Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

The potential effects of D5 on the dopamine receptor, estrogenic or androgenic activity were investigated. D5 was demonstrated to act as D2-receptor agonist but was without effect in all other cases.

In a two-generation reproductive toxicity study (WIL Research Laboratories Inc, 1999) conducted to appropriate EPA test guidelines and to GLP, no parental toxicity in the F0 and F1 generations was observed at exposure concentrations of 30, 70, and 160 ppm D5. F0 and F1 reproductive performance was not affected at any concentration. No test-substance-related total litter losses occurred, and no neonatal toxicity was evident in the F0 and F1 generations at concentrations of 30, 70, and 160 ppm. Based on the results of this study, the NOAEC for parental toxicity, reproductive toxicity, and neonatal toxicity is considered to be at least 160 ppm (actual 2496 mg/m3).

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22.10.1996 to 22.02.1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Qualifier:
according to
Guideline:
EPA OPP 83-6 (Developmental Neurotoxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Portage, Michigan.
- Age at study initiation: 45 days (F1 rats prior to pairing were 13-15 weeks old)
- Weight at study initiation: (P) Males: 138-232 g; Females: 115-193 g; (F1 prior to pairing) Males: 304-576 g; Females: 196-321 g
- Fasting period before study: No
- Housing: Wire-mesh cages or plastic maternity cages
- Diet (e.g. ad libitum): Ad libitum (except during exposure)
- Water (e.g. ad libitum): Ad libitum (except during exposure)
- Acclimation period: 15 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22± 2.2
- Humidity (%): 40-70
- Air changes (per hr): Approximately 10
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 25.10.1996 To: 21.08.1998
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Each group of animals was exposed in a 2 m3 stainless steel and glass whole body inhalation chamber.
- Method of holding animals in test chamber: cages
- Temperature, humidity, pressure in air chamber: 18-26oC, 30-60%
- Air change rate: 12 to 15 changes/hour
- Treatment of exhaust air: No data


TEST ATMOSPHERE
- Brief description of analytical method used: Gas chromatography
- Samples taken from breathing zone: yes
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after one unsuccessful attempt: no
- After successful mating each pregnant female was caged (how): plastic maternity cage
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Exposures within each chamber were measured 9 to 11 times during each daily exposure period by a validated gas chromatographic method.
Duration of treatment / exposure:
Exposure period: F0 and F1 males and females: At least 70 days prior to mating, throughout mating, gestation (to gestation day 20), lactation,
with the exception of lactation days 0-4, until scheduled euthanasia. F1 pups were exposed from weaning through sexual maturity, breeding, gestation.
Duration of test: ca. 18 months
Frequency of treatment:
6 hr/day, 7 days/week
Details on study schedule:
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: 13-15 weeks
Dose / conc.:
478 mg/m³ air (analytical)
Remarks:
31 ppm (analytical conc.)
Dose / conc.:
1 094 mg/m³ air (analytical)
Remarks:
71 ppm (analytical conc.)
Dose / conc.:
2 496 mg/m³ air (analytical)
Remarks:
162 ppm (analytical conc.)
No. of animals per sex per dose:
30/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: No data
- Rationale for animal assignment (if not random): Random
- F2 pups were randomly selected on PND 4 for neuropathological and/or neurobehavioural evaluations.
Positive control:
None
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for appearance, behaviour, moribundity and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly for all animals and on gestation day (GD) 0, 7, 10, 14, and 20 and PND 1, 4, 7, 14, and 21 for females in the F0 and F1 generations.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. FC was also recorded on GD 0, 7, 10, 14, and 20 and PND 1, 4, 7, 14, and 21 for females in the F0 and F1 generations.

WATER CONSUMPTION: No

FUNCTIONAL OBSERVATION BATTERY (FOB): On all females F1 rats on gestation day 10 and lactation day 20.
Oestrous cyclicity (parental animals):
Vaginal smears were prepared daily to determine the stage of oestrus cycle for each F0 and selected F1 females, beginning 21 days prior to pairing and continuing until evidence of copulation was observed. For females with no evidence of copulation, smearing was continued until termination of the mating period.
Sperm parameters (parental animals):
Parameters examined in all P, F1 and F2 male parental generations: testicular and epididymal sperm count, sperm production rates, sperm motility, sperm morphology
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED: When parturition was judged complete, litters were sexed and examined for gross malformations and the numbers of stillborn and live pups were recorded. Individual gestation durations were calculated using the date delivery initiated. Each litter was examined twice daily for survival, and all deaths were recorded. Litters were also examined for any adverse changes in appearance or behaviour. Each pup received a detailed physical examination on PND 1, 4, 7, 14 and 21, at weekly intervals thereafter until scheduled euthanasia. On PND 1, the anogenital distance was measured for all F1 pups in the control and high dose groups, and for all F2 pups. Pups were weighed on PND 1, 4, 7, 14 and 21. Individual body weights were also recorded for selected F2 rats at weekly intervals. Pup sexes were individually determined on PND 0, 4 and 21.

FOB: Thirty pups/sex/group from the F2 generation were selected for developmental landmarks, neurobehavioural testing, neuropathy, brain weights and/or brain dimension measurements.

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- All surviving F0 and F1 parental animals after the last litter of each generation was weaned.

GROSS NECROPSY: On all parental animals dying spontaneously, euthanised in extremis, or surviving to the scheduled sacrifice.
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS: Histopathology (on all F0 and F1 adult animals from the control and high-exposure groups and from all F0 and F1 parental animals dying spontaneously or euthanised in extremis): Adrenal glands, brain, cervix, coagulating gland, epididymis (right caput, corpus, and cauda), kidneys, liver, lungs, ovaries, penis, pituitary gland, prepuce, preputial gland, prostate, seminal vesicles, testis (right), thyroid, uterus, vagina, and vas deferens, All gross (internal) lesions. Organ weights: Adrenals, prostate, brain, seminal vesicles with epididymides (total and cauda), coagulating glands, heart, kidneys, spleen, liver, testes, lungs, thymus, ovaries, thyroid, pituitary, and uterus.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 or 28 days of age. Selected F2 rats not allocated for neuropathy and brain dimension measurements were necropsied on PND 70 and selected organs were weighed.
- These animals were subjected to a macroscopic examination, with emphasis placed on developmental morphology evaluations.

HISTOPATHOLOGY / ORGAN WEIGHTS: Including all pups that died after PND 4 and prior to weaning.

F2 NEUROPATHOLOGICAL EXAMINATION: Brain weights and brain region dissections: At the scheduled evaluations (PND 11 and 70), 10 and 16 F2 pups/sex/group, respectively, selected for neuropathology and/or brain weight measurements were euthanised. Brains and carcasses of F2 rats selected only for brain weight measurement were discarded following weighing of brain regions.
Neuropathology: At the PND 11 evaluation, 6 selected F2 pups/sex/group were allocated for neuropathological analysis. Brain tissue was subjected to histopathological examination. At study termination (PND 70), 6 selected F2 rats/sex/group were euthanised. The central and peripheral nerve tissues were dissected and preserved. Brain weights and dimensions were recorded. Any observable gross changes, abnormal coloration, or lesions of the brain and spinal cord were recorded. Nerve tissues were examined histopathologically.
Statistics:
Statistical methods:
Chi-Square test with Yates' correction factor for parental mating and fertility indices
One-way ANOVA with Dunnett's test for the following endpoints:
Pre-coital intervals, Parental weekly, gestational and lactational body weight and food consumption data,
gestation lengths, Sperm numbers, Sperm production rates, Organ weights*, Ovarian primordial follicle counts,
Numbers of pups born**, Live litter sizes**, Anogenital distances**, Pup body weights**, Continuous FOB data,
Startle response data, Balanopreputial separation, Vaginal patency

Fisher's Exact Test - Scalar/descriptive FOB data

Kruskal-Wallis test with Mann-Whitney U test - Sperm motility, Sperm morphology, Pup sexes at birth (% males per
litter)**, Postnatal survival**

Kolmogorov-Smimov test (one-tailed test) - Histopathological findings

* for PND 21 organ weights, the litter was the experimental unit of evaluation
**litter used as experimental unit
Reproductive indices:
Male (female) mating index (%): No. of males (females) with evidence of mating/No. of paired animals x100.
Male (female) fertility index: No. of males siring a litter (females confirmed pregnant)/No. of males (females) used for mating x100.

Because the protocol specified that each female was to be paired with only one male, the mating and fertility indices are identical for both sexes.
Offspring viability indices:
Live litter size: total viable pups Day 0/No. of litters with viable pups Day 0 x100.
postnatal survival between birth and PND 0 or PND 4 (pre-selection)(%/litter): Σ(viable pups per litter on PND 0 or 4/No. of pups born per litter)/No. of litters per group x 100.

Postnatal survival for all other intervals (%/litter): Σ(viable pups per litter at end of interval N/viable pups per litter at start of interval N)/ No. of litters per group. Where N = PND 0-1, 1-4, 4-7, 7-11, 11-14 or 4-21.
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Maternal data with dose level (with NOAEL value): No adverse effects were noted in any endpoint in the F0 or F1 parental
animals at any dose level tested.

• Mortality and day of death: No exposure-related increases in mortality were reported.
• Number pregnant per dose level: mating indices for the 0, 30, 70, and 160 ppm dose groups were 100%, 83.3%, 93.3%, and 93.1%
• Number aborting: no animals aborted
• Duration of Pregnancy: No exposure-related differences in time to coitus or in length of pregnancy were reported.
• Body weight: No exposure-related changes in body weight were reported.
• Food/water consumption: No exposure-related changes in food consumption were reported.
• Description, severity, time of onset and duration of clinical signs: No exposure-related changes in clinical signs were reported.
• Gross pathology incidence and severity: No exposure-related increase in gross pathological findings was
reported. Increased incidences of minimal alveolar histiocytosis were noted in the F0 females and the F1 males and females in the 30, 70, and 160 ppm groups. While the increased incidences in these groups were attributed to test article exposure, the finding was not considered to be an adverse effect as it was graded minimal in all groups (including the control group). Moreover, no associated histopathological lung lesions or differences in
mean lung weights were observed at any exposure level. This finding was considered to be a compensatory response to material delivered to the lungs via the inhalation route.
• Organ weight changes, particularly effects on total uterine weight: No exposure-related changes in organ
weights were reported.
• Histopathology incidence and severity: No exposure-related increase in histopathological findings was reported.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 160 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No parental toxicity, reproductive performance or neonatal toxicity in the P generation was seen at exposure concentrations of 30, 70, and 160 ppm.
Critical effects observed:
no
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Fetal data with dose level (with NOAEL value): No adverse effects were noted in any endpoint in the F1 or F2 generation
animals at any dose level tested.

• Pup data, provide at a minimum qualitative descriptions of responses where dose related effects were seen:
Ø Litter size and weights: Litter sizes were comparable across groups.
Ø Number viable (number alive and number dead): Pup survival was unaffected by exposure to D5. One F0 female in the 160 ppm group had total litter loss on lactation day 0. However, this female delivered only one pup. Because no exposure-related decreases in postnatal survival of the F1 and F2 litters were noted at any concentration, the single occurrence of total litter loss in the 160 ppm group was not attributed to test substance exposure. Mean pup body weights and the general physical condition of the F1 and F2 pups were similar in control, 30, 70, and 160 ppm groups both before and after weaning.
Ø Sex ratio: Sex ratios were comparable across groups in the F1 and F2 generations.
Ø Grossly visible abnormalities: No treatment-related gross external malformations were reported.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 160 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No parental toxicity, reproductive performance or neonatal toxicity in the F1 generation was seen at exposure concentrations of 30, 70, and 160 ppm.
Critical effects observed:
no
Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
>= 160 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No F2 developmental neurotoxicity was evident at any concentration.
Critical effects observed:
no
Reproductive effects observed:
no
Conclusions:
In a two-generation reproductive toxicity study (reliability score 1) conducted to appropriate EPA test guidelines and to GLP, no parental toxicity in the F0 and F1 generations was observed at exposure concentrations of 30, 70, and 160 ppm. F0 and F1 reproductive performance was not affected at any concentration. No test-substance-related total litter losses occurred, and no neonatal toxicity was evident in the F0 and F1 generations at concentrations of 30, 70, and 160 ppm. No F2 developmental neurotoxicity was evident at any concentration. Based on the results of this study, the NOAEL for parental toxicity, reproductive toxicity, neonatal toxicity, and developmental neurotoxicity is considered to be at least 160 ppm.
Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
2 420 mg/m³
Study duration:
subchronic
Species:
rat
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The key study for reproductive toxicity was conducted to EPA OPPTS 870.3800 (Reproduction and Fertility Effects) and to EPA OPP 83-6 (Developmental Neurotoxicity Study), which is similar to OECD test guideline 416 (WIL Research Laboratories Inc, 1999). Sprague-Dawley rats (30 animals/sex/dose) were exposed, whole body, to D5 vapour 6 hours/day, 7 days/week at concentrations of 30, 70 and 160 ppm (actual analytical concentrations of 478, 1094 and 2496 mg/m3). The F0 and F1 males and females were exposed to D5 at least 70 days prior to mating, throughout mating, gestation (to Day 20), lactation (except days 0-4), then until scheduled euthanasia. F1 pups were exposed from weaning, through sexual maturity, breeding and gestation. Observations and examinations were conducted according to the guideline. In addition, a neuropathological examination of the brain and spinal cords of animals from the F2 generation were conducted on postnatal days 11 and 70. There were no signs toxicity in adult animals of the F0 and F1 generations, nor was there any adverse effect on the reproductive performance. For offspring, litter sizes and weights were comparable across the doses, as were the number of viable pups and the sex ratios. In addition, there were no treatment-related gross external malformations observed. The neuropathological examination did not discover any adverse effects of D5 on the brain region weights, and there were no observable gross changes, abnormal colouration, or lesions of the brains, spinal cords and nerve tissues. Based on the results of this study, the NOAEC for parental toxicity, reproductive toxicity, and neonatal toxicity is considered to be at least 160 ppm (actual 2496 mg/m3).

There is one additional reproductive toxicity study (WIL Research Laboratories Inc, 1996) that supports the findings of the key study in that no adverse effects on reproductive parameters were observed up to an exposure concentration of 132 ppm (2034 mg/m³).


Effects on developmental toxicity

Description of key information

In an inhalation prenatal developmental toxicity study in rats, conducted according to OECD Test Guideline 414 and in compliance with GLP, the concluded NOAEC for maternal and developmental effects was greater than 160 ppm (2427 mg/m3), the highest achievable vapour concentration. There were no adverse effects reported in the study (The Dow Chemical Company (Toxicology and Environmental Research and Consulting), 2020).

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 October 2019 to 6 November 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2018
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
other: JMAFF, Guideline 2-1-18, Teratogenicity Study
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (CRL) (Raleigh, North Carolina)
- Age at study initiation: Sexually mature adults.
- Weight at study initiation: 200-246 g
- Fasting period before study: No
- Housing: Upon arrival and after assignment, animals were housed one per cage in plastic cages. Cages had solid floors with corncob bedding. Cages contained a feed crock and a pressure activated Lixit ® valve-type watering system. Animals were housed in the same room; however, cages were contained in separate racks by dose group.
- Diet (e.g. ad libitum): LabDiet Certified Rodent Diet #5002 in meal form, ad libitum except during the six-hour exposure period
- Water (e.g. ad libitum): municipal water, ad libitum except during the six-hour exposure period
- Acclimation period: at least four days
- Feed and water analysis: There were no contaminants in either the feed or water at levels that would have adversely impacted the results or interpretation of this study. Copies of these analyses are maintained in the study file.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C with a range of 20°C-24°C
- Humidity (%): 50% with a range of 30-70%
- Air changes (per hr): 10-15 times/hour (average)
- Photoperiod (hrs dark / hrs light): 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)

Route of administration:
inhalation: vapour
Vehicle:
unchanged (no vehicle)
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 2 cubic meter stainless steel and glass Rochester-type inhalation whole-body exposure chambers [1.3 meters (m) x 1.2 m wide x 1.2 m deep with pyramidal top and bottom] under dynamic airflow conditions. The D5 exposure chambers were operated at a slightly negative pressure relative to the surrounding area. The control (0 ppm) chamber was operated at a pressure that was slightly positive to the surrounding area to minimize the potential for accidental exposure of the control animals to the test material in the case of fugitive emissions.
- Method of holding animals in test chamber: Test animals were housed singly in wire mesh cages to minimize crowding during the exposure.
- Source and rate of air: D5 vapors were generated using a glass J-tube method (Miller et al., 1980). Liquid test material was metered into the glass J-tube(s) assembly and vaporized by passing a preheated stream of nitrogen gas, approximately 30 - 50 litres per minute, through a bead bed in the glass J-tube. Glass wool filters were placed at the exit of the J-tubes and HEPA filters were placed at the inlet of each whole-body exposure chamber. The control chamber (filtered air) received the same volumetric flow of nitrogen as the exposure chambers. The nitrogen was heated with a flameless heat torch (FHT-4, Master Appliance Corporation, Racine, Wisconsin) to the minimum extent necessary to vaporize the test material. The generation system was electrically grounded and the J-tubes were changed as needed. Chamber airflow was maintained at rate of approximately 450-600 L/min, which was sufficient to provide the normal concentration of oxygen to the animals.
- Method of conditioning air: The vaporized D5 in nitrogen gas was rapidly diluted and mixed with filtered chamber supply air prior to passing through the HEPA filters and entering the chambers to achieve approximately 450-550 litres per minute total flow rate and the desired D5 chamber concentration. On the day of the first exposure, the chambers were monitored to ensure sufficient CO2 and O2 levels.
- System of generating particulates/aerosols: not applicable
- Temperature, humidity, pressure in air chamber: Chamber temperature and relative humidity were displayed and monitored continuously, and recorded approximately every hour using a resistance temperature device (RTD) and a humidity sensor (Omega HX94C, Omega Engineering Inc., Norwalk, Connecticut), respectively. Calibration of the temperature and relative humidity sensors were conducted pre-exposure. Chamber temperature and relative humidity were maintained at approximately 22 ± 2 °C and 40-60%, respectively.
- Air flow rate: Airflow through each exposure chamber was displayed and monitored continuously, and recorded approximately every hour using a calibrated differential pressure transducer (Model 264, Setra System, Boxborough, Massachusetts) coupled to a calibrated orifice plate.
- Air change rate: approximately 12 - 18 calculated air changes per hour.
- Method of particle size determination: not applicable
- Treatment of exhaust air: not applicable

TEST ATMOSPHERE
- Brief description of analytical method used: Chamber concentrations of D5 vapor were measured with a sampling probe located approximately in the centre of the chamber within the breathing zone of the animals and were determined at least twice per hour during each exposure period using an Agilent Gas Chromatograph 6890A with flame ionization detector (GC/FID). D5 peak area (height) was determined using integration by ChemStation software (Agilent Technologies, Inc., Palo Alto, California). The exposure chambers were monitored with a real-time laser aerosol monitor (Casella CEL-712 Microdust Pro, Casella Inc., Buffalo, NY) for the presence of aerosol throughout the study and the concentration of aerosol was measured at least three times per exposure day for the treated chambers and once/week for the control chamber. During the pre-inlife phase of the study, prior to exposure, the chambers were checked to ensure that a uniform distribution of D5 vapor was present from at least five sample points in the breathing zone of the animals. The GC/FID was calibrated, and a standard curve compiled pre-exposure using air standards of D5 vapor by vaporizing measured volumes of D5 vapor into Tedlar sampling bags (SKC, Eighty Four, Pennsylvania) along with metered volumes of HEPA-filtered room air. The analytical (D5) vapor concentration during each exposure was interpolated from the standard curve and was reported as the definitive exposure concentration. The analytical system was checked prior to and after each exposure with a D5 vapor standard of known concentration. The nominal concentration of test material in each chamber was calculated based on the amount of test material fed into the inhalation apparatus each day and the total chamber airflow through the chamber for each exposure period.
- Samples taken from breathing zone: Yes. Chamber concentrations of D5 vapor were measured with a sampling probe located approximately in the center of the chamber within the breathing zone of the animals and were determined at least twice per hour during each exposure period . During the pre-inlife phase of the study, prior to exposure, the chambers were checked to ensure that a uniform distribution of D5 vapor was present from at least five sample points in the breathing zone of the animals.


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber concentrations of D5 vapor were measured with a sampling probe located approximately in the centre of the chamber within the breathing zone of the animals and were determined at least twice per hour during each exposure period using an Agilent Gas Chromatograph 6890A with flame ionization detector (GC/FID).
Details on mating procedure:
- Impregnation procedure: Sexually mature virgin females were naturally mated with males of the same strain (one male:one female) at CRL
- M/F ratio per cage: one male:one female
- Length of cohabitation: Females were checked for in situ copulation plugs the following morning and those found with such a plug were removed from the males' cages.
- Further matings after two unsuccessful attempts: not specified
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: Females were checked for in situ copulation plugs the following morning and those found with such a plug were removed from the males' cages. The day on which a vaginal plug was detected was considered GD 0. GD 0 body weights were provided by the supplier, and maintained in the study file. Rats arrived in the test laboratory on GD 1 or 2.
- Any other deviations from standard protocol: no
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
7 days/week
Duration of test:
20 days
Dose / conc.:
0 ppm
Remarks:
Filtered air, control group
Dose / conc.:
32 ppm (analytical)
Remarks:
32 ± 1.1 ppm (analytically-determined mean chamber concentrations over the course of the study)
Dose / conc.:
74 ppm (analytical)
Remarks:
74 ± 2.0 ppm (analytically-determined mean chamber concentrations over the course of the study)
Dose / conc.:
161 ppm (analytical)
Remarks:
161 ± 3.8 ppm (analytically-determined mean chamber concentrations over the course of the study)
Dose / conc.:
29 ppm (nominal)
Remarks:
29.1 ± 0.6 ppm (nominal, based on amount of test material fed into inhalation apparatus each day and total chamber airflow for exposure period); equivalent to 455 mg/m3
Dose / conc.:
81.2 ppm (nominal)
Remarks:
81.2 ± 2.1 ppm (nominal, based on amount of test material fed into inhalation apparatus each day and total chamber airflow for exposure period); equivalent to 1062 mg/m3
Dose / conc.:
169.7 ppm (nominal)
Remarks:
169.7 ± 5.6 ppm (nominal, based on amount of test material fed into inhalation apparatus each day and total chamber airflow for exposure period); equivalent to 2427 mg/m3
No. of animals per sex per dose:
24
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: Exposure levels for this study were selected on the basis of the method development study (Bell and Hotchkiss, 2020) and the two-generation reproduction study (Stump, 1999). The high exposure concentration was the highest attainable, stable, vapor concentration without the presence of a significant amount of aerosol (~ 0.1 mg/m3) in the test atmosphere. The lower exposure levels were selected to provide exposure response data for any toxicity that may have been observed among the high exposure level rats.
- Rationale for animal assignment (if not random): randomised
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A cage-side examination was conducted twice daily, approximately at the same time each day.
- Cage side observations checked: significant clinical abnormalities that are clearly visible upon a limited examination, and to monitor the general health of the animals. Significant abnormalities that could have been observed included, but were not limited to: decreased/increased activity, repetitive behaviour, vocalization, incoordination/limping, injury, neuromuscular function (convulsion, fasciculation, tremor, twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), alterations in faecal consistency, and faecal/urine quantity. In addition, all animals were observed for morbidity, mortality, and the availability of feed and water at least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Upon arrival, clinical observations were conducted on all animals at least once daily at approximately the same time each day (prior to exposure period). Clinical observations included a careful, hand-held examination of the animal with an evaluation of abnormalities in the eyes, urine, faeces, gastrointestinal tract, extremities, movement, posture, reproductive system, respiration, skin/hair-coat, and mucous membranes, as well as an assessment of general behaviour, injuries or palpable mass/swellings.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on GD 0 by the supplier, on GD 3, and prior to exposure on GD 6, 9, 12, 15, 18, and prior to necropsy (GD 21). Statistical analysis of body weights was performed using data collected on GD 0, 3, 6, 9, 12, 15, 18, and 21. Statistical analysis of body weight gains was conducted for the following intervals: GD 0-6, 6-9, 9-12, 12-15, 15-18, 18-21, 6-21, and 0-21.

FOOD CONSUMPTION: Yes
The amount of feed consumed was recorded and statistically analyzed for all animals every 3 days from GD 3-21 by weighing feed containers at the start and end of a measurement cycle and consumption was calculated using the following equation:
Feed consumption (g/day) = (initial weight of feed container - final weight of feed container) / (# of days in measurement cycle)

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: The maternal necropsy included an examination of the external tissues and all orifices. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera were examined. The liver and kidneys were dissected from the carcass and were incised. The thyroid with parathyroids were also dissected from the carcass. Any obvious gross pathologic alterations were recorded, and the weight of the liver, kidneys, thyroid with parathyroids (weighed after fixation) and gravid uterus (the gravid uterus for dam #341 was inadvertently not recorded) were recorded. The ratios of organ weights to terminal body weight were calculated (only absolute weight for gravid uterus). The thyroid with parathyroids and representative portions of liver, kidneys, and gross lesions were preserved in neutral, phosphate-buffered 10% formalin. Microscopic examination of liver, kidneys, and gross lesions were not conducted. Transponders were removed and saved with the preserved tissues.

OTHER:
Serum for Thyroid Hormone: Blood samples were obtained from the orbital sinus following anaesthesia with a mixture of isoflurane vapours and medical oxygen at the scheduled necropsy. Terminal blood samples were collected from all females. Approximately 2.5 mL of blood was collected for hormone analysis (T3, T4, and TSH) in tubes with no anticoagulant, and allowed to clot at room temperature for ~ 30 minutes. Analyses of blood for T3, T4, and TSH was performed from all pregnant animals on study (n=95 animals; all dams were pregnant with the exception of one animal (#420) in the 161 ppm (analytical) group.). Histopathological examination of the thyroid was conducted on all pregnant rats.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: All fetuses were given an external examination which included observations on body proportions, the head and face (including closure of the palate), abdomen, spine, extremities, genitalia, rectum and tail.
- Soft tissue examinations: Yes: At least one half of all the fetuses in each litter were chosen randomly via computer for visceral examination conducted by dissection under a low power stereomicroscope for evidence of visceral alterations. The visceral examination included observation of the thymus, trachea, esophagus, lungs, great vessels, heart (external and internal), liver, gastrointestinal tract, pancreas, spleen, kidney (sectioned), adrenal glands, ureters, bladder, and reproductive organs.
- Skeletal examinations: Yes: The second half of all the fetuses in each litter not selected for visceral examination were skinned, eviscerated, preserved in alcohol, and double stained with Alcian Blue and Alizarin Red S for cartilage and bone examination. A thorough evaluation of the fetal skeleton was conducted on the stained fetuses with the exception of one fetus from dam #421 that could not be evaluated for a skeletal examination due to an error during the fetal staining process.
- Head examinations: Yes: The heads of the fetuses used for visceral examinations were removed, placed in Bouin’s fixative and serially sectioned to allow for inspection of the eyes, brain, nasal passages, and tongue.
- Anogenital distance: Anogenital distance (AGD; absolute and relative to the cube root of fetal body weight) was measured on all live fetuses prior to euthanasia using a digital calliper. The sequence of the data collection was conducted without knowledge of treatment group and counterbalanced across groups to the extent possible on each day to control for potential confounding influences of collection timing.
Statistics:
The litter, rather than the pup, was considered as the experimental unit. Maternal body weights, corrected by body weight, maternal body weight gains, organ weights, T3, T4, TSH, anogenital distance, fetal body weights and feed consumption were evaluated by Bartlett’s test (alpha = 0.01) for homogeneity of variance. Based on the outcome of Bartlett's test, a parametric or non-parametric analysis of variance (ANOVA) was performed. If the ANOVA was significant at alpha = 0.05, analysis by Dunnett's test (alpha = 0.05) or the Wilcoxon Rank-Sum test (alpha = 0.05) with Bonferroni's correction were performed, respectively. Feed consumption values were excluded from analysis if the feed was spilled or scratched.
Frequency of pre- and post-implantation loss, and fetal alterations were analyzed using a censored Wilcoxon test with Bonferroni’s correction applied when the incidence was greater than 5%. The number of corpora lutea, implantations and litter size were evaluated using a non-parametric ANOVA (alpha = 0.05) followed by the Wilcoxon Rank-Sum test (alpha = 0.05) with Bonferroni's correction. Pregnancy rates were analyzed using the Fisher exact probability test (alpha = 0.05) with Bonferroni’s correction. Fetal sex ratios - using a binomial distribution test. Non-pregnant females, females with resorptions only, or females found to be pregnant after staining of their uteri were excluded from the appropriate analyses. Statistical outliers were identified, using a sequential method (alpha = 0.02) and, if excluded, were excluded for sound scientific reasons. Dunnett’s test and Bonferroni’s correction corrected for multiple comparisons to the control and were reported at the corrected alpha level. Means and standard deviations were calculated for descriptive purposes for chamber concentration, chamber aerosol concentration, chamber temperature, humidity, and airflow.
Indices:
Not specified
Historical control data:
Yes
Clinical signs:
no effects observed
Description (incidence and severity):
Examinations performed on all animals revealed no treatment-related findings.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no treatment-related or statistically significant differences in the body weights, body weights corrected for gravid uterine weight, or body weight gains of any treated groups when compared to their respective controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no treatment-related or statistically significant differences in the amount of feed consumed by any treated groups when compared to their respective controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Dams in the 161 ppm (analytical) group had a slight statistically significant increase in absolute (10.9%) and relative (9.3%) liver weights compared to controls. These were considered treatment related but non-adverse due to the small magnitude of change compared to controls. Kidney and thyroid gland weights for all treated groups and liver weights for the 32 and 74 ppm (analytical) groups were similar to their respective controls.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no maternal gross observations.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No test substance-related microscopic findings were noted. The microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of Sprague Dawley rats, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to exposure of rats to D5.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
There were no treatment-related or statistically significant differences on T3, T4, or TSH serum concentrations in any treated groups when compared to controls.
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
There were no treatment-related or statistically significant effects on pregnancy rates, resorption rates, litter size, numbers of corpora lutea or implantations, percent preimplantation loss, percent postimplantation loss or gravid uterine weights at any exposure concentration level. All dams were pregnant with the exception of one animal (#420) in the 161 ppm (analytical) group.
Dose descriptor:
NOAEC
Effect level:
>= 161 ppm (analytical)
Based on:
test mat.
Basis for effect level:
other: No adverse effects observed.
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related or statistically significant external alterations in any treated group. Incidental findings bearing no relationship to treatment included the malformations anasarca, domed skull, macroglossia, shortened muzzle, short trunk, polydactyly, and ankylodactyly. These observations were considered unrelated to treatment as they were all confined to one litter (Dam #392) in the 74 ppm (analytical) group and lack of an exposure-response relationship.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related skeletal alterations in any treated group. Incidental findings bearing no relationship to treatment included the variations supernumerary skull bone, delayed ossification (DO) parietal, DO cervical centra, DO sternebrae, DO thoracic centra, irregular pattern of ossification sternebrae, wavy ribs, calloused ribs, extra 1st lumbar rib, DO lumbar centra and the malformations fused ribs, missing lumbar centra, missing lumbar vertebrae, extra metacarpals, extra phalanges, and extra metatarsals. These observations were considered unrelated to treatment due to the low incidence, occurrence in the control group, and/or lack of a dose response relationship. Fetuses in the 161 ppm (analytical) treatment group had statistically-identified lower incidence of DO thoracic centra which was considered unrelated to treatment as a treatment-related effect in this parameter is expected to be a higher value than control.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related or statistically significant visceral alterations in any treated group. Incidental findings bearing no relationship to treatment included the variations fused lung, missing caudal lung lobe, supernumerary hepatic liver lobule, petechial haemorrhage adrenal, and bifurcated renal vein, and the malformations situs inversus, hydronephrosis, and hypoplastic testis. These observations were considered unrelated to treatment due to the low incidence, occurrence in the control group, and/or lack of a dose response relationship.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related or statistically significant craniofacial alterations in any treated group. An incidental finding bearing no relationship to treatment included the malformation hydrocephaly in the 74 ppm (analytical) group (Dam #392). This observation was considered unrelated to treatment due to the isolated occurrence and lack of a dose-response relationship.
Details on embryotoxic / teratogenic effects:
There were no treatment-related or statistically significant effects on fetal sex ratios, fetal body weights absolute and relative anogenital distance at any exposure concentration level. All dams were pregnant with the exception of one animal (#420) in the 161 ppm (analytical) group. There were no treatment-related differences in the incidence of any fetal alteration in any of the treated groups compared to controls. The small number of alterations observed in fetuses from dams either occurred at low frequencies, in the control group, and/or were not dose related.
Key result
Dose descriptor:
NOAEC
Effect level:
>= 161 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed.
Abnormalities:
no effects observed
Developmental effects observed:
no

- Concentration analysis: Analytically-determined mean chamber D5 vapor concentration values were 0.0 ± 0.0, 32 ± 1.1, 74 ± 2.0, and 161 ± 3.8 ppm over the course of the study.  

The nominal concentration of test material in each chamber was calculated based on the amount of test material fed into the inhalation apparatus each day and the total chamber airflow through the chamber for each exposure period. The nominal concentrations were 29.1 ± 0.6, 81.2 ± 2.1, and 169.7 ± 5.6 for D5 exposure chambers. 

Chamber aerosol concentrations were 0.000 ± 0.0000, 0.004 ± 0.006, 0.036 ± 0.011, and 0.083 ± 0.021 mg/m3 for the 0, 32, 74, and 161 ppm (analytical) D5 exposure chambers, respectively.  

- Chamber temperature: Mean chamber temperature values for all chambers were maintained between 22.4 and 24.4 °C.  

- Chamber humidity: Mean chamber relative humidity was maintained in the range of 35.3 - 38.7% for all exposure chambers.  

- Chamber airflow: The mean chamber airflow in all four exposure chambers was maintained between 463 and 569 L/min.

See attachments for detailed result tables.

Conclusions:
In the inhalation prenatal developmental toxicity study, conducted according to OECD Test Guideline 414 and in compliance with GLP, the concluded NOAEC for maternal and developmental effects was greater than 161 ppm (analytical mean measured, equivalent to 2427 mg/m3), the highest achievable vapour concentration. There were no adverse effects reported in the study.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
2 427 mg/m³
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In an inhalation prenatal developmental toxicity study, conducted according to OECD Test Guideline 414 and in compliance with GLP, groups of 24, time-mated female Crl:CD(SD) rats were exposed to D5 vapour for 6 hours a day, 7 days a week in whole body exposure chambers at concentrations of 0 (filtered air), 30, 70, or 160 ppm on gestation days, (GD) 6-20 (The Dow Chemical Company (Toxicology and Environmental Research and Consulting), 2020).  In-life parameters evaluated for all groups included:  clinical observations, body weight, body weight gain, and feed consumption.  On GD 21 all rats were euthanised, examined for gross pathologic alterations, and blood was collected for thyroid hormone analyses.  Liver, kidneys, thyroid (post-fixation) and gravid uterine weights were recorded, along with the number of corpora lutea, uterine implantations, resorptions, and live/dead fetuses.  Histopathological examination of thyroid was conducted on all pregnant rats.  All fetuses were weighed, sexed, anogenital distance recorded, and examined for external alterations.  Approximately one half of the fetuses were examined for visceral and craniofacial alterations while skeletal examinations were conducted on the remaining fetuses.

Inhalation exposure to D5 vapour up to a concentration of 160 ppm resulted in no treatment-related maternal toxicity.

There were no treatment-related effects on pregnancy rates, resorptions/litters with resorptions, litter size, numbers of corpora lutea or implantations, percent preimplantation loss, percent postimplantation loss, fetal sex ratios, fetal body weights, anogenital distance or gravid uterine weights at any exposure concentration.  There were no treatment-related differences in the incidence of any fetal alteration in any of the treated groups compared to controls.

Therefore, under the conditions of this study, the no-observed-adverse-effect concentration (NOAEC) for maternal and developmental toxicity was 160 ppm (2427 mg/m³), the highest achievable vapour concentration.

Toxicity to reproduction: other studies

Additional information

The potential for D5 to act at the dopamine receptor was investigated in a reliability 2 study (Dow corning Corporation, 2005) which demonstrated that it acts as a dopamine D2-receptor agonist. The estrongenic activity of D5 was investigated in ovariectomised Fischer F344 or Sprague-Dawley rats in reliability 2 studies (Dow Corning Corporation, 2004a; Dow Corning Corporation, 2004b). Both studies demonstrated that D5 did not have any estrogenic, or anti-estrogenic activity.

An in vitro reliability 2 study (Dow Corning Corporation, 2005) evaluated the potential of D5 to act as a progesterone receptor ligand, and found no evidence that it did. Two further reliability 4 studies demonstrated that D5 did not produce an androgenic or anti-androgenic response (Dow Corning, 2003) and that D5 was not a direct competitor at the active estrogen receptor alpha site (Dow Corning, 2002).

Justification for classification or non-classification

Based on the available data D5 does not require classification for toxicity to reproduction according to Regulation (EC) No. 1272/2008.