group during PND 35–42; however, these differences were transient, did not affect mean body weights, and were not considered test substance-related. Other differences from the control group were slight and not statistically significant. No test substance-related effects on mean body weights, body weight gains, and cumulative body weight gains were noted for males or females in the 5000 and 7500 ppm groups. A statistically significantly higher mean body weight gain was noted for females in the 7500 ppm group during PND 35–42; however, this was transient, did not affect mean body weights, and was not considered test substance-related. Other differences from the control group were slight and not statistically significant. Mean maternal body weights, body weight gains, and cumulative body weight gains for F1 females were unaffected by test substance exposure during gestation. Mean body weight gains in the 7500 and 12500 ppm groups were generally slightly higher than the control group during gestation; differences from the control group were statistically significant when the entire gestation period (Gestation Days 0–20) was evaluated. As a result, mean body weights in these groups were statistically significantly higher (6.7% and 8.8%, respectively) than the control group on Gestation Day 20. Similar increases in mean body weight gain and body weight were not noted for females in the F0 generation during the pre-mating period and were considered potentially related to test substance administration, but not adverse. Mean maternal body weights, body weight gains, and cumulative body weight gains for F1 females were unaffected by test substance exposure during lactation. Differences between the control, 5000, 7500, and 12500 ppm groups were slight and not statistically significant.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Test substance-related lower mean food consumption was noted for males and females in the 7500 and 12500 ppm group following the initiation of exposure (PND 28–35), corresponding to lower mean body weight gains for males and females in the 12500 ppm during this period. These effects on food consumption were transient, did not correspond to effects on mean body weight, and were not considered adverse. No other test substance-related effects were noted on food consumption or food efficiency. Statistically significantly higher mean food consumption or food efficiency was noted sporadically for males and females in the test substance-exposed groups compared to the control group; however, these differences were transient, did not occur in a dose-related manner, and/or did not correspond to effects on mean body weight gain and were not considered test substance-related.
Mean maternal food consumption, evaluated as g/animal/day, and food efficiency for P1 females were unaffected by test substance exposure during gestation. Higher mean food consumption was generally noted for females in all test substance-exposed group throughout gestation when compared to the control group, corresponding to higher mean body weight gains for females in the 7500 and 12500 ppm groups; differences from the control group were statistically significant during Gestation Days 17–20 (5000 ppm), 0–4 and 7–11 (7500 ppm), 7-11 and 14–17 (12500 ppm), and when the entire gestation period was evaluated (Gestation Days 0–20; all exposure concentrations). Similar increases in mean food consumption were not noted for females in the F0 generation during the pre-mating period and were considered potentially related to test substance administration, but not adverse.
Mean maternal food consumption, evaluated as g/animal/day, and food efficiency for P1 females were unaffected by test substance exposure during lactation. Statistically significantly higher mean food consumption was noted for females in the 7500 and 12500 ppm groups during Lactation Days 17–21; however, this was transient and did not affect mean body weight gains. Other differences between the control, 5000, 7500, and 12500 ppm groups were slight and not statistically significant.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related higher mean prostate weights (absolute and relative to brain weight) were noted in the 12500-ppm group males. Test substance-related higher mean liver weights (absolute) were noted in the 12500-ppm group females. Test substance-related lower mean thymus to final body weight were noted in the 7500 and 12500 ppm group females. Histology of the liver and thymus was not performed on most of the animals, so a histologic correlation could not be made. All of the weight differences were minimal to mild, and within historical control ranges, and so were therefore considered to be nonadverse.Some organ weight differences were statistically significant when compared to the control group but were considered to be a result of a test substance-related effect on final body weight. The mean kidney to final body weight and lung to final body weight were statistically significantly lower in the 12500-ppm group females, but were considered to be a result of non-significantly increased mean final body weight in the 12500-ppm group females. There were no other test substance-related effects on organ weights.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related yellow and/or white areas were noted in the prostates of 0, 5000, 7500, and 12500-ppm group males, with a mildly higher incidence in the 5000 and 12500 ppm groups compared to the control group. These correlated microscopically with infiltrates of neutrophils in the prostates. The findings were not severe and therefore considered to be non adverse. No test substance-related effects were observed on the number of former implantation sites and the number of unaccounted-for sites. The differences between the control and test substanceexposed groups were slight and not statistically significant.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related microscopic findings were noted in the prostate of the 5000, 7500, and 12500-ppm group males. The number of male rats with neutrophil infiltrate was 9/29, 13/30, 12/30 and 16/27 for the control group and the groups exposed to 5000, 7500 and 12500 ppm, respectively. The severity was increase in a dose-related way: 0/29, 2/30, 0/30 and 4/27 rats had moderate infiltration (2/29, 4/30, 5/30 and 8/27 had mild and 7/29, 7/30, 7/30 and 4/27 had minimal infiltration) in the control group and the groups exposed to 5000, 7500 and 12500 ppm, respectively. The incidence and severity of neutrophilic infiltrates were characterized by infiltrates of neutrophils, mostly within the acinar lumens and a few infiltrating in the adjacent interstitium and through the epithelium of the acini. As these findings were common in control rats, and did not result in a decrease in fertility, the findings were not considered adverse.
There were statistically significantly higher numbers of primordial follicles in the 7500 ppm group females (p < 0.05, N = 4) and the 12500-ppm group females (p < 0.01, N = 30) than in the control group females. This was considered to be toxicologically irrelevant. There were no other test substance-related histologic changes. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test substance. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
There were no microscopic findings in females that would be expected to result in reduced fertility. There were histologic changes of neutrophilic infiltrates in the prostate that may have contributed to reduced fertility in some males. In males with reduced fertility, this finding was noted in 4/7 matings (0/1 control group, 1/1 5000 ppm group, 2/4 7500 ppm group, and 1/1 12500 ppm group). This microscopic finding was noted in many animals without reduced fertility, so the change was not likely to have resulted in significant loss of fertility by itself. However, neutrophilic infiltrates in the prostate were noted with increased incidence and severity in the 5000, 7500, and12500 ppm group males compared to control group males. One male in the 7500 ppm group with reduced fertility had decreased thickness of the muscle layer in 1 of the vas deferens, which could have contributed to reduced fertility.

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No test substance-related effects were observed on P1 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 results of P1 reproductive performance are included in the table below. No test substance-related effects on P1 reproductive performance were observed at any exposure level. No statistically significant differences were noted between the control and test substance-exposed groups. One, 1, 4, and 1 males in the control, 5000, 7500, and 12500 ppm groups, respectively, did not sire a litter. One, 1, 4, and 1 females in these same respective groups were determined to be nongravid. 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.

Details on results (P1)

No test substance-related effects were noted on mean gestation lengths or the process of parturition at any exposure level. Mean P1 gestation lengths in the test substance-exposed groups were similar to the control group value. Differences were slight and were not statistically significant. The mean gestation lengths in the 5000, 7500, and 12500 ppm groups were 21.7, 21.5, and 21.7 days, respectively, compared to mean gestation lengths of 21.9 days in the concurrent control group and 21.7 days in the Charles River Ashland historical control data. No signs of dystocia were noted at any exposure level.

Effect levels (P1)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 7 500 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

Target system / organ toxicity (P1)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Description (incidence and severity):

The general physical condition (defined as the occurrence and severity of clinical observations) of all F1 pups in this study was unaffected by parental test substance exposure.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

One total litter loss was noted in the 12,500-ppm group on PND 8. This was limited to a single litter and there was no other evidence of effects on pup survival; therefore, this was not considered test substance-related. Eleven (6), 30(9), 5(5), and 11(5) pups (litters) in the control, 5000, 7500, and 12500 ppm groups, respectively, were found dead. Seven (4), 6(6), 5(3), and 5(4) pups (litters) in the same respective groups were missing and presumed to have been cannibalized.
Overall, 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, 21–24, 24–28, and from birth to PND 4 (pre-selection) and PND 4 (post-selection)–28 were unaffected by administration of the test substance to the F0 parental animals at all exposure levels.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean male and female pup body weights and body weight changes in the 5000, 7500, and 12500 ppm groups were unaffected by test substance administration throughout the postnatal period. No statistically significant differences from the control group were noted.

Sexual maturation:

no effects observed

Description (incidence and severity):

Mean ages of attainment of balanopreputial separation and mean body weights at the age of attainment were unaffected by test substance administration. The mean ages of attainment of balanopreputial separation were 43.9, 44.2, and 44.3 days in the 5000, 7500, and 12500 ppm groups, respectively, compared to 44.1 in the control group. Mean body weights at the age of attainment were 217.8 g, 217.5 g, and 215.4 g in the same respective groups compared to 214.4 g in the control group. None of the differences from the control group were statistically significant.
Mean ages of attainment of vaginal patency and mean body weights at the age of attainment were unaffected by test substance administration. The mean ages of attainment of vaginal patency were 37.8, 36.9, and 37.1 days in the 5000, 7500, and 12500 ppm groups, respectively, when compared to 36.3 days in the control group. Mean body weights at the age of attainment were 134.4 g, 127.6 g, and 130.7 g in the same respective groups compared to 127.4 g in the control group. None of the differences from the control group were statistically significant.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No test substance-related effects on organ weights (absolute, relative to final body weight, and relative to brain weight) were observed at any exposure level when the test substance-exposed groups were compared to the control group.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No internal findings that could be attributed to parental administration with the test substance were noted at the necropsy of weanlings euthanized on PND 28 or pups euthanized due to death of the dam. Internal findings in the test substance-exposed groups included dark red discoloration of the lungs for one pup in the 5000 ppm group and one pup in the 12500-ppm group; however, this finding was noted for single pups in each group.
At the PND 28 necropsy of F1 weanlings selected for organ weights, internal findings were limited to dark red discoloration of the lungs for one female pup in the 5000 ppm group; however, this finding was noted for a single pup and was not noted in a dose-related manner. No other internal findings were noted.
No internal findings that could be attributed to parental test substance administration were noted at the necropsies of pups that were found dead. Six malformations and 1 variation were noted for one pup in the 5000 ppm group. Craniorachischisis was noted for this pup,
consisting of a small cranial vault with skin reflected (anterior), cranial portions open (posterior), and vertebral column open (thoracic through sacral regions); skeletally, this finding consisted of absent frontal (bilateral), parietal (bilateral), interparietal, and supraoccipital bones and the dorsal aspect of thoracic arch No. 7 through sacral arch No. 4 reflected more laterally then normal (bilateral). In addition, this pup had rib anomaly (right rib Nos. 9 and 10 fused and left rib Nos. 10 and 11 fused), cleft lip (upper left), microphthalmia (bilateral; skeletally consisting of an orbit smaller than normal), cleft jaw (upper, right; skeletally consisting of small and misshapen right premaxilla and maxilla), and a high-arched palate (medial and cranial portions). The variation of 14th rudimentary rib was also noted for this pup. These findings were noted for a single pup and were not noted in a dose-related manner. Aside from the absence of milk in the stomach, no other internal findings were noted.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 12 500 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects seen in F1 pups up to PND 21 up to and including highest dose tested (12,500 ppm)

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The general physical condition (defined as the occurrence and severity of clinical observations) of all F2 pups in this study was unaffected by parental test substance exposure. Sixteen (6), 5(3), 8(1), and 5(3) pups (litters) in the control, 5000, 7500, and 12500 ppm groups, respectively, were found dead. Three (2), 5(5), 6(4), and 1(1) pups (litters) in the same respective groups were missing and presumed to have been cannibalized.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

A total litter loss was noted for one litter in the 7500 ppm group on PND 12. However, this was noted for only 1 litter, did not occur in a dose-related manner, and total litter loss was also noted for 1 control group litter (on PND 0). Therefore, this was not considered test substance-related. 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, 21–24, 24–28, and from birth to PND 4 (pre selection), and PND 4 (post-selection)–28 were unaffected by administration of the test substance to the F1 parental animals at all exposure levels. Differences from the control group were slight, were not statistically significant, and/or did not occur in a dose-related manner.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean male and female pup body weights and body weight changes in the 5000, 7500, and 12500 ppm groups were unaffected by parental test substance exposure throughout the postnatal period. Statistically significantly lower mean pup body weight gains were noted for males and females in the 5000 ppm group during PND 24–28, for males and females in the 7500 ppm group during PND 4–7, for females in the 12,500 ppm group during PND 11–14, and for males in the 12500 ppm group during PND 24–28. Mean body weight for females in the 7500 ppm group were statistically significantly lower (7.5%) than the control group on PND 21. However, these differences were transient, did not occur in a dose-related manner, and/or did not affect mean body weights. Therefore, these changes were not considered test substance-related. Other differences from the control group were slight and not statistically significant.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No test substance-related effects on organ weights (absolute, relative to final body weight, and relative to brain weight) were observed at any exposure level when the test substance-exposed groups were compared to the control group. None of the differences from the control group were statistically significant.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the PND 28 necropsy of F2 weanlings selected for organ weights, no test substance-related internal findings were observed at any exposure level. Internal findings in the test substance-exposed groups included dilated renal pelvis for one pup in the 7500 ppm group, a depressed area of the liver for one pup in the 7500 ppm group, and a misshapen thymus for one pup in the 5000 ppm group. However, these findings were noted in single pups, were not noted in a dose-related manner, and/or were also observed for pups in the control group.
Sixteen (6), 5(3), 8(1), and 5(3) pups (litters) in the control, 5000, 7500, and 12500 ppm groups, respectively, were found dead from PND 0 to 28. No internal findings that could be attributed to parental test substance administration were noted at the necropsies of pups that were found dead. Aside from the absence of milk in the stomach, no other internal findings were noted.
At the PND 28 necropsy of F2 weanlings not selected for organ weights and pups euthanized due to death of the dam, no internal findings were observed at any test substance exposure level.

Effect levels (F2)

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

>= 12 500 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects seen up to and including the highest dose tested (12,500 ppm)

Target system / organ toxicity (F2)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Exposure atmosphere homogeneity asessment and exposure concentrations:

Based on mean differences from the reference location, spatial homogeneity was considered acceptable for all exposure chambers. 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.

The overall mean exposure concentrations of the P0 Generation were analysed to be 0, 4994, 7490 and 12420 ppm and for the P1 generation 0 , 5000 ppm, 7455 and 12433 ppm.

Results of P0 reproductive performance:

 

Parameter	Dosage level (ppm)
	0	5000	7500	12500
Male Mating Index (%)	100	100	96.7	96.7
Female Mating Index (%)	100	100	96.7	96.7
Male Fertility Index (%)	96.7	93.3	90	96.7
Female Fertility Index (%)	96.7	93.3	90	96.7
Male Copulation Index (%)	96.7	93.3	93.1	100
Female Conception Index (%)	96.7	93.3	93.1	100
Estrous Cycle Length (days)	4.8	4.3	4.8	4.8
Precoital Interval (days)	2.6	2.6	3.0	3.5

 

Results of P1 reproductive performance

 

Parameter	Dosage level (ppm)
	0	5000	7500	12500
Male Mating Index (%)	100	100	96.7	100
Female Mating Index (%)	100	100	96.7	100
Male Fertility Index (%)	96.6	96.6	86.7	96.6
Female Fertility Index (%)	96.7	96.6	86.7	96.7
Male Copulation Index (%)	96.6	96.6	89.7	96.6
Female Conception Index (%)	96.7	96.6	89.7	96.7
Estrous Cycle Length (days)	4.6	4.6	4.2	4.5
Precoital Interval (days)	2.7	2.8	2.5	3.2

 

Applicant's summary and conclusion

Conclusions:

Based on the results of a two-generation study with exposure via the inhalation route, performed according to OECD Guideline 416 and the EPA Guideline OPPTS 870.3800 and following GLP principles, the parental NOAEL for HFO-1336mzz-E was found to be at least 7500 ppm based on clinical signs observed at the highest dose tested (12500 ppm), whereas the NOAEL for reproduction was found to be at least 12500 ppm.

Executive summary:

A two-generation study was performed with HFO-1336mzz-E according to OECD Guideline 416 and the EPA Guideline OPPTS 870.3800 and following GLP principles. Male and female rats (P0 and P1 generation; n=30) were exposed to 0, 5000, 7500 or 12500 ppm via whole body inhalation for 6 hours daily for at least 70 consecutive days prior to mating and continuing through the day prior to euthanasia. Exposure was suspended from Gestation Day 21 through Lactation Day 4 for gravid and presumed gravid females. Exposure for the offspring selected to constitute the P1 parental generation began on the day of weaning, PND 28. Exposure atmosphere analyses verified that the test atmosphere was homogeneous and that the animals were exposed to target concentrations. Mortality was seen in the high dose group of the P0 generation, two males were euthanized in extremis and one male was found dead. Based on the clinical findings for these males (including head tilt, hypoactivity, pale extremities, shallow respiration, unkempt appearance, red material around the nose, mouth, and/or facial area, and/or yellow material on the urogenital area at approximately 1 hour post exposure on the day of death/euthanasia), this mortality/moribundity was likely related to test substance exposure. No other test substance-related effects on survival were noted for the P0 or P1 males and females. Test substance-related clinical findings in the high dose groups including tremors (P0 and P1 males and females) and clonic convulsions (P0 females) were considered adverse. Test substance-related lower mean body weight gains, with corresponding effects on food consumption and/or food efficiency, were noted for P0 males and P1 males and females in the 12500-ppm group following the initiation of exposure (Study Day 0–7 or PND 28–35), resulting in lower (up to 6.4%) mean body weights for P0 males in the 12500-ppm group. These findings were transient and were not considered adverse. Increased mean body weight gains, with corresponding effects on food consumption, were noted for P0 females in the 12500-ppm group during the premating period, resulting in higher (up to 6.6%) mean body weight in this group; these findings were potentially related to test substance exposure, but the magnitude of change was minimal and the changes were not considered adverse. No other test substance-related effects were noted on P0 or P1 parental body weights, body weight gains, food consumption, or food efficiency at any exposure concentration. No test substance-related effects were noted on P0 or P1 reproductive performance (mating, fertility, copulation, or conception), estrous cyclicity, gestation lengths, the process of parturition, precoital intervals, or spermatogenesis at any exposure concentration. There were no test substance -related effects on attainment of balanopreputial separation and vaginal patency or body weights at attainment of these developmental landmarks in the F1 pups at any exposure level. There were no test substance-related effects noted on F1 and F2 mean numbers of pups born, live litter size on PND 0, percentage of males, or postnatal survival. Mean F1 and F2 male and female pup body weights and body weight changes in the 5000, 7500, and 12500 ppm groups were unaffected by test substance exposure throughout the postnatal period. There were no test substance -related macroscopic findings noted at any dosage level in the F1 and F2 pups that were found dead or pups that were euthanized at the scheduled necropsy on PND 28. No test substance-related effects on organ weights were observed for F1 and F2 pups on PND 28. No test substance-related adverse macroscopic or microscopic findings or organ weight alterations were noted for P0 and P1 males or females at any exposure level.

Based on the effects on survival for P0 males at 12500-ppm and adverse clinical findings of tremors noted for P0 and P1 males and females at 12500-ppm, the NOAEL was considered to be at least 7500 ppm. Since no effects were observed on reproductive parameters or survival and growth of the offspring during the pre-weaning period, the NOAEL for parental reproductive toxicity and neonatal toxicity was concluded to be at least 12500-ppm, the highest exposure level evaluated.