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Effects on fertility

Description of key information

Data on RA substance 124-40-3 (DMA)


OECD 422: NOAEL reproduction = 75 ppm (highest dose tested)


OECD 443: NOAEL reproduction = 30 ppm (highest dose tested)

Link to relevant study records

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Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Principles of method if other than guideline:
The design of this study is based on OECD Test Guideline 422 with the addition of a 14-day direct exposure of the weanling pups.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: Airgas Specialty Products
- Lot No.: A180338919
- Expiration date: 2020-03-01
- Purity, including information on contaminants, isomers, etc.: 100%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Kept in a controlled temperature area set to maintain 18°C to 24°C
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: CRL
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 12 wks
- Weight at study initiation: 220-423g
- Fasting period before study: no
- Housing: groups up to 3 prior to mating, single afterwards
- Diet (e.g. ad libitum): ad lib. (withheld during exposures)
- Water (e.g. ad libitum): ad lib (withheld during exposures)
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26°C
- Humidity (%): 30-70%
- Air changes (per hr): >=10
- Photoperiod (hrs dark / hrs light): 12h/12h
Route of administration:
inhalation: gas
Type of inhalation exposure (if applicable):
whole body
Vehicle:
clean air
Details on exposure:
Exposures were conducted in four, 2000-L stainless-steel and glass whole-body exposure chambers. One chamber was used for the filtered air control group and 1 chamber was used for each test substance groups. The chambers were operated under dynamic conditions, at a slight negative pressure, and with a minimum of 12 air changes per hour. Air supplied to the whole-body chambers was provided from an in-house compressed nitrogen source and a HEPA- and charcoal-filtered, temperature- and humidity-controlled supply air source. The mean temperature and relative humidity of the exposure atmospheres were to be 19°C to 25°C and 30% to 70%, respectively. Oxygen content of the exposure atmospheres was measured during the method development phase and was 20.9% for all groups.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Exposure concentrations were determined at app. 60 min intervals using a gas chromatograph. Samples were collected from the approximate animal breathing zone.
Duration of treatment / exposure:
6h / day for 28 days (males), 58-65 days (pregnant females), 39-52 days (females that failed to deliver)
Frequency of treatment:
daily (except during parturition from GD 20 to PND 4)
Dose / conc.:
75 ppm
Remarks:
mean analyzed concentration F0: 75.9ppm, F1: 73.9ppm
Dose / conc.:
25 ppm
Remarks:
mean analyzed concentrations F0: 26.9ppm, F1: 25.2ppm
Dose / conc.:
8 ppm
Remarks:
mean analyzed concentration F0: 9.2ppm, F1: 7.6ppm
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dosage levels were determined from results of a previous tolerability/range finding study. In that study, DMA was administered via whole-body inhalation exposure to up to 250 ppm in rats for 14 days (6 hours per day). No test substance-related adverse clinical signs, body weight, or food consumption effects were noted at any exposure level. However, at the histopathology evaluation, degeneration of the transitional and respiratory epithelium, multifocal mixed inflammation, and ulceration with bone atrophy of the nasoturbinates and maxilloturbinates were noted at ≥ 50 ppm. Changes were noted in an exposure-dependent manner and were of minimal grade at the low-exposure level (50 ppm). Based on the corrosive nature of DMA gas and the
findings from the previous tolerability/range finding study, port of entry effects were expected in the current study. Therefore, exposure levels of 8, 25, and 75 ppm were selected for the current study.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily, prior to exposure.
Additional observations were also recorded 0.5-3h after exposure.

BODY WEIGHT and FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly (twice weekly during gestation and lactation)

OTHER: FOB, motor activity, clinical pathology, hematology, serum chemistry, thyroid hormone analysis, implantation sites
Oestrous cyclicity (parental animals):
Vaginal lavages were performed daily from two weeks prior to randomization until mating was observed and on the day of necropsy.
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
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups, thyroid hormone analysis.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: Study day 28
- Maternal animals: Lactation day 29

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs were weighed: Adrenal glands, Brain, Epididymidesa, Heart, Kidneys, Liver, Lungs, Ovaries (with oviducts), Pituitary gland, Prostate gland, Seminal vesicle (with coagulating gland and fluid), Spleen, Testes, Thymus gland, Thyroids (with parathyroids)

The following tissues were prepared for microscopic examination:
Adrenal glands, Aorta, Bone with marrow (sternebrae), Brain, Coagulating glands, Eyes with optic nerve, Gastrointestinal tract, Esophagus, Stomach, Duodenum, Peyer’s Patches, Jejunum, Ileum, Cecum, Colon, Rectum, Heart, Kidneys, Liver (sections of 2 lobes), Lymph node, Axillary, Mandibular, Mesenteric, Lungs (including bronchi, fixed by, inflation with fixative), Nasal cavities with turbinatesb, Ovaries and oviducts, Pancreas, Peripheral nerve (sciatic), Pituitary gland, Prostate gland, Salivary gland (mandibular), Seminal vesicles , Skeletal muscle (quadriceps), Skin with mammary glandd, Spinal cord (cervical), Spleen, Testes with epididymidese and vas deferens, Thymus gland, Thyroids (with parathyroids, if present), Trachea, Urinary bladder, Uterusf with cervix and vagina, All gross lesions
Postmortem examinations (offspring):
SACRIFICE
PND 28 (10males and 10 females were selected and directly exposed to the test substance until PND40)

GROSS NECROPSY
- One pub/sex/litter and all F1 animals sacrificed on PND40 were subjected to a complete necropsy examination, with emphasis on developmental morphology and organs of the reproductive system. All other animals were discarded without examination.

HISTOPATHOLOGY / ORGAN WEIGTHS
The following organs were weighed: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Lungs, Ovaries (with oviducts), Pituitary gland, Prostate gland, Seminal vesicle (with coagulating gland and fluid), Spleen, Testes, Thymus gland, Thyroids (with parathyroids)

The following tissues were prepared for microscopic examination: Thyroid (with parathyroids) (only PND28), nasal cavities with turbinates, all gross lesions
Reproductive indices:
Male and Female mating index
Male and Female fertility index
Male and Female Copulation index
Female conception index
estrous cycle length
pre-coital interval
Offspring viability indices:
Pre-, post implantation loss
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Endocrine findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related microscopic findings were noted in the nasal cavity (Level II) of the 8, 25, and 75 ppm F0 group males and females. Test substance-related lesions within Level II of the nasal cavity included minimal or mild hyperplasia of the respiratory and transitional epithelium and minimal or mild mixed cell inflammation. The transitional and respiratory epithelial hyperplasia was haracterized by a focal disorganized, thickened epithelial surface. There was loss or clustering of goblet cells and absence of cilia in the affected respiratory epithelium. In some animals the hyperplasia was associated with mixed cell inflammation characterized by the presence of variable numbers of neutrophils, lymphocytes, and plasma cells.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
average cycle length varied between 4 and 4.5 days
Reproductive performance:
no effects observed
Description (incidence and severity):
2 females each in the low dose and control group did not become pregnant. All other mating pairs produced offspring. Gestation length was similar in all groups.
Dose descriptor:
LOAEC
Remarks:
local
Effect level:
8 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEC
Remarks:
systemic and reproduction
Effect level:
75 ppm
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
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 test substance exposure. Two (2), 4(4), 5(4), and 6(3) pups (litters) in the control, 8, 25, and 75 ppm groups, respectively, were found dead. Two (2), 0(0), 1(1), and 3(3) pups (litters) in the same respective groups were missing and presumed to have been cannibalized.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weight gain in high dose females was 10% lower compared to controls from PND 25-28.
Mean body weight gain in high dose males and females was lower from PND28-40, resulting in lower absolute body weights up to 5% and 6.3%, respectively.
Sexual maturation:
no effects observed
Anogenital distance (AGD):
no effects observed
Nipple retention in male pups:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related microscopic findings were noted in the nasal cavity (Level I, II, III, and/or IV) of the 8, 25, and 75 ppm F1 group male and females. Test substance-related lesions observed at Levels I through IV were more prominent at Level II and included minimal to marked degeneration of the respiratory, mild to severe degeneration of the transitional epithelium, and minimal to moderate mixed cell inflammation. The degeneration of the transitional and respiratory epithelium was characterized by a disorganized epithelium (Levels I through IV) composed of enlarged, usually vacuolated cells (Levels I through III). There was loss or clustering of goblet cells and absence of cilia in the affected respiratory epithelium. In some animals, the degeneration was associated with ulceration and mixed cell inflammation characterized by the presence of variable numbers of neutrophils, lymphocytes, and plasma cells. Ulcerated surfaces were often covered with a serofibrinous or serocellular exudate and in some areas the adjacent epithelial extended across the defect.
Dose descriptor:
LOAEC
Remarks:
local
Generation:
F1
Effect level:
8 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
systemic / developmental
Generation:
F1
Effect level:
75 ppm
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Endpoint:
extended one-generation reproductive toxicity - with F2 generation (Cohorts 1A, and 1B with extension)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
GLP compliance:
yes (incl. QA statement)
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS [please address all points below]:

- Premating exposure duration for parental (P0) animals: 10 weeks
- Basis for dose level selection: based on OECD422 (for details, see below)
- Inclusion of extension of Cohort 1B: Due to observation of lower mean fertility and copulation/conception indices for F0 males and females, F1 animals assigned to Cohort 1B were bred to obtain reproductive performance data for the F1 generation.
- Termination time for F2: Gestation day 15, because there were no observations related to the progress of parturition or gestation lenghts in the F0 generation nor any effects on F1 pup weights or survival, and hence further assessments of the same endpoints in the next generation were not warranted.
- Exclusion of developmental neurotoxicity Cohorts 2A and 2B: no hints for neurotoxicity in existing rep. dose toxicity studies nor in the current OECD 443
- Exclusion of developmental immunotoxicity Cohort 3: no hints for effects on the immune system in existing rep. dose toxicity studies nor in the current OECD 443
- Route of administration: The route of administration was whole-body inhalation exposure because inhalation would be a likely route of unintended human exposure for this gaseous test substance.
- Other considerations: number of animals: The number of animals selected for this study was based on the OECD Guideline for the Testing of Chemicals, Guideline 443, Extended One-Generation Reproductive Toxicity Study, June 2018, which recommends including a sufficient number of mating pairs to yield at least
20 pregnant females per dose group. Given the possibility of nongravid animals, unexpected deaths, total litter losses, or treatment-related moribundity and/or mortality in each generation of the study, 24 rats/sex/group was an appropriate number of animals to achieve the desired number of animals in each generation.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: Airgas Specialty Products
- Lot No.: A180338919
- Expiration date: 2020-03-01
- Purity, including information on contaminants, isomers, etc.: 100%
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: CRL
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6 wks
- Weight at study initiation: 167-280g
- Fasting period before study: no
- Housing: groups up to 3 prior to mating, single afterwards
- Diet (e.g. ad libitum): ad lib. (withheld during exposures)
- Water (e.g. ad libitum): ad lib (withheld during exposures)
- Acclimation period: yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26°C
- Humidity (%): 30-70%
- Air changes (per hr): >=10
- Photoperiod (hrs dark / hrs light): 12h/12h
Route of administration:
inhalation: gas
Type of inhalation exposure (if applicable):
whole body
Vehicle:
clean air
Details on exposure:
Exposures were conducted in four, 2000-L stainless-steel and glass whole-body exposure chambers.
Oxygen content of the exposure atmospheres was measured during the method development phase of the study using a Dräger PAC III equipped with a calibrated oxygen sensor (Serial No. ERRH-0143, Draeger Safety Inc.; Pittsburgh, PA) and was 20.9% for all groups.
Neat test substance (1,000,000 ppm) was delivered from the original cylinder to a 25-L Tedlar® bag using a 2-stage regulator (Matheson; Montgomeryville, PA). The neat test substance bag was placed into a box that was heated to approximately 50°C using an Omega® heat tape controlled, J-type thermocouple, and temperature controller (Model No. CN370, Omega Engineering, Inc.; Stamford, CT).
Generation bags (250,000 ppm) were prepared by injecting 2 L of the neat test substance into a 10-L Tedlar® bag that was filled with 6 L of nitrogen. The volume of compressed nitrogen was measured using a dry test meter (DTM-200A, Elster American Meter Co.; Nebraska City, NE). The generation bags were placed into a sealed polycarbonate box, one for each test substance chamber. The box was heated to approximately 35°C using a heat pad, J-type thermocouple, and Omega® temperature controller (Model No. 370). Using a regulator (model no. 8802K, Coilhose Pneumatics; East Brunswick, NJ) and a Dwyer rotameter-type flowmeter (Model No. VFB-69-BV), dry compressed air was added to pressurize the generation box. A Dwyer Magnehelic® Indicating Transmitter pressure gauge was used to monitor the box pressure. The positive pressure within the box forced the test substance in the generation bag to be delivered to the chamber inlet using a flowmeter (Model No. 10, Barnant Co. / Gilmont Instruments; Barrington, IL) and needle valve. The gas entered the exposure chamber inlet via 1/8” Teflon tubing where it mixed with dilution supply air prior to entering the exposure chamber.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug or 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): single
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test substance concentration within the exposure chambers were sampled and analyzed at approximately 60-minute intervals using a gas chromatograph. Samples were collected from the approximate animal-breathing zone of the exposure chamber via 1/8-inch Teflon® tubing.
Exposure concentrations were very close to the nominal concentrations.

target analytic result
4ppm 4.3 (F0), 4.2 (F1) ppm
12ppm 11.9 (F0), 11.9 (F1) ppm
30ppm 30.7 (F0), 31.1 (F1) ppm

Homogeneous distribution of the test substance was shown.
Duration of treatment / exposure:
6h/day
Frequency of treatment:
daily
Exposure was suspended from gestation day 21 to PND4.
Details on study schedule:
- Selection of parents from F1 generation: prior to weaning
- Age at mating of the mated animals in the study: 90-120 days
Dose / conc.:
4 ppm
Dose / conc.:
12 ppm
Dose / conc.:
30 ppm
No. of animals per sex per dose:
24 (F0)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The exposure concentrations were determined from results of the previous OECD 422 rat study. Histopathological examination revealed dimethylamine-related adverse findings at all exposure levels in both the F0 and F1 generations. At 8 and 25 ppm, histological findings were generally limited to nasal cavity Level II and included respiratory and transitional epithelial degeneration and/or mixed cell inflammation, without ulceration. In the 75 ppm groups, the changes were consistently observed at multiple nasal cavity levels and ulceration was prominent at Level II. Based on these histopathological findings, the 75 ppm exposure level was considered excessive for an extended one-generation reproductive study and dose levels of 4, 12 and 30 ppm were selected.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: general health, mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily, prior to exposure

BODY WEIGHT: Yes
- Time schedule for examinations: twice weekly, after mating, females were weighted on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and on Lactation Days 1, 4, 7, 11, 14, 21, and 28.

FOOD CONSUMPTION:
- Food consumption for each animal determined weekly, except during mating. For females, food consumption after mating was recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and on Lactation Days 1, 4, 7, 11, 14, 21, and 28.
Food efficiency was calculated.

OTHER:
Thyroid hormone analysis (total T4, TSH): 10 animals / sex / group in week 19 prior to fasting
Hematology and serum chemistry, urinalysis: 10 animals / sex / group in week 19 after fasting
Oestrous cyclicity (parental animals):
F0: Vaginal lavages were performed daily until evidence of mating and on the day of necropsy.
F1: Vaginal lavages were performed daily from the day of vaginal opening to first estrus
cohort 1A: additionally between PND 75 and 91
cohort 1B: additionally 14 days prior to cohabitation until evidence of mating was observed

- Regular cycling (RC): the animal has at least 6 days of data collected and displays 1 complete cycle with no cycles greater than 5 days in duration, no cycles with 3 or more consecutive days of P and/or E, and no cycles less than 4 days in duration.

- Irregular cycling (IC): the animal does not display 1 complete cycle but has at least 1 E and/or P and a partial cycle greater than 5 days, the animal has at least one E present and either at least 1 cycle (complete or partial) greater than 5 days in duration or at least 1 cycle with 3 or more consecutive days of P and/or E, at least 1 cycle less than 4 days in duration, or 1 irregular cycle and 1 regular cycle.

- Non-cycling (NC): no E or P present on any days of estrous cycle determination and at least 5 days of data collected.

- Insufficient data (ID): the animal does not display at least 1 complete cycle but has at least 1 E and/or P and a partial cycle of 5 days or fewer, no E present on any days of estrous cycle determination and 4 or fewer days of data collected, or at least 1 E or P present and only 1-4 days of data collected.
Sperm parameters (parental animals):
Parameters examined in F0 and F1 (cohort 1A) male generations:
testis weight, epididymis weight, sperm count, sperm motility, sperm morphology, sperm production rate, staging
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)

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups, thyroid hormones [PND4 - culled pubs and PND28 - nonselected pubs, PND91 - cohort 1A], balanopreputial separation, vaginal patency, clinical pathology [cohort 1A, PND 91]

GROSS EXAMINATION OF DEAD PUPS:
[no / yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead]

Postmortem examinations (parental animals):
SACRIFICE
F0: study days 134-137

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. The number of former implantation sites was recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
All tissues listed below were examined in the control and high dose. For mid and low dose animals, only gross lesions, target tissues and - for animals with reduced reproductive performance or altered estrous cycle/sperm parameters - reproductive tissues were examined.
Organs weighed: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Lungs, Ovaries (with oviducts), Pituitary gland, Prostate gland, Seminal vesicle (with coagulating gland and fluid), Spleen, Testes, Thymus gland, Thyroids (with parathyroids), Uterus with oviducts and cervix, Levator ani and bulbocavernosus (LABC) muscle group
Organs examined histopathologically: Adrenal glands, Aorta, Bone with marrow (sternebrae), Brain, Coagulating glands, Eyes with optic nerve, Gastrointestinal tract, Esophagus, Stomach, Duodenum, Peyer’s Patches, Jejunum, Ileum, Cecum, Colon, Rectum, Heart, Kidneys, Lacrimal/Harderian glands, Liver (sections of 2 lobes), Lymph node, Axillary, Mandibular, Mesenteric, Levator ani and bulbocavernosus (LABC) muscle group, Lungs (including bronchi, fixed by, inflation with fixative), Nasal cavities with turbinatesb, Ovaries and oviducts, Pancreas, Peripheral nerve (sciatic), Pituitary gland, Pharynx, Prostate, Salivary gland (mandibular), Seminal vesicles , Skeletal muscle (quadriceps), Skin with mammary gland, Spinal cord (cervical), Spleen, Testes with epididymides and vas deferens, Thymus, Thyroids (with parathyroids, if present), Trachea, Urinary bladder, Uterus with cervix and vagina, All gross lesions
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 91 days of age.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
PND4: 1 culled pub/sex/litter with special attention on heart and major vessels
PND28: non-selected pubs with special attention on reproductive organs
PND 91 (cohort 1A)
PND 133-140 (males), GD15 (females): cohort 1B

HISTOPATHOLOGY / ORGAN WEIGTHS
PND4: culled pubs:
tissues preserved: trachea, all gross lesions
PND28 - non-selected pubs:
organs weighed: brain, liver, spleen, thymus, thyroid
tissues preserved: brain, liver, nasal cavities, ovaries, skin with mammary gland, spleen, testes, thymus, thyroid, all gross lesions
PND 91 - cohort 1A
Organ weights and histopathology performed as for F0 generation
cohort 1B
females: laparohysterectomy: number of corpora lutea, number of viable and noncviable embryos, early resorptions, total number of implantations
males and females:
organs weighed: brain, epididymides, levator ani band bulbocavernosus muscle group, pituitary gland, prostate, seminal vesicles, testes
The same tissues as for cohort 1A were preserved, but no histopathology performed
Statistics:
Were applicable, the litter was used as the experimental unit.
Reproductive indices:
Male and female mating index
male and female fertility index
male copulation index
female conception index
estrous cycle length
pre-coital interval
Offspring viability indices:
Mean live litter size
Postnatal survival (PND 0-1, PND 0-4, PND 1-4, PND 4-7, PND 7-14, PND 14-28, PND 4-28)
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
1 high dose male was found dead on study day 46
1 high dose female was found dead on study day 133
Microscopic examination did not reveal a cause of death, but in the absence of any other evidence of systemic toxicity in this dose group, relation to treatment is considered unlikely.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Endocrine findings:
no effects observed
Description (incidence and severity):
Thyroid hormone analysis:
Higher T4 levels noted for F0 males (54.8% and 87.9% respectively) and F0 females (68.7 and 65.3% respectively) in the 12 and 30 ppm groups were considered incidental based on the lack of a dose response (females), atypically low control group mean values in comparison with Charles River Historical Control Data, and the lack of an effect on TSH levels in both males and females. In addition, mean values across all treated groups were within the range of the Historical Control
Data. In the control group, only 5 and 6 (of 10) samples were available for analysis (due to insufficient serum quantity), for both males and females, which may have contributed to the noted differences. Lastly, there were no noted differences in thyroid organ weights or corresponding changes in thyroid histopathology.
Urinalysis findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Dimethylamine was associated with microscopic findings in the nasal cavity (level II) at ≥ 4 ppm in males and females. There was a dose-related increase in incidence and/or severity of respiratory epithelial hyperplasia and transitional epithelial hyperplasia in males and females, and mixed cell inflammation in the males. The mixed cell inflammation was similar in females at 4 and 12 ppm and at a higher incidence in the 30 ppm group.
Histologically, the transitional and respiratory epithelial hyperplasia was characterized by focal to multifocal increased numbers of cells and disorganization. There was loss or clustering of goblet cells and/or decreased cilia in the affected respiratory epithelium. Inflammation, when present, was associated with the hyperplasia and included variable numbers of neutrophils, lymphocytes, and plasma cells. These histologic lesions were considered locally adverse at ≥ 4 ppm based on the presence of inflammation and epithelial hyperplasia.
Histopathological findings: neoplastic:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
A decrease in male and female fertility and copulation/conception was noted in the 30 ppm group, where 5 of 24 mating pairs did not sire a litter. There were no microscopic or macroscopic lesions to account for the reduced fertility with the exceptions of 2 females with abnormally long estrous cycles and histopathological findings of early senescence, which is occasionally observed in this strain of rats. One male was paired with a second female and successfully sired a litter.
Based on these findings, cohort 1B animals were bred to obtain data on reproductive performance. Since no effects on fertility were observed in this cohort, the finding was considered incidental and not related to test substance administration.

Of the remaining groups, none (control), 3 (low dose), and 1 (mid dose) mating pairs did not produce offspring.

No further differences in gestation length, parturition, estrous cycle length, time to mating, implantation sites, and number of offspring were observed.
Dose descriptor:
NOAEC
Remarks:
systemic
Effect level:
30 ppm
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
LOAEC
Remarks:
local
Effect level:
4 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
no
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One cohort 1B male animal in the mid dose was found dead on PND63. No other deaths occurred in all doses including the high dose, so this deaths was considered incidental.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Endocrine findings:
no effects observed
Urinalysis findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Dimethylamine was associated with microscopic findings in the nasal cavity (level II) in all test substance groups for males and females of the F1 Cohort 1A. There was a dose-related increase in incidence and/or severity of respiratory epithelial hyperplasia, transitional epithelial hyperplasia, vacuolar degeneration of the respiratory epithelium, and vacuolar degeneration of the transitional epithelium in males and females at ≥ 4 ppm. Inflammation was similar in all dose groups in males and showed a slightly increased incidence at 12 and 30 ppm in females compared to 4 ppm.

Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
There were no dimethylamine-related effects on primordial/small growing ovarian follicle counts between control and 30 ppm F1 females from Cohort 1A.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The mean ages at the first occurrence of estrus in the 4, 12, and 30 ppm groups (35.8, 36.3, and 36.4 days, respectively) were generally comparable to the control group (36.0 days). In addition, the duration from vaginal opening to first estrus in these same respective groups (2.8, 2.9, and 2.8 days) was generally comparable to the control group (2.7 days). None of the differences were statistically significant.
The mean lengths of estrous cycles in the test substance-treated groups from PND 75-91 were also comparable to the control group value. None of these differences were statistically significant.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Mean testicular and epididymal sperm numbers and sperm production rate, motility, progressive motility, and morphology were similar in all groups.
Reproductive performance:
no effects observed
Description (incidence and severity):
No test substance-related effects on F1 reproductive performance were observed at any exposure concentration. Males that did not sire a litter numbered 0, 2, 1, and 1 in the control, 4, 12, and 30 ppm groups, respectively. Females that had evidence of mating but were nongravid numbered 0, 1, 1, and 1 in the same respective groups.
The mean numbers of days between pairing and coitus in the test substance-exposed groups were comparable to the control group value. The statistically significantly higher precoital interval noted in the 4 ppm group was attributed to a single mating pair in this group that had a precoital interval of 12 days, and successfully produced a litter. The mean lengths of estrous cycles in these groups were also comparable to the control group value. None of these differences were statistically significant.

Mean numbers of corpora lutea, implantation sites, pre- / post implantation loss, and intrauterine survival of the F2 embryos were unaffected by test substance exposure to F1 animals at exposure concentrations of 4, 12, and 30 ppm.
Dose descriptor:
NOAEC
Remarks:
systemic
Effect level:
30 ppm
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
LOAEC
Remarks:
local
Effect level:
4 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
no
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Live litter size and postnatal survival between birth and PND 0 (relative to number born), PND 0–1, 1–4 (pre-selection), 4 (post-selection)–7, 7–14, 14–21, 21-28, and from birth to PND 4 (preselection) and PND 4 (post-selection)–28 were unaffected by the test substance at all dosage concentrations.
Nineteen (8), 25 (8), 8 (4), and 7 (4) pups (litters) in the control, 4, 12, and 30 ppm groups, respectively, were found dead or euthanized in extremis. Six (6), 9 (4), 3 (3), and 3 (3) pups (litters) in the same respective groups were missing and presumed to have been cannibalized.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Lower mean body weight gains were noted across all test substance exposure groups during PND 21-28 resulting in mean pup body weights that were 3.3 to 6.1% lower than the control group on PND 28 for both males and females; differences were dose responsive for females only. Based on the low magnitude of the noted differences, and the transient nature of the deficits versus controls (recovery was noted for both males and females by PND 35), these differences were considered test substance related, but nonadverse.
Sexual maturation:
no effects observed
Anogenital distance (AGD):
no effects observed
Nipple retention in male pups:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
1 pub found dead in the control group had several external malformations.
Kidney cysts and dilated renal pelvis were observed in one pub each in the control, mid and high dose.
Findings on mulitple internal organs were observed in a single mid dose pub on PND 28.
Other effects:
no effects observed
Description (incidence and severity):
No test substance-related difference in thyroid hormone levels were noted for culled pups on PND 4 or for non-selected pubs on PND28 at any exposure level.
Dose descriptor:
NOAEC
Remarks:
systemic
Generation:
F1
Effect level:
30 ppm
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
LOAEC
Remarks:
local
Generation:
F1
Effect level:
4 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Endpoint:
extended one-generation reproductive toxicity - with F2 generation (Cohorts 1A, and 1B with extension)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
See justification attached below
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
GLP compliance:
yes
Limit test:
no
Species:
rat
Sex:
male/female
Route of administration:
inhalation: gas
Type of inhalation exposure (if applicable):
whole body
Dose descriptor:
NOAEC
Remarks:
Reproduction
Effect level:
30 ppm
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Dose descriptor:
NOAEC
Remarks:
Reproduction
Effect level:
30 ppm
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Dose descriptor:
NOAEC
Remarks:
Reproduction
Generation:
F1
Effect level:
30 ppm
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Reproductive effects observed:
no
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Additional information

OECD 422


 


In a GLP study according to OECD 422 (CRL 2020), animals were exposed via whole-body inhalation for 6 hours daily to gas concentrations of 8, 25, and 75 ppm. Males were exposed for 14 days prior to mating and continuing throughout mating for a total of 28 days. Females were exposed for 14 days prior to mating and continuing through Gestation Day 20; exposure resumed on Lactation Day 5 and continued until Lactation Day 28 for a total of 58–65 days. Selected F1 pups were directly exposed from Postnatal Day (PND) 28–40. All F0 animals survived to the scheduled necropsies. There were no test substance-related clinical observations noted at the daily examinations. No test substance-related effects were noted on mean body weights, body weight changes, food consumption, motor activity, serum chemistry, and hematology. Reproductive performance was also unaffected by treatment. Histopathological findings were limited to the nasal cavity (port of entry): Test substance-related lesions within Level II of the nasal cavity in the 8, 25, and 75 ppm group F0 males and females consisted of minimal or mild transitional and/or respiratory epithelial hyperplasia and mixed cell type inflammation in all test substance exposure groups. All F1 animals displayed transitional epithelial degeneration and/or ulceration, respiratory epithelial degeneration, and/or mixed cell inflammation on PND 40. At 8 and 25 ppm, findings were generally limited to nasal cavity Level II and there was no ulceration, while at 75 ppm, changes were consistently observed at multiple levels, ulceration was prominent at Level II.


In conclusion, the NOAECs for systemic toxicity and reproduction were set to 75 ppm, since no adverse effects were observed. For local effects, a NOAEC could not be identified. 8 ppm was thus the LOAEC.


 


OECD 433


In an extended one-generation reproduction toxicity study according to OECD 443, rats were exposed to DMA (124-40-3) via whole body inhalation to concentrations of 4, 12, and 30 ppm. DNT and DIT cohorts were not included, but cohort 1B animals were mated to clarify finding in the F0 generation. In the high dose, five (5) of 24 mating pairs did not produce a litter as opposed to 0, 3, and 1 mating pairs in the control, low, and mid dose groups, respectively. There were no correlating effects on estrous cyclicity, gestation lengths, the process of parturition or on sperm morphology, or histopathology of the reproductive organs. The mean number of days between pairing and coitus were comparable across all groups. F1 animals assigned to Cohort 1B for follow-up reproductive assessments were euthanized on Gestation Day 15 because there were no observations related to the process of parturition or gestation lengths in the F0 generation nor any effects on F1 pup birth weights or survival, and hence further assessments of the same endpoints in the next generation were not warranted. Upon evaluation, there were no test substance-related effects observed in the F1 generation at any exposure concentration. Thus, based on the lack of any effects on reproductive performance of the F1 generation, it was concluded that the lower male and female fertility, copulation and conception indices noted for animals in the 30 ppm group in the F0 generation were incidental and hence unrelated to test substance exposure.


No systemic toxicity was noted in all dose groups with the exception of a slight and transient reduction in body weight in high dose F1 offspring during PND 21-28, mainly in females. Based on the low magnitude (-3.3 and -6.1%) and recovery by PND 35, this finding was considered test substance related, but non-adverse.


Local changes were noted in the nasal cavity (level II) of F0 generation and F1 Cohort 1A males and females at all exposure concentrations. There was transitional and/or respiratory epithelial hyperplasia and mixed inflammation in both generations, along with vacuolar degeneration of the transitional and/or respiratory epithelium of the F1 Cohort 1A animals. These histologic lesions were considered locally adverse at all exposure concentrations based on the presence of inflammation and epithelial hyperplasia.


In conclusion, no NOAEC for local effects was identified. (LOAEC = 4 ppm). In the absence of adverse systemic toxicity and no effects on reproductive performance, sperm measurements, estrous cyclicity, and number, survival, and development of offspring, the NOAECs for systemic toxicity and toxicity to reproduction were set to 30 ppm, the highest dose tested.

Effects on developmental toxicity

Description of key information

No embryotoxic or teratogenic effects observed in rats and rabbits.

Read across was performed to dimethylamine and its salt.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Remarks:
US EPA EPA GLP Standards 40 CFR Part 160 and 40 CFR Part 792 (16-Oct-1989 and 18-Sep-1989, respectively) and the OECD Principles of GLP [C(97) 186/Final] (26-Nov-1997)
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain: New Zealand White [Hra:(NZW)SPF]
- Source: Covance Research Products, Inc., Greenfield, IN
- Age at study initiation: approximately 6 months old upon receipt.
- Weight at study initiation: Body weight values ranged from 2900 g to 3878 g on gestation day 0.
- Fasting period before study: no, food was withheld only during exposure periods.
- Housing: Upon arrival, all rabbits were housed individually in clean, stainless steel cages suspended above ground corncob bedding (Pel O’Cobs®; The Andersons, Cob Products Division, Maumee, OH). During exposure period, animals were individually housed in stainless steel wire mesh caging.
- Diet (e.g. ad libitum): The basal diet: PMI Nutrition International, LLC Certified Rabbit LabDiet® 5322. The basal diet was offered in 25-g increments 3 times per day on the day of arrival and in increased amounts over the next few days, until the animals gradually achieved ad libitum status prior to the exposure period; basal diet was offered ad libitum throughout the study, except during the exposure periods when food was withheld.

Kale (1 leaf at each occasion) was provided to each animal daily for environmental enrichment and to aid in maintaining the animal's gastrointestinal health, beginning upon animal receipt and continuing throughout the duration of the study. Kale present in the cage was discarded at the time of providing a new leaf.
Use of non-certified kale did not have an adverse impact on the quality or integrity of the data or the outcome of the study as kale is commonly regarded as safe and is intended for human consumption.

- Water (e.g. ad libitum): Municipal water. Reverse osmosis purified (on site) drinking water, delivered by an automatic watering system, was provided ad libitum throughout the study, except during the exposure periods when water was withheld.
- Acclimation period: not specified. However, it is reported that animals were received on gestation day 3 or 4 and the exposure started from gestation day 7.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19°C ± 3° (66°F ± 5°F)
- Humidity (%): 50% ± 20%,
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
21-Sep-2015 to 13-Oct-2015 (Test substance exposure period (Phase I))
02-Nov-2015 to 24-Nov-2015 (Test substance exposure period (Phase II))
Route of administration:
inhalation: gas
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: humidified filtered air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Exposures were conducted using four 1500-L glass and stainless steel whole-body exposure chambers. One 1500-L chamber was dedicated to the filtered-air control group and three 1500-L chambers were dedicated to each of the 3 exposure levels. One chamber was dedicated for each group for the duration of the study. Chamber supply air was provided from a HEPA- and charcoal-filtered, temperature- and humidity-controlled source. All exposure chamber exhaust passed through the facility exhaust system that included charcoal- and HEPA-filtration units.

- Method of holding animals in test chamber: Animals were individually housed in stainless steel wire mesh caging and food and water were withheld during the exposure periods. Animals were housed in a normal animal colony room during non-exposure hours. Prior to each exposure, the animals were transferred to exposure caging and transported to the exposure room. Animals were then exposed for the requisite duration and returned to their home cages in the animal colony room. Animals were housed individually in standard exposure batteries of appropriate size for the whole-body chamber in use during exposure periods. To ensure a similar exposure for all animals, the exposure batteries were rotated daily amongst 3 chamber positions.

- Source and rate of air: Test substance atmosphere was generated by releasing test substance gas (1,000,000 ppm) from the original cylinder. The gas cylinder was heated using heating pads controlled by temperature controllers and J-Type thermocouples. A 2 stage regulator with pressure gauges was used to control test substance flow from the cylinder to a needle valve through 1/8-inch stainless steel tubing. Test substance from the regulator was delivered to a manifold system equipped with a pressure gauge to monitor manifold pressure. The manifold delivery line was heated using a heat tape with a temperature controller and a J Type thermocouple. The manifold was used to distribute test substance to each exposure chamber through 1/8-inch Teflon® tubing. The test substance gas flow to the manifold was controlled using a needle valve and metered using rotameter-type flowmeters.

- Temperature, humidity, pressure in air chamber: Temperature, relative humidity, chamber ventilation rate, and negative pressure within the exposure chambers were continually monitored and recorded approximately every 45 minutes during the 6-hour exposure periods. The mean temperature and mean relative humidity were to be between 16°C to 22°C and 30% to 70%, respectively.

- Air flow rate: Chamber airflow rates were monitored using a sharp edge orifice meter and Dwyer Magnehelic® Indicating Transmitter pressure gauge (Dwyer Instruments, Inc.; Michigan City, IN). Each gauge was calibrated for conversion from pressure to airflow in standard liters per minute through the use of a Fox Gas Mass Flowmeter Transmitter (Model FT2, Fox Thermal Instruments; Marina, CA).

- Air change rate: at least 12 air changes per hour.

- Treatment of exhaust air: Test substance gas was directed to the chamber inlet through a 3-way valve where it was mixed and diluted with facility dilution supply air. The 3-way valve was used to divert the flow of the test substance gas from the chamber directly to facility exhaust, if necessary. The test substance delivery lines from the 3-way bypass valve to the chamber inlet were 1/4 inch stainless-steel tubing heated using a heat tape with temperature controllers and J-Type thermocouples.
- Oxygen content was measured during the method development phase and was 20.9% for all chambers.

TEST ATMOSPHERE
- Brief description of analytical method used: GC
- Samples taken from breathing zone: yes.

VEHICLE (if applicable)
The control substance used for exposure of the control group (Group 1) was humidified, filtered air.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Due to the use of a single cylinder of test substance for generation of all test substance atmospheres, nominal concentrations were not calculated. Test substance usage was documented in the study records.

Analyzed exposure concentrations were determined at approximately 45-minute intervals using an appropriate gas chromatography (GC) method. Samples were collected from the approximate animal breathing zone of each exposure chamber via 1/8-inch Teflon® tubing. Exposure atmosphere samples were collected automatically using an external multi position valve. Gas sample injection into the chromatography column occurred via an internal gas-sampling valve with a sample loop. The chromatograph was displayed, the area under the sample peak was calculated and stored, and the concentration in parts per million (ppm) was calculated.
Duration of treatment / exposure:
6 hours per day
Frequency of treatment:
once a day
Duration of test:
during gestation days 7-28
Dose / conc.:
0 ppm (analytical)
Remarks:
Due to the use of a single cylinder of test substance for generation of all test substance atmospheres, nominal concentrations were not calculated. Test substance usage was documented in the study records.
Dose / conc.:
50 ppm (analytical)
Remarks:
Due to the use of a single cylinder of test substance for generation of all test substance atmospheres, nominal concentrations were not calculated. Test substance usage was documented in the study records.
Dose / conc.:
100 ppm (analytical)
Remarks:
Due to the use of a single cylinder of test substance for generation of all test substance atmospheres, nominal concentrations were not calculated. Test substance usage was documented in the study records.
Dose / conc.:
250 ppm (analytical)
Remarks:
Due to the use of a single cylinder of test substance for generation of all test substance atmospheres, nominal concentrations were not calculated. Test substance usage was documented in the study records.
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: pre-test:
In WIL 235503 (Weinberg, 2015), nonpregnant rabbits were exposed to 50, 100, 150, 300, and 700 ppm of the test substance atmospheres for 6-hours (whole-body) per day for 10 consecutive days. Exposure of 700 ppm was not tolerated and the animals were euthanized prior to the end of the first day of exposure. On study day 1, observations of labored respiration were noted at the 300 ppm exposure concentration, but all rabbits survived and this observation was not noted following the first day of exposure. There were no gross observations at necropsy from the rabbits that survived to scheduled euthanasia. Based on these data, exposure concentrations of 50, 150, 200, and 250 ppm were selected for a range-finding study in pregnant rabbits (Charlap, 2015, WIL-235501). All animals survived to scheduled euthanasia. Decreased respiration was noted in animals at the approximate mid-point of exposure observation from 150 ppm through 250 ppm (highest exposure concentration). Additional observations at the approximate mid-point of exposure observation included clear nasal discharge in the 250 ppm group and wet clear material around the nose in the 150 ppm group (although only a single incidence) through the 250 ppm group. Clear material around the mouth was also noted in 200 and 250 ppm groups. Observations at 1 2 hours following exposure included rales in the 200 and 250 ppm groups and clear material around the nose in the 150, 200, and 250 ppm groups. Body weight and food consumption data were generally comparable across groups.
Based on these data, target exposure concentrations of 0, 50, 100, and 250 ppm were selected for this definitive study.

- Rationale for animal assignment (if not random): each animal judged to be in good health and meeting acceptable gestation day 0 body weight requirements was selected for use in the computerized randomization procedure based on body weight stratification in a block design.
Separate randomizations were conducted for Phases I and II. Replacement animals were arbitrarily assigned based on body weight prior to the initiation of exposure

- Other: the study was conducted in two phases:
The number of animals selected for this study (24 females/group) was based on the United States EPA Health Effects Test Guidelines: OPPTS 870.3700, Prenatal Development Toxicity Study, Aug-1998 and the OECD Guidelines for the Testing of Chemicals Guideline 414, Prenatal Developmental Toxicity Study, Jan 2001, which recommend evaluation of approximately 20 females with implantation sites at necropsy. Given the possibility of nongravid animals, unexpected deaths, or treatment-related moribundity and/or mortality, this was an appropriate number of animals to obtain a sample size of 20 at termination. In addition, due to limitation of exposure chamber size, only 12 rabbits could be placed into each exposure chamber; therefore, 24 rabbits was the maximum number of animals per group that could be placed on study to allow completion of the study in 2 phases.
Fifty-two time-mated female New Zealand White rabbits were received in good health from Covance Research Products, Inc., Greenfield, IN, on 18-Sep-2015 (first shipment; Phase I). An additional 52 rabbits of the same strain were received from the same supplier on 30-Oct-2015 (second shipment; Phase II). The time-mated rabbits were received on gestation day 3 or 4; a breeding record was provided by the supplier and is maintained in the study records.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All rabbits were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Individual clinical observations were recorded daily from the day of receipt through gestation day 29 (prior to exposure during the treatment period). Animals were also observed for signs of toxicity at the approximate midpoint of exposure and 1-2 hours following exposure. The absence or presence of findings was recorded for individual animals at observations conducted 1-2 hours following exposure. Only significant findings were recorded at the approximate midpoint of exposure.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual maternal body weights were recorded on gestation days 0 (by supplier under conditions that were not compliant with GLPs, but in accordance with the supplier’s SOPs), 4, and 7-29 (daily). Group mean body weights were calculated for each of these days. Mean body weight changes were calculated for each corresponding interval and also for gestation days 7-10, 10-13, 13-20, 20-29, and 7-29.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Individual food consumption was recorded on gestation days 4-29.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Food intake was reported as g/animal/day and g/kg/day for the corresponding body weight change intervals.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

POST-MORTEM EXAMINATIONS: Yes
The laparohysterectomies and macroscopic examinations were performed blind to treatment group.
- Sacrifice on gestation day 29
- Organs examined: The thoracic, abdominal, and pelvic cavities were opened by a ventral mid line incision, and the contents were examined.

OTHER: Ocular Irritation Observations
Examination of ocular irritation in both eyes of all animals was performed on gestation day 3 or 4 (day of animal receipt) and gestation day 28 in accordance with the method of Draize (1965) and was facilitated by use of a direct ophthalmoscope, as necessary. Sodium fluorescein was used to aid in revealing possible corneal injury at all examinations.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
The thoracic, abdominal, and pelvic cavities were opened by a ventral mid line incision, and the contents were examined. In all instances, the postmortem findings were correlated with the antemortem observations, and any abnormalities were recorded. The uterus and ovaries were then exposed and excised.
Examinations included:
The number of corpora lutea on each ovary was recorded. The trimmed uterus was weighed and opened, and the number and location of all fetuses, early and late resorptions, and the total number of implantation sites were recorded. The placentae were also examined. The individual uterine distribution of implantation sites was documented using the following procedure. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn.
- Gravid uterus weight: Yes
Gravid uterine weight was collected and net body weight (the gestation day 29 body weight exclusive of the weight of the uterus and contents) and net body weight change (the gestation day 0 29 body weight change exclusive of the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy.

- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss (Salewski, 1964).
Maternal tissues were preserved in 10% neutral buffered formalin for possible future histopathologic examination only as indicated by the gross findings. Representative sections of corresponding organs from a sufficient number of control animals were retained for comparison. The carcass of each female was then discarded.
Fetal examinations:
- External examinations: Yes: [all per litter]: each viable fetus
Fetal examinations were performed blind to exposure group. Each viable fetus was examined externally, individually weighed, euthanized by a subcutaneous injection of sodium pentobarbital in the scapular region, and tagged for identification. Fetal tags contained the WIL Research study number, the female number, and the fetus number. The detailed external examination of each fetus included, but was not limited to, an examination of the eyes, palate, and external orifices, and each finding was recorded. Crown rump measurements, degrees of autolysis and gross examinations, if possible, were recorded for late resorptions, and the tissues were discarded.

- Soft tissue examinations: Yes: [all per litter]: each viable fetus
Each viable fetus was subjected to a visceral examination using a modification of the Stuckhardt and Poppe fresh dissection technique to include the heart and major blood vessels (Stuckhardt and Poppe, 1984). The sex of each fetus was determined by internal examination. Fetal kidneys were examined and graded for renal papillae development (Woo and Hoar, 1972).

- Skeletal examinations: Yes: [all per litter]
Following fixation in alcohol, each fetus was stained with Alizarin Red S (Dawson, 1926) and Alcian Blue (Inouye, 1976). Fetuses were then examined for skeletal malformations and developmental variations.

- Head examinations: Yes: [half per litter]
Heads from approximately one half of the fetuses in each litter were placed in Harrison’s fixative for subsequent soft tissue examination by the Wilson sectioning technique (Wilson, 1965). The heads from the remaining one half of the fetuses were examined by a midcoronal slice. All carcasses were eviscerated and fixed in 100% ethyl alcohol.
Statistics:
All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance exposed group to the control group. Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Data obtained from nongravid animals were excluded from statistical analyses.
Maternal body weights (absolute and net), body weight changes (absolute and net), and food consumption, gravid uterine weights, numbers of corpora lutea, implantation sites, and viable fetuses, and fetal body weights (separately by sex and combined) were subjected to a parametric one way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance exposed groups to the control group. Mean litter proportions (percent per litter) of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, pre and post implantation loss, and fetal sex distribution), total fetal malformations and developmental variations (external, visceral, skeletal, and combined), and each particular external, visceral, and skeletal malformation or variation were subjected to the Kruskal Wallis nonparametric ANOVA test (Kruskal and Wallis, 1952) to determine intergroup differences. If the nonparametric ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test (Dunn, 1964) was used to compare the test substance exposed groups to the control group.
Indices:
Intrauterine data were summarized using 2 methods of calculation:
- Postimplantation Loss/Litter based on 1) group mean litter basis and on 2) proportional litter basis (in %);
- Sum of postimplantation losses per group was calculated based on proportional litter basis (please see below table 1).

The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison and calculating the number of affected fetuses in a litter on a proportional basis as presented in table 2 (please see below).
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: the higher incidence clinical observations (rales, elevated head, and clear material findings), mean body weight losses, and lower food consumption in the 250 ppm group.

Details on maternal toxic effects:
All females in the control and test substance-exposed groups survived to the scheduled necropsy on gestation day 29.
Test substance-related adverse rales were noted in 250 ppm group at the daily examinations and at 1-2 hours following exposure throughout the exposure period. An increase in the incidence of elevated head and clear material around the mouth was noted at the approximate mid-point of each daily exposure for the 250 ppm group throughout the exposure period compared to the control group. Clear material around the eyes was noted primarily for the 250 ppm group at 1-2 hours following exposure and at the daily examinations beginning on gestation day 14 and continuing through euthanasia. Clear material around the nose was noted for all test substance-exposed groups at 1-2 hours following exposure throughout the exposure period; this finding was also noted for the majority of females in the 250 ppm group at the daily examinations and the mid-point of exposure.
Other clinical findings, including brown, red, and or yellow material on various body surfaces (nose and/or anogenital region), occurred infrequently and/or in a manner that was not exposure-related.

A test substance-related significant (p<0.01) mean body weight loss was noted in the 250 ppm group during the gestation day 7-10 interval compared to a mean body weight gain in the control group. Mean body weight gains in the 250 ppm group were similar to the control group throughout the remainder of the treatment period (gestation days 10-29). The initial mean body weight loss resulted in a significantly (p<0.05) lower mean body weight gain for the entire exposure period (gestation day 7-29) at 250 ppm. However, the mean body weight loss was not of sufficient magnitude to result in a lower mean body weight at the end of the exposure period. In addition, a larger mean net body weight loss (not statistically significant) was noted in the 250 ppm group when compared to the control group. Mean net body weight and gravid uterine weight in this group were similar to the control group.
Mean maternal body weights, body weight gains, net body weights, net body weight gains, and gravid uterine weights in the 50 and 100 ppm groups were unaffected by test substance exposure. Differences from the control group were slight and not statistically significant, with the following exception. A significantly (p<0.05) higher mean body weight gain was noted in the 100 ppm group during gestation day 21-22. This difference was transient and not dose-responsive.

Test substance-related lower mean food consumption (g/animal/day and g/kg/day) was noted in the 250 ppm group during the first 5 days of exposure (gestation days 7-12; differences were generally significant [p<0.05 or p<0.01]). For the remainder of the exposure period slightly lower mean food consumption was noted for the 250 ppm group compared to the control group; differences were significant (p<0.05) during gestation days 19-20 and 20-21 (g/animal/day only). Lower mean food consumption noted in the 250 ppm group correlated to lower mean body weight gains during the exposure period.
Food consumption, evaluated as g/animal/day and g/kg/day, in the 50 and 100 ppm groups was unaffected by test substance exposure. Differences from the control group were slight and not statistically significant.

Details on ocular irritation observations, maternal necropsy data and on laparohysterectomy on gestation day 29 are presented below in the "Any other information on results incl.tables".
Dose descriptor:
NOAEC
Effect level:
100 ppm (analytical)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects. Remark: no statistically significant malformations or developmental variations were atributed to the test substance.

Details on embryotoxic / teratogenic effects:
The numbers of fetuses (litters) available for morphological evaluation were 209(21), 191(22), 211(23), and 195(21) in the control, 50, 100, and 250 ppm groups, respectively. Malformations were observed in 7(7), 3(3), 7(4), and 6(3) fetuses (litters) in these same respective exposure groups and were considered spontaneous in origin. When the total malformations and developmental variations were evaluated on a proportional basis, no statistically significant differences from the control group were noted. Fetal malformations and developmental variations, when observed in the test substance exposed groups, occurred infrequently or at a frequency similar to that in the control group, did not occur in an exposure-related manner, and/or were within the WIL Research historical control data ranges (Appendix F). Based on these data, no fetal malformations or developmental variations were attributed to the test substance.

Dose descriptor:
NOAEC
Effect level:
250 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Embryotoxic / teratogenic effects:no effects.
Abnormalities:
no effects observed
Developmental effects observed:
no

The overall mean analyzed concentrations for each group during Phases I and II are presented below:

Table 1. Mean Analyzed Exposure Concentration (Phase I)

Exposure System:

1

2

3

4

Target Concentration (ppm):

0

50

100

250

Study Mean Concentration (ppm):

0

50

103

251

Standard Deviation:

0.0

5.4

4.3

7.9

N:

23

23

23

23

 

Table 2. Mean Analyzed Exposure Concentration (Phase II)

Exposure System:

1

2

3

4

Target Concentration (ppm):

0

50

100

250

Study Mean Concentration (ppm):

0

50

100

247

Standard Deviation:

0.0

3.0

2.7

7.2

N:

23

23

23

23

Maternal data

Ocular Irritation Observations

There were no test substance-related effects on the eyes for any animals. There were some animals that were noted with conjunctival hyperemia/congestion or corneal opacity in the test substance groups. However, the findings were noted in a manner that was not exposure-related, occurred in single animals, and/or occurred at a similar incidence in the control group or during the pre-exposure assessment on gestation day 3 or 4.

 

At the scheduled necropsy on gestation day 29, no test substancerelated internal findings were observed at any exposure level. Macroscopic findings observed in the test substanceexposed groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not exposurerelated. All females were gravid with the exception of 3, 2, 1, and 3 female(s) in the control, 50, 100, and 250 ppm groups, respectively.

 

Gestation Day 29 Laparohysterectomy

Intrauterine growth and survival were unaffected by test substance exposure at exposure levels of 50, 100, and 250 ppm. Parameters evaluated included post implantation loss, live litter size, mean fetal body weights, and fetal sex ratios. Mean numbers of corpora lutea and implantation sites were similar across all groups. Mean litter proportions of pre-implantation loss were higher in the test substance-exposed groups (9.9%, 10.5%, and 7.7% for the 50, 100, and 250 ppm groups, respectively) compared to the control group (2.3%) but were not consider test substance-related because the initiation of exposure started after implantation and the values in the test substance groups were similar to the WIL Research historical control mean (8.1 ± 2.95% per litter; Appendix F). All other differences from the control group were slight and not statistically significant.

 

Fetal morphological data

The numbers of fetuses (litters) available for morphological evaluation were 209(21), 191(22), 211(23), and 195(21) in the control, 50, 100, and 250 ppm groups, respectively. Malformations were observed in 7(7), 3(3), 7(4), and 6(3) fetuses (litters) in these same respective dosage groups and were considered spontaneous in origin.

 

External Malformations and Variations

External malformations were limited to 2 fetuses in the 100 ppm group and 1 fetus in the 250 ppm group. Fetus nos. 3178-02 and 3178-07 in the 100 ppm group were noted with disseminated subcutaneous hemorrhage. In the 250 ppm group, fetus no. 5220-09 was noted with a short tail; skeletally, this finding consisted of absent, fused, small, and misshapen caudal vertebra. These malformations were noted in single fetuses or litters and/or in a manner that was not exposure-related, and therefore were not considered test substancerelated.

There were no external developmental variations noted for fetuses at any exposure level.

 

Visceral Malformations and Variations

Visceral malformations were observed in 7(7), 3(3), 1(1), and 3(2) fetuses (litters) in the control, 50, 100, and 250 ppm groups, respectively. A visceral malformation of diaphragmatic hernia (portion of left lobe of liver and stomach protruded into the thoracic cavity though an opening in the diaphragm) was noted for 2 fetuses(nos. 5220-06 and 5220-09) from the same litter in the 250 ppm group. The incidence of this malformation at 250 ppm(1.2% per litter) was slightly outside of the WIL Research historical data range (0% to 1.0% per litter; Appendix F). However, the incidence was not statistically significant when compared to the concurrent control group, diaphragmatic hernia has been previously observed in 2 fetuses from 1 control group in the WIL Research historical control data (version 4.3 full), and this finding is the second most common visceral malformation observed in the WIL Research historical control data (Appendix F). In addition, no other effects on fetal morphology were observed in the 250 ppm group. Therefore, the 2 diaphragmatic hernias observed in a single litter in the 250 ppm group were not considered to be test substance-related.

Lobular agenesis of the lungs (right accessory lobe absent) was noted for 6(6), 2(2), 1(1), and 1(1) fetuses (litters) in the control, 50, 100, and 250 ppm groups, respectively. An enlarged heart (all chambers), interventricular septal defect (an opening in the anterior portion of the septum), and misshapen papillary muscles were noted for fetus no. 5214-01 in the 50 ppm group. The aforementioned visceral malformations were not consisted test substance-related because they occurred in single fetuses, similarly in the control group, and/or in a manner that was not dose-related. Fetus no. 5232-09 in the control group was noted with folded retina.

No test item-related visceral developmental variations were noted. Findings observed in the test itemtreated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the WIL Research historical control data (Appendix F).Red fluid abdominal cavity was noted for a single fetus (no. 5214-01) in the 50 ppm group. This finding was not classified as either a malformation or developmental variation, was not included on the summary tables, and was not considered to be test substance-related because it occurred in single fetus and in a manner that was not exposure-related. Skeletal Malformations and Variations Skeletal malformations were noted for 4(2) and 4(3) fetuses (litters) in the 100 and 250 ppm groups, respectively. Fetus no. 3156-10 in the 100 ppm group and fetus nos. 3168-03 and 5220-06 in the 250 ppm group were noted with vertebral anomalies without associated rib anomalies(absent, extra, fused, mal positioned, or mal proportioned arches or fused, extra, misshapen, mal positioned, or mal proportioned centrum). Fetus nos. 3156-04 and 5235-01 in the 100 ppm group and fetus no. 3168-08 in the 250 ppm group were noted with vertebral central anomalies(bipartite, fused, mal positioned, or mal proportioned centrum). Fetus no. 3156-13 in the 100 ppm group was noted with severely maligned sternebra(e). Fetus no. 5194-02 in the 250 ppm group was noted with a rib anomaly (forked rib and mal positioned costal cartilage) and costal cartilage anomaly (right costal cartilage no. 7 bifurcates, rejoins and associates with sternum in normal no. 7 position). The aforementioned malformations were noted in a single fetus and/or mean litter proportions were within the WIL Research historical control data ranges(Appendix F)and therefore they were not considered test substancerelated. No test substance-related skeletal developmental variations were noted. Findings observed in the test substanceexposed groups were noted infrequently, similarly in the control group, were not observed in an exposure-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the WIL Research historical control data (Appendix F).

Conclusions:
Based on the higher incidence clinical observations (rales, elevated head, and clear material findings), mean body weight losses, and lower food consumption in the 250 ppm group, an exposure level of 100 ppm was considered to be the no observed adverse effect concentration (NOAEC) for maternal toxicity. There were no test substance-related effects on intrauterine growth, survival, and fetal morphology at any exposure concentration; therefore, the NOAEC for embryo/fetal development was 250 ppm when dimethylamine was administered via whole body inhalation exposure for 6 hours per day from gestation days 7 through 28 to time-mated New Zealand White rabbits.
Executive summary:

The objectives of the study were to determine the potential of the test substance, dimethylamine, to induce developmental toxicity after maternal exposure from implantation to 1 day prior to expected parturition, to characterize maternal toxicity at the exposure levels tested, and to determine a no-observed-adverse-effect concentration (NOAEC) for maternal toxicity and developmental toxicity (WIL Research, 2016).

Dimethylamine (DMA) was administered via whole-body inhalation exposure to 3 groups of 24 time-mated female New Zealand White [Hra:(NZW)] rabbits for 6 hours per day from gestation days 7 through 28. Target exposure concentrations were 50, 100, and 250 ppm for Groups 2, 3, and 4, respectively. A concurrent control group (Group 1) composed of 24 time-mated females was exposed to humidified, filtered air on a comparable regimen. Due to limitation of exposure chamber size, only 12 rabbits could be placed into each exposure chamber; therefore, this study was conducted in 2 phases, with 12 animals/group in each phase. The females were approximately 6 months of age at the initiation of dose administration. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. The eyes of all animals were examined for ocular irritation on gestation day 3or 4 (day of animal receipt) and gestation day 28. On gestation day 29, a laparohysterectomy was performed on each female. The uteri, placentae, and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

All females in the control and test substance-exposed groups survived to the scheduled necropsy on gestation day 29.

Rales were noted in 250 ppm group at the daily examinations and at 12 hours following exposure throughout the exposure period. An increase in the incidence of elevated head was also noted in the 250 ppm group at the mid-point of exposure throughout the exposure period. Clear material around the eyes, mouth, and nose were noted at the daily examinations, approximate mid-point of each daily exposure, and/or 12 hours following exposure for the 250 ppm group throughout the exposure period compared to the control group. The aforementioned clinical observations at 250 ppm were considered test substance-related and adverse. In addition, test substance-related clear material around the nose was noted for 50 and 100 ppm groups at 12 hours following exposure throughout the exposure period. In the absence of other signs of toxicity at 50 and 100 ppm, and because the material findings did not persist to the daily examinations, they were not considered adverse at these exposure levels.

Test substance-related mean body weight losses were noted in the 250 ppm group during gestation days 7-10 compared to mean body weight gains in the control group. Corresponding lower mean food consumption was noted in this group generally throughout the exposure period. The initial mean body weight losses in the 250 ppm group were not of sufficient magnitude to result in lower mean body weight at the end of the exposure period. A mean net body weight loss was noted in the 250 ppm group. Mean maternal body weights, net body weights, and gravid uterine weights in all test substance-exposed groups and mean body weight gains and net body weight changes in the 50 and 100 ppm groups were unaffected by test substance exposure.

There were no remarkable ocular or macroscopic findings in any exposure group.

Intrauterine growth, survival, and fetal morphology (external, visceral, and skeletal) were unaffected by test substance exposure at all exposure levels.

Based on the higher incidence clinical observations (rales, elevated head, and clear material findings), mean body weight losses, and lower food consumption in the 250 ppm group, an exposure level of 100 ppm was considered to be the noobservedadverseeffect concentration (NOAEC) for maternal toxicity. There were no test substance-related effects on intrauterine growth, survival, and fetal morphology at any exposure concentration; therefore, the NOAEC for embryo/fetal development was 250 ppm when dimethylamine was administered via wholebody inhalation exposure for 6 hours per day from gestation days 7 through 28 to time-mated New Zealand White rabbits.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP compliant
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
yes
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 10-15 weeks
- Housing: singly
- Diet (e.g. ad libitum): ad libitum (Kliva maintenance diet mouse/rat "GLP"; PROVIMI KLIBA SA
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): 10 times
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
The test substance solutions were prepared at the beginning of the administration period and thereafter at intervals of 3 or 7 days, which took into account the analytical results of the stability verification.
For the preparation of the solutions, appropriate amounts of the test substance was weighed in graduated measuring flasks, topped up with drinkingwater and subsequently intensely shaken.

The drinking water was regularly assayed for chemical contaminants both by the municipal
authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring of
BASF SE as well as for bacteria by a contract laboratory.
On the basis of the analytical findings, the drinking water was found to be suitable. German
Drinking Water Regulation (“Trinkwasserverordnung”) served as a guideline for maximum
tolerable contaminants.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analyses were carried out as a separate study at the test facility Analytical Department of
BASF SE, Ludwigshafen, Germany, under the responsibility of the Study Director of this test
facility. The study was carried out in compliance with the Principles of Good Laboratory
Practice. The analytical reports can be found in PART III (Supplement).
Analytical verifications of the stability of the test substance in drinking water for a period of at
least 10 days at room temperature were carried out before the study was initiated (analytical
report 07L00385).
Samples of the test substance solutions were sent to the analytical laboratory twice during
the study period (at the beginning and towards the end) for verification of the concentrations.
Given that the test substance is completely miscible with water, solutions were considered to
be homogenous without further analysis.
Details on mating procedure:
The animals were paired by the breeder ("time-mated") and supplied on GD 0 (= detecion of vaginal plugs/sperm).
Duration of treatment / exposure:
Gesation day 6 - 19
Frequency of treatment:
Once daily
Duration of test:
On GD 20 all surviving females were sacrificed.
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg b.w.
Basis:
nominal in water
No. of animals per sex per dose:
25 females per dose group
Control animals:
yes, concurrent vehicle
Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- determined for GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations:

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20


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:
Each fetus was weighed, sexed, and external tissues and all orifices were examined macroscopically.

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
Statistics:
DUNNETT-test
FISHER`S EXACT test
WILCOXON-test
Historical control data:
yes
Details on maternal toxic effects:
Details on maternal toxic effects:
The test substance did not cause any mortality. Test substance-related relevant clinical effects were only seen in the high-dose dams (1000 mg/kg bw/d), i.e. salivation after treatment and decreased food consumption, although the latter did not affect body weight, body weight gain, net body weight gain and uterus weight. At necropsy, no test substance-related findings findings were noted in any of the dams. The temporary salivation was likely to be induced by the taste of the test substance or by local irritation of the upper digestive tract. It was not considered to be a sign of systemic toxicity. All animals of the low-, mid- and high-dose groups showed yellowish discolored urine which occurred from GD 8 onwards and persisted until the end of the study. The urine discoloration was a sign of systemic availability of the test substance and happened most likely due to the excreted test compound or its metabolite(s). This finding has been considered to be treatment-related but was not assessed as an adverse effect. No differences of toxicological relevance between the control and the dose groups were determined for reproductive parameters such as conception rate, mean number of corpora lutea, mean number of implantations, pre- and postimplantation losses, live fetuses and fetal sex ratio.
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Basis for effect level:
food consumption and compound intake
Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
Examination of the fetuses revealed incidental fetal external, soft tissue and skeletal malformations in individual litters of the low- and the mid-dose groups as well as the control. Since malformations only occurred in one litter of the low- and one litter of the mid-dose group, it is reasonable to consider a spontaneous background in single animals rather than a test substance-induced effect. A consistent pattern and a dose-response relationship were missing. Thus, a test substance-related effect on ontogeny is not assumed. No external variation was noted. Four soft tissue and a broad range of skeletal variations occurred in every test group including the control. All of these variations are documented at a comparable frequency in the historical control data (Part III, Supplement). A spontaneous origin is also assumed for the unclassified cartilage observations, which were recorded for fetuses of all dose-groups including the control. Character as well as distribution of all of these findings did not suggest a relation to treatment. In summary, there was no evidence of an adverse effect of Dimethylamine hydrochloride on fetal morphology at any dose level tested.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed
Abnormalities:
no effects observed
Developmental effects observed:
no

Ther were no test substance-related mortalities in any of the female animals in any of the groups.

The following females were excluded from the above-mentioned calculations:

Testgroup 0 (0 mg/kg bw/d): female No. 4 - not pregnant

Testgroup 1 (100 mg/kg bw/d): female No. 37 - not pregnant

Testgroup 2 (300 mg/kg bw/d): females Nos. 60 and 74 - not pregnant

Conclusions:
In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 300 mg/kg bw/d based on decreased food consumption and salivation after treatment in the high-dose dams (1000 mg/kg bw/d).
The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 1000 mg/kg bw/d because there was no evidence of an adverse effect of the test compound on fetal morphology.
Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
See attached read-across justification
Reason / purpose for cross-reference:
read-across source
Dose descriptor:
NOAEC
Effect level:
100 ppm
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
Dose descriptor:
NOAEC
Effect level:
250 ppm
Sex:
male/female
Basis for effect level:
other: no effects observed
Abnormalities:
no effects observed
Developmental effects observed:
no
Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
See attached read-across justification
Reason / purpose for cross-reference:
read-across source
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Basis for effect level:
food consumption and compound intake
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed
Abnormalities:
no effects observed
Developmental effects observed:
no
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Additional information

No data available concerning reproduction toxicity for Diethylamine CAS 109-89-7. Read across is proposed to Dimethylamin CAS No. 124 -40 -3.

In a developmental toxicity study (OECD 414) (ACC, 2009) for the Hydrochloride salt of the structural analogue Dimethylamine (CAS No. 124-40-3), the test substance was administered to pregnant Wistar rats daily by gavage from implantation (GD 6) to one day prior to the expected day of parturition (GD 19). The test substance did not cause any mortality. Test substance-related relevant clinical effects were only seen in the high-dose dams (1000 mg/kg bw/d), i.e. salivation after treatment and decreased food consumption, although the latter did not affect body weight, body weight gain, net body weight gain and uterus weight. At necropsy, no test substance related findings were noted in any of the dams. The temporary salivation was likely to be induced by the taste of the test substance or by local irritation of the upper digestive tract. It was not considered to be a sign of systemic toxicity. No differences of toxicological relevance between the control and the dose groups were determined for reproductive parameters such as conception rate, mean number of corpora lutea, mean number of implantations, pre- and postimplantation losses, live fetuses and fetal sex ratio. In summary, there was no evidence of an adverse effect of Dimethylamine hydrochloride on fetal morphology at any dose level tested.

In a second study according to OECD 414 (Wil 2016), the potential of dimethylamine to induce developmental toxicity was evaluated in rabbits. Dimethylamine (DMA) was administered via whole-body inhalation exposure to 3 groups of 24 time-mated female New Zealand White [Hra:(NZW)] rabbits for 6 hours per day from gestation days 7 through 28. Target exposure concentrations were 0, 50, 100, and 250 ppm for Groups 2, 3, and 4, respectively. Due to limitation of exposure chamber size, only 12 rabbits could be placed into each exposure chamber; therefore, this study was conducted in 2 phases, with 12 animals/group in each phase. The females were approximately 6 months of age at the initiation of dose administration. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. The eyes of all animals were examined for ocular irritation on gestation day 3or 4 (day of animal receipt) and gestation day 28. On gestation day 29, a laparohysterectomy was performed on each female. The uteri, placentae, and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

All females in the control and test substance-exposed groups survived to the scheduled necropsy on gestation day 29.

Rales were noted in 250 ppm group at the daily examinations and at 1‑2 hours following exposure throughout the exposure period. An increase in the incidence of elevated head was also noted in the 250 ppm group at the mid-point of exposure throughout the exposure period. Clear material around the eyes, mouth, and nose were noted at the daily examinations, approximate mid-point of each daily exposure, and/or 1‑2 hours following exposure for the 250 ppm group throughout the exposure period compared to the control group. The aforementioned clinical observations at 250 ppm were considered test substance-related and adverse. In addition, test substance-related clear material around the nose was noted for 50 and 100 ppm groups at 1‑2 hours following exposure throughout the exposure period. In the absence of other signs of toxicity at 50 and 100 ppm, and because the material findings did not persist to the daily examinations, they were not considered adverse at these exposure levels.

Test substance-related mean body weight losses were noted in the 250 ppm group during gestation days 7-10 compared to mean body weight gains in the control group. Corresponding lower mean food consumption was noted in this group generally throughout the exposure period. The initial mean body weight losses in the 250 ppm group were not of sufficient magnitude to result in lower mean body weight at the end of the exposure period. A mean net body weight loss was noted in the 250 ppm group. Mean maternal body weights, net body weights, and gravid uterine weights in all test substance-exposed groups and mean body weight gains and net body weight changes in the 50 and 100 ppm groups were unaffected by test substance exposure.

There were no remarkable ocular or macroscopic findings in any exposure group.

Intrauterine growth, survival, and fetal morphology (external, visceral, and skeletal) were unaffected by test substance exposure at all exposure levels.

Based on the higher incidence clinical observations (rales, elevated head, and clear material findings), mean body weight losses, and lower food consumption in the 250 ppm group, an exposure level of 100 ppm was considered to be the no‑observed‑adverse‑effect concentration (NOAEC) for maternal toxicity. There were no test substance-related effects on intrauterine growth, survival, and fetal morphology at any exposure concentration; therefore, the NOAEC for embryo/fetal development was 250 ppm.

Justification for classification or non-classification

Diethylamin CAS No. 109 -89 -7 is not warranted for classification and labelling according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.


No effects were observed for the structural analogue.

Additional information