Dimethylamine

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23-Jan-2019 - 04-Aug-2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29-Jul-2019 - 20-Nov-2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EU Method B.56 (Extended One-Generation Reproductive Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

- Premating exposure duration for parental (P0) animals: 10 weeks (according to ECHA Guidance, the starting point for deciding on the length of premating exposure period should be ten weeks to cover the full spermatogenesis and folliculogenesis before the mating, allowing meaningful assessment of the effects on fertility)
- Basis for dose level selection: preliminary dose range finder (the highest dose level shall aim to induce some toxicity to allow comparison of effect levels and effects of reproductive toxicity with those of systemic toxicity)
- exclusion of extension of Cohort 1B (the conditions to include the extension of Cohort 1B are currently not met (please also refer to the summary of effects observed depicted below)
- Termination time for F2: not applicable
- exclusion of developmental neurotoxicity Cohorts 2A and 2B (No triggers for the inclusion of Cohorts 2A and 2B (developmental neurotoxicity) were identified, please also refer to the summary of effects observed depicted below)
- exclusion of developmental immunotoxicity Cohort 3 (No triggers for the inclusion of Cohort 3 (developmental immunotoxicity) were identified, please also refer to the summary of effects observed depicted below)
- Route of administration: inhalation (gas)
- Other considerations, e.g. on choice of species, strain, vehicle and number of animals: not applicable


Background information used to conclude on the study design:
- Uses and exposure assessment for professional workers and consumers: no consumer uses identified, several professional uses identified, which do not lead to significant exposure
- Genotoxicity: the substance has been shown to be not genotoxic (negative results in a bacterial reverse mutation assay (Ames test); negative results in an multiple-endpoint mutagenesis test with Chinese hamster ovary cells; negatvie results In an in vivo Mammalian Chromosome Aberration Assay; and in conclusion no classification as mutagenic)
- Bioaccumulaiton: there are no indications that an extended exposure durcation is needed to reach the steady state kinetics, as the substance has been shown to have a very low potential for bioaccumulation in aquatic and terrestrial organisms. The very low Bioconcentration factor (BCF = 3.2), derived based on an octanol-water coefficient of only -0.71 (BASF, 1988), clearly shows that persistence of the test substance in environmental compartments or organisms can be ruled out.
- Repeated-dose toxicity: The olfactory sensory cell is highly sensitive to the toxic effects of DMA, with minor lesions being produced in rodents even at the current threshold limit value of 10 ppm. A LOAEC can be set at 10 ppm (18.7 mg/m³), since only a few animals were affected with slight alterations.
- Neurotoxicity: there is no data avaialble which rises a concern for dimethylamine and its potential to induce neurotoxicity (please also refer to the summary of effects observed depicted below)
- Immunotoxicity: there is no data avaialble which rises a concern for dimethylamine and its potential to induce immunotoxicity (please also refer to the summary of effects observed depicted below)
- Endocrine effects and modes of action: there is no data available which rises a concern for dimethylamine and its potential to induce endocrine effects (please also refer to the summary of effects observed depicted below). The available repeated dose toxicity and reproductive toxicity studies do not provide indication of endocrine disrupting modes of action. Furthermore, no data rising concerns related to endocrine disruption is available which is derived from in vivo assays, from non-animal approaches for prediction to (endocrine disrupting modes of action, from eco-toxicity testing for predicting endocrine (disrupting) modes of action.

The available results for dimethylamine and its read-across substances can be summarised as follows:
The results of two chronic inhalation studies and two recent GLP guideline developmental studies showed that the target substance does not to produce adverse effects on reproductive organs or tissues and therefore no concern exists in relation with reproductive performance. In details, the toxicity of Dimethylamine was investigated in F-344 rats and B6C3F1 mice following a one-year inhalation exposure to 0, 10, 50, or 175 ppm (Buckley et al., 1985). After 6 or 12 month exposure period, animals were sacrificed and their organs and tissues, including reproductive organs, were examined for gross abnormalities. Any gross lesions were then collected and examined histopathologically. The only treatment-related changes were concentration-related lesions in the nasal passages: the respiratory epithelium and olfactory epithelium. The study results were confirmed by another chronic two-year inhalation study in F-344 rats (Gross et al., 1987). The only treatment-related lesions were abnormalities related to destruction of the nasal tissues. In the OECD 414 drinking water study conducted with Dimethyl hydrochloride (CAS 506-59-2) and rats, the NOAEL for maternal toxicity was 300 mg/kg bw/d, mid dose, based on decreased food consumption and salivation after treatment in the high dose dams (1000 mg/kg bw/d) (BASF, 2009). No test substance-related and/or biologically relevant differences with regard to conception rate, mean number of corpora lutea, implantation sites, pre- and postimplantation loss and resorptions (total, early and late) were observed. At necropsy, no test substance-related findings were observed in the dams. Reproductive organs examined grossly were similar to those of controls. The NOAEL for prenatal developmental toxicity was 1000 mg/kg bw/d because there was no evidence of an adverse effect of the test compound on fetal morphology. In the OECD 414 inhalation study with Dimethylamine and rabbits, there were no treatment-related effects on reproductive performance and morphology and histology of reproductive organs of dams (WIL Research, 2016). Gravid uterine weights in all treatment groups were similar to controls. Intrauterine growth and survival were unaffected by test substance exposure at all exposure levels. 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.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL : Dimethylamine (CAS No. 124-40-3)
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Airgas Specialty Products
- Purity: pure

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
- Stability of the test material under the storage conditions provided by the Sponsor: The test substance is considered to be stable under the storage conditions provided by the Sponsor.
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: see below
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: see lebow
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: see below
- Reactivity of the test material with the incubation material used (e.g. plastic ware): not specified

Exposure Atmosphere Homogeneity Assessment
Homogeneity of exposure atmospheres was evaluated during the method development phase of the study. Four test locations and a reference location were used for sampling. Samples were collected and analyzed on the GC as rapidly as possible alternating from the reference and test location. For each location, the measured concentration was calculated as a percent difference from the reference location. Homogeneity was performed in triplicate for each test substance chamber. Based on mean differences from the reference location, spatial homogeneity was considered acceptable for all exposure chambers. Temporal stability was evaluated using the concentrations from the reference location. The variability during the homogeneity evaluation was acceptable for all exposure chambers. The generation and exposure trial performed before the start of animal exposure also demonstrated acceptable concentration stability for each exposure chambers.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): yes - heated up tp 50 °C (For the test substance-treated groups, a gaseous exposure was generated from the primary cylinder. The exposure atmospheres were characterized by analyzed exposure concentrations using a gas chromatograph. 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).
- Preliminary purification step (if any): not applicable
- Final concentration of a dissolved solid, stock liquid or gel: 0 ppm, 4 ppm, 12 ppm or 30 ppm

OTHER SPECIFICS
- Receipt Date: 15 Oct 2018
- Physical Description: Clear, colorless gas

Species:

rat

Strain:

Crj: CD(SD)

Remarks:

Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Crl:CD(SD) rats were received from Charles River Laboratories, Inc., Raleigh, NC
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation: approximately 6 weeks old
- Weight at study initiation: between 167 and 280 g at the initiation of exposure
- Fasting period before study: none (Food and water were withheld during the exposure periods.)
- Housing: On arrival (F0) or following weaning (F1), animals were group housed (2 to 3 animals of the same sex) until cohabitation. During cohabitation, the animals were paired for mating in the home cage of the male. Following the breeding period, animals were individually housed. Animals were housed in solid-bottom cages containing appropriate bedding equipped with an automatic watering valve.
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum): PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 (meal) was provided ad libitum throughout the study, except during designated procedures.
- Water (e.g. ad libitum): Municipal tap water after treatment by reverse osmosis was freely available to each animal via an automatic watering system, except during designated procedures. Water bottles were provided, if required. [Animals were housed in solid-bottom cages containing appropriate bedding equipped with an automatic watering valve.]
- Acclimation period: After receipt at the Testing Facility, the Crl:CD(SD) rats were acclimated prior to initiation of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 68 °F to 78 °F (20 °C to 26 °C)
- Humidity (%): 30 % to 70 %
- Air changes (per hr): Ten or greater air changes per hour with 100 % fresh air (no air recirculation) were maintained in the animal rooms.
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle
IN-LIFE DATES: From: 12 Jul 2019 To: 17 Mar 2020

Upon receipt, each animal was identified using a subcutaneously implanted electronic identification chip (BMDS system). Offspring were identified by tattoo markings applied to the digits after parturition and by microchip starting from PND 21. Pups selected for the F1 generation retained the dam number, followed by a hyphen "-" and the digit tattoo marking (i.e., 9999 01).

Animals were separated during designated procedures/activities. Each cage was clearly labeled with a color-coded cage card indicating study, group, animal, cage number(s), dosage level, and sex. Cages were arranged on the racks in group order.

Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining the animals’ oral health.

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

Route of administration:

inhalation: gas

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Remarks:

Filtered, humidified air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:
For the filtered air control group, dry compressed air was mixed to achieve a similar humidity, airflow, and/or oxygen content to that in the test substance-exposed groups.
For the test substance-treated groups, a gaseous exposure was generated from the primary cylinder.
The exposure atmospheres were characterized by analyzed exposure concentrations using a gas chromatograph.

Exposure Atmosphere Generation Methods
For the atmosphere generation and exposure setup used for test substance exposures 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.

- Method of holding animals in test chamber: as it is a whole body inhalation procedure, the animals were kept freely in the exposure chamber. Exposures will be conducted using four 2000-L glass and stainless-steel whole-body exposure chambers. One chamber will be dedicated for the filtered air group (0 ppm, Group 1) and one chamber will be dedicated for each of the three test substance-exposed groups.
- Source and rate of air: dry compressed air (supplied from a breathing quality in-house compressed air source); moreover, air supplied to the whole-body chambers was provided from the Inhalation Department HEPA and charcoal-filtered, temperature- and humidity-controlled supply air source.
- Method of conditioning air: braught to a tmeperature of 35 °C
- System of generating particulates/aerosols: not applicable
- Temperature, humidity, pressure in air chamber: 35 °C; the control chamber was placed under positive pressure to prevent any further potential contamination from the test substance; the other chambers will be operated under dynamic conditions and at a slight negative pressure.
- Air flow rate: as such as the below mentionned air change range
- Air change rate: a minimum of 12 air changes per hour
- Method of particle size determination: not applicable
- Treatment of exhaust air: All chambers exhaust was directed to the facility exhaust system, which consists of redundant exhaust blowers preceded by activated-charcoal and HEPA-filtration units.

TEST ATMOSPHERE
- Brief description of analytical method used: Test substance concentration within the exposure chambers were sampled and analyzed at
approximately 60-minute intervals using a gas chromatograph (GC, Model No. 7890B, Agilent Technologies Inc.; Santa Clara, CA). Samples were collected from the approximate animal-breathing zone of the exposure chamber via 1/8-inch Teflon® tubing. Sampling of the exposure chambers occurred under the control of Chromeleon data acquisition software (version 7.2, ThermoFisher Scientific, Waltham, MA), sampling and analyses were performed as follows. The program controls an external multi-position valve (Cheminert®, Model No. 19P-0105L, Valco Instruments Co., Inc. Houston, TX) that permitted sequential sampling from the exposure room and each exposure chamber. Gas sampling injection onto the chromatography column occurred via an internal gas-sampling valve with a sample loop, the chromatogram was displayed and the area under the sample peak was calculated and stored. The Chromeleon program used an equation based on the GC calibration curve to calculate the measured concentration in ppm. WINH then acquired the calculated concentration to monitor and report values.
- Samples taken from breathing zone: yes (Samples were collected from the approximate animal-breathing zone of the exposure chamber via 1/8-inch Teflon® tubing.)

Administration of Test Material:
F0 males were exposed for 6 hours daily for 70 consecutive days prior to mating and continuing through the day prior to euthanasia. F0 females were exposed for 6 hours daily for 70 consecutive days prior to mating and continuing throughout mating, gestation, and lactation, through the day prior to euthanasia. For females with evidence of mating, exposures during gestation were conducted up to and including Gestation Day 20, at which time exposure was suspended through Lactation Day 4, to avoid confounding effects on parturition and nursing behavior. Exposures resumed on Lactation Day 5 and continued until 1 day prior to scheduled euthanasia. For females with evidence of mating that failed to deliver, exposures were resumed on Postmating Day 25. The offspring selected for the F1 generation began exposure following weaning until the day prior to euthanasia (PND 91 [Cohort 1A] or following the reproductive assessment [Cohort 1B]). All animals were exposed at approximately the same time each day.

Details on mating procedure:

- M/F ratio per cage: 1:1 [During cohabitation, the animals were paired for mating in the home cage of the male.]
- Length of cohabitation: 14 days
- Proof of pregnancy: [either vaginal plug or sperm in vaginal smear] referred to as [day 0] of pregnancy
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.: not conducted
- Further matings after two unsuccessful attempts: [no / yes (explain)] not specified
- After successful mating each pregnant female was caged (how): Following the breeding period, animals were individually housed.
- Any other deviations from standard protocol:

Breeding Procedures
The F0 animals were paired on a 1:1 basis within each treatment group after 70 days of exposure. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage. If evidence of mating was not apparent after 14 days, the animals were separated, with no further opportunity for mating. Animals cohabited over a 12 hour dark cycle were considered to have been paired for 1 day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The exposure atmospheres were characterized by analyzed exposure concentrations using a gas chromatograph.
The Overall Mean Exposure Concentrations - F0 Generation (number of exposures = 137) were 0, 4.3 (+/- 0.89), 11.9 (+/- 0.89) and 30.7 (+/- 2.24) ppm
The Overall Mean Exposure Concentrations - F1 Generation (number of exposures = 113) were 0, 4.2 (+/- 0.80), 11.9 (+/- 1.36) and 31.1 (+/- 3.62) ppm

Duration of treatment / exposure:

6 hours

Frequency of treatment:

daily for 70 consequtive days prior to mating and continuing throughout mating, gestation, and lactation, through the day prior to euthanasia [For females with evidence of mating, exposures during gestation were conducted up to and including Gestation Day 20, at which time exposure was suspended through Lactation Day 4, to avoid confounding effects on parturition and nursing behavior. Exposures resumed on Lactation Day 5 and continued until 1 day prior to scheduled euthanasia. For females with evidence of mating that failed to deliver, exposures were resumed on Postmating Day 25. The offspring selected for the F1 generation began exposure following weaning until the day prior to euthanasia (PND 91 [Cohort 1A] or following the reproductive assessment [Cohort 1B]).]

Details on study schedule:

Control (filtered air) and test substance atmospheres were administered as a daily, 6-hour, whole body inhalation exposure. To accommodate protocol-specified activities, exposures were staggered as necessary.
Prior to each exposure, the selected animals were transferred to appropriate caging and transported to the exposure room. Following completion of daily exposures, the animals were returned to their home cages. Food and water were withheld during the exposure periods.
For the filtered air control group, dry compressed air was mixed to achieve a similar humidity, airflow, and/or oxygen content to that in the test substance-exposed groups.
For the test substance-treated groups, a gaseous exposure was generated from the primary cylinder.

- F1 parental animals not mated until [...] weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were [4] days of age.
- Age at mating of the mated animals in the study: [between 90 and 120 days of age]

To reduce variability among the F1 litters, 8 pups/litter, 4 pups/sex when possible, were randomly selected on PND 4. Standardization of litter size was not performed on litters with fewer than 8 pups.
For the F1 generation, 2 F1 pups/sex/litter from all available litters (≥ 20 litters/group) were randomly selected prior to weaning and were assigned to the following cohorts: Cohorts 1A and 1B were assigned to reproductive/developmental toxicity testing. Animals assigned to Cohort 1B were maintained on study for breeding when the animals were between 90 and 120 days of age. Assignment of same-sex littermates to a particular cohort was avoided whenever possible. In addition, if there were an insufficient number of pups to fill a designated cohort, the following prioritization plan was used (highest to lowest priority): Cohort 1A, Cohort 1B.

Dose / conc.:

0 ppm

Dose / conc.:

4 ppm

Dose / conc.:

12 ppm

Dose / conc.:

30 ppm

No. of animals per sex per dose:

24 rats/sex/group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The exposure concentrations were determined from results of previous studies and were provided by the Sponsor. In a previous modified OECD 422 rat study (Wang, 2020), dimethylamine was administered via whole-body inhalation exposure at 8, 25, and 75 ppm in F0 male and female rats during the premating, mating, gestation, and lactation periods. In addition, F1 rats were exposed to the same concentrations from PND 28–40. Systemic toxicity was limited to lower body weights and body weight gains in the F1 generation, and only at the 75 ppm exposure concentration. Histopathological examination revealed dimethylamine-related adverse findings at all exposure levels in both the F0 and F1 generations. At the same dimethylamine exposure levels, nasal cavity injury was more prominent in F1 animals. In the F0 generation, lesions were restricted to Level II of the nasal cavity in all test substance-exposure groups. For F1 animals in the 8 and 25 ppm exposure groups, histological findings were generally limited to nasal cavity Level II and included respiratory and transitional epithelial degeneration and/or mixed cell inflammation, without ulceration. These findings were considered locally adverse based on the concurrent presence of inflammation and degeneration or regeneration. 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.
- Rationale for animal assignment (if not random): F0 animals were assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights. Males and females were randomized separately. Animals in poor health or at extremes of body weight range were not assigned to groups.

To reduce variability among the F1 litters, 8 pups/litter, 4 pups/sex when possible, were randomly selected on PND 4. Standardization of litter size was not performed on litters with fewer than 8 pups.
For the F1 generation, 2 F1 pups/sex/litter from all available litters (≥20 litters/group) were randomly selected prior to weaning and were assigned to the following cohorts: Cohorts 1A and 1B were assigned to reproductive/developmental toxicity testing. Animals assigned to Cohort 1B were maintained on study for breeding when the animals were between 90 and 120 days of age. Assignment of same-sex littermates to a particular cohort was avoided whenever possible. In addition, if there were an insufficient number of pups to fill a designated cohort, the following prioritization plan was used (highest to lowest priority): Cohort 1A, Cohort 1B.

- Fasting period before blood sampling for clinical biochemistry: Animals were fasted overnight prior to blood collection.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, once in the morning and once in the afternoon. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were removed from the cage, and a detailed clinical observation was performed once daily throughout the study. During the exposure period, these observations were performed prior to exposure. On exposure days, clinical observations were also recorded 0.5- 3 hours postexposure.
During social housing, some observations (e.g., fecal observations) may not have been attributable to an individual animal.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually twice weekly throughout the study and prior to the scheduled necropsy. Once evidence of mating was observed, female body weights were recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and on Lactation Days 1, 4, 7, 11, 14, 21, and 28. A fasted weight was recorded on the day of necropsy. Terminal body weights were not collected from animals found dead.

FOOD CONSUMPTION:
Food consumption was quantitatively measured weekly throughout the study, except during the mating period. Once evidence of mating was observed, female food consumption was recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and Lactation Days 1, 4, 7, 14, 21, and 28. Food efficiency (body weight gained as a percentage of food consumed) was calculated and reported.

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

OTHER:
Sample Collection for Clinical Pathology was conducted in Week 19.
Animals were fasted overnight prior to blood collection.
Urine was collected overnight using metabolism cages.
Blood samples for hematology and serum chemistry were collected from the jugular vein.
Blood samples for coagulation parameters were collected by necropsy personnel from the inferior vena cava at the time of euthanasia from animals euthanized via carbon dioxide inhalation.
K2EDTA was used for the anticoagulant on samples collected for hematology.
Sodium citrate was used for samples collected for clotting determinations.
Samples for serum chemistry were collected without anticoagulants.

Hematology
Blood samples were analyzed for the following parameters:
Total leukocyte count (WBC); Erythrocyte count (RBC); Hemoglobin (HGB); Hematocrit (HCT); Mean corpuscular volume (MCV); Mean corpuscular hemoglobin (MCH); Mean corpuscular hemoglobin concentration (MCHC); Platelet count (Platelet); Reticulocyte count; Percent (RETIC); Absolute (RETIC Absolute), Differential leukocyte count: - Percent and absolute; -Neutrophil (NEU); -Lymphocyte (LYMPH)
-Monocyte (MONO); -Eosinophil (EOS); -Basophil (BASO); -Large unstained cell (LUC); Red cell distribution width (RDW); Hemoglobin distribution width (HDW); Platelet estimate; Red cell morphology (RBC Morphology)

Coagulation
Blood samples were processed for plasma, and the plasma was analyzed for the following parameters: Activated partial thromboplastin time (APTT), Prothrombin time (PT).

Serum Chemistry
Blood samples were processed for serum, and the serum was analyzed for the following parameters: Albumin; Total protein; Globulin [by calculation]; Albumin/globulin ratio (A/G Ratio) [by calculation];Total bilirubin (Total BILI); Urea nitrogen; Creatinine; Alkaline phosphatase (ALP); Alanine aminotransferase (ALT); Aspartate aminotransferase (AST); Gamma glutamyltransferase (GGT); Glucose; Total cholesterol (Cholesterol); Calcium; Chloride; Phosphorus; Potassium; Sodium; Sorbitol dehydrogenase (SDH); Triglycerides (Triglyceride); Bile acid; Appearance

Urinalysis
Urine samples were processed and analyzed for the following parameters: Specific gravity (SG); pH; Urobilinogen (URO); Total volume (TVOL); Color (COL); Clarity (CLA); Protein (PRO); Glucose (GLU) ; Ketones (KET); Microscopy of sediment; Bilirubin (BIL); Occult blood (BLD); Leukocytes (LEU)

Oestrous cyclicity (parental animals):

Slides of the daily Vaginal lavages were evaluated microscopically to determine estrous cycle stage of each F0 female for 14 days prior to cohabitation and continuing until evidence of mating was observed or until the end of the mating period. The average cycle length was calculated for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [D] from estrus [E] or proestrus [P], beginning 14 days prior to initiation of the mating period and continuing until the detection of evidence of mating). Estrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in a subsequent cycle. The cycle during which evidence of mating was observed for a given animal was not included in the mean individual estrous cycle length calculation. Vaginal lavages were also performed on the day of necropsy to determine the stage of the estrous cycle.
At the end of the study, the overall pattern of each female was characterized as regularly cycling, irregularly cycling, not cycling, or insufficient data.

Sperm parameters (parental animals):

Parameters examined in [all/P/F1/F2] male parental generations:
[testis weight, epididymis weight, daily sperm production, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology, other:]

Sperm Evaluations
Immediately upon euthanasia, the reproductive tract of each male was exposed via a ventral mid line incision. The right cauda epididymis was excised and weighed. An incision was made in the distal region of the right cauda epididymis, and it was then placed in Dulbecco's phosphate buffered saline (maintained at approximately 37 °C) with 10 mg/mL BSA. After a minimum 10 minute incubation period, a sample of sperm was loaded onto a slide with a 100 µm chamber depth for determination of sperm motility. Because sperm motility can be affected by temperature shock, all pipettes, slides, and diluents were warmed in an incubator, and motility determinations were performed under constant temperature (approximately 37 °C). Analysis of a minimum of 200 motile and nonmotile spermatozoa per animal (if possible) in all groups was performed by the analyzer.
The motility score (percent) for motile (showing motion only) and progressively motile (showing net forward motion) sperm was reported:
Percent Motile (or Progressively Motile) Sperm = (Number of Motile (or Progressively Motile) Sperm/ Total Number of Sperm Counted) * 100

The right epididymis was then placed in modified Davidson’s solution for subsequent microscopic examination. Sperm morphology was evaluated by light microscopy via a modification of the wet mount evaluation technique (Linder et al., 1992). Abnormal forms of sperm (double heads, double tails, microcephalic, or megacephalic, etc.) from a differential count of 200 spermatozoa per animal, if possible, were recorded.
The left testis and cauda epididymis from all males were weighed, stored frozen, homogenized, and analyzed for determination of homogenization resistant spermatid count and calculation of sperm production rate (Blazak et al., 1985). An aliquot of each sample was added to a solution containing a DNA specific fluorescent dye (the dye stains DNA that is present in the head of the sperm). For analysis, each sample was mixed, and an aliquot was placed on a slide with a 20 µm chamber depth. Illumination from a xenon lamp within the analyzer allowed for the visualization and quantitation of the sperm. A minimum of 200 cells, if possible, or up to 20 fields were counted for each sample. The sperm production rate was calculated as follows:
Sperm Production Rate = (No. of Sperm Per Gram of Tissue/ 6.1. days)
* where 6.1 days is the rate of turnover of the germinal epithelium

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 / F2 / F3] 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, other. Particular attention should be paid to the external reproductive genitals which should be examined for signs of altered development; gross evaluation of external genitalia]
- Viability: Litters were observed for general health/mortality and moribundity twice daily, once in the morning and once in the afternoon. A daily record of litter size was maintained. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.
- Observations: The animals were removed from the cage, and a detailed clinical observation was performed on PND 1, 4, 7, 14, 21, and 28. Any abnormalities in nesting and nursing behavior were recorded
- Sex Determination: Pups were individually sexed on PND 0, 4, 14, 21, and 28.
- Body Weights: Pups were weighed individually on PND 1, 4 (before culling), 7, 14, 21, and 28.

Preweaning Developmental Landmarks
Anogenital Distance: The anogenital distance of all pups was measured on PND 1. Anogenital distance was defined as the distance from the caudal margin of the anus to the caudal margin of the genital tubercle (Gallavan et al., 1999).
Assessment of Areolas/Nipple Anlagen: On PND 13, all male pups were evaluated for the presence of thoracic nipples/areola (Gray et al., 1999). The number of nipples was recorded.

Thyroid Hormone Analysis
Sample Collection: Blood samples for thyroid hormone analyses were collected (on PND4 and PND 28; each time prior to 1200 hours in order to avoid normal diurnal fluctuation in thyroid hormone levels) via cardiac puncture of animals anesthetized by inhalation of isoflurane (PND 4 culled pups) or via the jugular vein (PND 28 nonselected pups) into tubes without anticoagulants.
Details on samples collected on PND4: Samples were collected from culled pups and pooled by litter until a total of 10 samples/dosage level were obtained. To the extent possible, samples from the first 10 litters at each dosage level with sufficient numbers of culled pups were used.
Details on samples collected on PND28: Samples collected from nonselected pups (10/sex/group).
Sample Processing: Blood samples were maintained at room temperature and allowed to clot. Serum was isolated in a refrigerated centrifuge and stored in a freezer set to maintain a target of -70 °C.
Sample Analysis: Blood samples were analyzed for the followingparameters: Thyroxine (Total T4); Thyroid Stimulating Hormone (TSH)
Samples to be analyzed for T4 were transferred to the Charles River Ashland Bioanalytical Chemistry Department; analyses were performed using a validated UHPLC/MS/MS assay. Samples to be analyzed for TSH were transferred to the Charles River Ashland ADME/DMPK Department; analyses were performed using a qualified radioimmunoassay.

F1 Developmental Landmarks
Balanopreputial Separation: Each male was observed for balanopreputial separation beginning on PND 35 (Korenbrot et al., 1977). Examination of the males was continued daily until balanopreputial separation was present, and the age of attainment was recorded. Body weights were recorded at the age of attainment of this landmark. Any abnormalities of genital organs (e.g., persistent preputial threads) were noted.
Vaginal Patency: Each female was observed for vaginal perforation beginning on PND 25 (Adams et al., 1985). Examination of the females was continued daily until vaginal patency was present, and the age of attainment was recorded. Body weights were recorded at the age of attainment of this landmark. Any abnormalities of genital organs (e.g., persistent vaginal threads) were noted.

GROSS EXAMINATION OF DEAD PUPS:
[yes] : Moribund pups will be euthanized by an intraperitoneal injection of sodium pentobarbital. Stillborn pups, pups found dead and any pups that are euthanized in extremis will be dissected (including the heart and brain examined by a mid-coronal slice) by a technique described by Stuckhardt and Poppe.6 If a skeletal anomaly is suspected, the pups will be eviscerated, cleared, and stained with Alizarin Red S as described by Dawson7 and examined. Cannibalized pups will be discarded without necropsy. Any findings will be recorded as either developmental variations
or malformations. Representative specimens with malformations may be preserved in 10 % neutral-buffered formalin at the discretion of the Study Director.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals [describe when, e.g. as soon as possible after the last litters in each generation were produced.]
- Maternal animals: All surviving animals [describe when, e.g. after the last litter of each generation was weaned.]

Unscheduled Death: A necropsy was conducted for animals that died on study, and specified tissues were saved. For females that delivered, the number of former implantation sites were recorded.
Scheduled Euthanasia: All surviving animals, including females that failed to deliver, were euthanized by carbon dioxide inhalation.

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]
All animals were subjected to a complete necropsy examination, which included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal, and pelvic cavities, including viscera. Special attention was paid to the organs of the reproductive system. The numbers of former implantation sites were recorded for females that delivered. The number of unaccounted-for sites was calculated for each female by subtracting the number of pups born from the number of former implantation sites observed. For females that failed to deliver, a pregnancy status was determined, and specific emphasis was placed on anatomic or pathologic findings that may have interfered with pregnancy.

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated below were collected, prepared for microscopic examination and weighed, respectively.
Representative samples of the tissues indicated below were collected from all animals and preserved in 10% neutral buffered formalin, unless otherwise indicated.
Tissues examined: Adrenals (2); Aorta; Bone with marrow (sternebrae); Brain; Coagulating glands (2); Eyes with optic nerve (2); Gastrointestinal tract (Esophagus; Stomach; Duodenum; Peyer’s Patches; Jejunum; Ileum; Cecum; Colon; Rectum); Heart; Kidneys (2); Lacrimal/Harderian glands; Liver (sections of 2 lobes); Lymph node; Axillary (2); Mandibular (2); Mesenteric; Levator ani and bulbocavernosus (LABC) muscle group ; Larynx; Lungs (including bronchi, fixed by inflation with fixative); Nasal cavities with turbinates; Ovariesb and oviduct (2); Pancreas; Peripheral nerve (sciatic); Pituitary; Pharynx; Prostate; Mandibular salivary glands (2); Seminal vesicles (2); Skeletal muscle (quadriceps); Skin with mammary gland; Spinal cord (cervical); Spleen; Testes with epididymides (2) and vas deferens; Thymus; Thyroids (with parathyroids if present [2]); Trachea; Urinary bladder; Uterus with cervix and vagina; All gross lesions (all groups)

The organs indicated below were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals found dead. Paired organs were weighed together, unless otherwise noted. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated.
Organs examined: Adrenal glands; Brain; Epididymidesa (total and cauda); Heart; Kidneys; Levator ani and bulbocavernosus (LABC) muscle group; Liver; Lungs; Ovaries; Pituitary gland; Prostate gland;
Seminal vesicles with coagulating glands (with accessory fluids); Spleen; Testes; Thyroids with parathyroids; Thymus gland; Uterus with oviducts and cervix

Histology
Ovarian tissues for quantitative assessment were processed at PAI Durham. All other tissues were processed at the Testing Facility. Tissues listed above from all animals in the control and high-dose groups and from all animals found dead, as well as nasal section Level II and gross lesions from all animals in all groups, and reproductive organs (ovaries, oviducts, uterus, cervix, and vagina of females; testes, epididymides, prostate gland, seminal vesicles, coagulating glands, and levator ani/bulbocavernosus [LABC] muscle group of males) of all animals suspected of reduced fertility (e.g., those that failed to mate, conceive, sire or deliver healthy offspring, or for which estrous cyclicity or sperm number, motility or morphology were affected) were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. Processing of the testes, epididymides, and ovaries were performed as noted below.
Sections of 2–4 microns of the testis (transverse) and epididymis (longitudinal) were stained with PAS and hematoxylin staining in addition to the routine hematoxylin and eosin (H&E) staining. Testes and epididymides from males that were found dead were stained with H&E only. The following regions of the epididymis were embedded in paraffin: caput, corpus, and cauda; the vas deferens was examined when possible.
Five (5) sections were taken approximately 100 µm apart from the inner third of each ovary from any F0 females suspected of reduced fertility, e.g., those that failed to mate, conceive, sire or deliver healthy offspring, or for which estrous cyclicity or sperm number, motility or morphology were affected. In addition, a single section was taken from remaining F0 females for a qualitative bilateral evaluation of each ovary. For females found dead, a single section from each ovary was qualitatively evaluated.

Histopathology
Pathological evaluation was performed by a board-certified veterinary pathologist. Tissues were selected for microscopic examination from all animals in the control and high dose groups and from all animals found dead. Nasal section level two and gross lesions were examined from all animals in all groups.
In addition, reproductive organs (ovaries, oviducts, uterus, cervix, and vagina of females; testes, epididymides, prostate gland, seminal vesicles, coagulating glands, and levator ani/bulbocavernosus [LABC] muscle group of males) of all animals suspected of reduced fertility (e.g., those that failed to mate, conceive, sire or deliver healthy offspring, or for which estrous cyclicity or sperm number, motility or morphology were affected) were subjected to a histopathologic evaluation.
Histopathological examination of the testis included a qualitative assessment of the stages of spermatogenesis (Russell et al., 1990). For males that survived to the scheduled necropsy, microscopic evaluation included a qualitative assessment of the relationships between spermatogonia, spermatocytes, spermatids, and spermatozoa seen in cross-sections of the seminiferous tubules. The progression of these cellular associations defines the cycle of spermatogenesis. In addition, sections of both testes were examined for the presence of degenerative changes (e.g., vacuolation of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis).
For any F0 female suspected of reduced fertility, e.g., those that failed to mate, conceive, sire or deliver healthy offspring, or for which estrous cyclicity or sperm number, motility or morphology were affected, a quantitative histopathologic evaluation of multiple sections was conducted. This examination included enumeration of the total number of primordial follicles (Bolon et al., 1997; Bucci et al., 1997; Picut et al., 2008).

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [Study Days 134-137] days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

LItters - Unscheduled Deaths: A necropsy was conducted for animals that died on study, and specified tissues were saved.
If necessary, for humane reasons, animals were euthanized as per Testing Facility SOPs. These animals underwent necropsy, and specified tissues were retained.
Intact offspring that were found dead or euthanized for humane reasons during PND 0–4 were necropsied using a fresh dissection technique, which included examination of the heart and major vessels (Stuckhardt and Poppe, 1984). Pups with external abnormalities that would warrant further skeletal examination were eviscerated and stained for subsequent skeletal evaluation (Dawson, 1926). Findings were recorded as developmental variations or malformations, as appropriate. A gross necropsy was performed on any pup found dead after PND 4.

Litters . Scheduled Euthanasia: On PND 4, culled pups were euthanized by exsanguination (those pups used for blood/thyroid collection) or an intraperitoneal injection of sodium pentobarbital.
On PND 28, nonselected pups were euthanized by carbon dioxide inhalation.

Litters: Terminal procedures were conducted on PND 4 for Groups 1-4 (No. of animals: 119; 94, 126 and 105, respectively) and on PND 28 for groups 1-4 (No. of animals: 99, 79, 98 and 75, respectively).

LITTERS - GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]
On PND 4, 1 culled pup/sex/litter was subjected to a complete necropsy examination. Pups were necropsied using a fresh dissection technique, which included examination of the heart and major vessels (Stuckhardt and Poppe, 1984). All remaining culled pups were discarded without examination.
On PND 28, nonselected pups were subjected to a complete necropsy examination, with emphasis on developmental morphology and organs of the reproductive system.

LITTERS - HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated below were collected rom 1 culled pup/sex/litter on PND 4 and preserved in 10% neutral buffered formalin, prepared for microscopic examination and weighed, respectively.
Tissues examined: Trachea (with thyroid gland); All gross lesions
Representative samples of the tissues identified below were collected from 1 nonselected F1 pup/sex/litter on PND 28 and preserved in 10 % neutral buffered formalin:
Brain; Liver; Nasal cavities with turbinates; Ovaries (2); Skin with mammary gland, Spleen Testes (2); Thymus; Thyroids; All gross lesions

Representative specimens with malformations and gross lesions from offspring found dead or euthanized for humane reasons were preserved in 10 % neutral buffered formalin.

The organs ispecified below were weighed at necropsy from 1 nonselected F1 pup/sex/litter on PND 28. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated.
Organs examined: Brain; Liver; Spleen; Thymus; Thyroid

F1 Cohort 1A: Terminal procedures were conducted on PND 91 for Groups 1-4 (No. of animals: 20 males and 20 females for group 1-3 and 19 males and 19 females for group 4) and for Males on PND 133-140 (No. of animals: 23, 20, 22 and 19, respectively) and for Females on Gestation Day 15d (No. of animals: 24, 21, 23 and 19, respectively).

F1 Cohort 1A:
Unscheduled Deaths: A necropsy was conducted for animals that died on study, and specified tissues were saved.
Scheduled Euthanasia: All surviving animals were euthanized by exsanguination following isoflurane inhalation.

F1 Cohort 1A - GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]
All animals were subjected to a complete necropsy examination, which included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal, and pelvic cavities, including viscera. Special attention was paid to the organs of the reproductive system.
Cohort 1B females were subjected to a laparohysterectomy and macroscopic examination (animals with evidence of mating or appearing to be gravid). The number of corpora lutea on each ovary were recorded and the number of viable and nonviable embryos, early resorptions, and the total number of implantations was recorded, if possible. 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).

F1 Cohort 1A - HISTOPATHOLOGY / ORGAN WEIGTHS
The organs indicated below were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals found dead. Paired organs were weighed together, unless otherwise noted. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated.
Organs examined: Adrenal glands, Brain; Epididymides (total and cauda); Heart, Kidneys, Levator ani and bulbocavernosus (LABC) muscle group, Liver, Lungs, Ovaries, Pituitary gland, Prostate gland,
Seminal vesicles with coagulating glands (with accessory fluids), Spleen, Testes, Thyroids with parathyroids, Thymus gland, Uterus with oviducts and cervix

Representative samples of the tissues identified below were collected from all animals and preserved in 10% neutral buffered formalin:
Adrenal glands (2); Aorta; Bone with marrow (sternebrae); Brain; Coagulating glands (2); Eyes with optic nerve (2), Gastrointestinal tract (Esophagus, Stomach, Duodenum, Peyer’s Patches, Jejunum, Ileum, Cecum, Colon, Rectum, Heart, Kidneys (2), Lacrimal/Harderian glands, Levator ani and bulbocavernosus (LABC) muscle group, Liver (section of 2 lobes), Lungs (including bronchi, fixed by
inflation with fixative), Lymph node (axillary [2], mandibular [2], and mesenteric); Ovariesb and oviducts (2); Pancreas; Peripheral nerve (tibial); Pituitary; Prostate; Mandibular salivary gland (2), Seminal vesicles (2); Skeletal muscle (rectus femoris), Skin with mammary gland and subcutis, Spinal cord (cervical, thoracic, lumbar), Spleen, Testes with epididymides (2) and vas deferens, Tongue, Thymus, Thyroids (with parathyroids if present [2]), Trachea, Urinary bladder, Uterus with cervix and vagina, All gross lesions (all groups)

Histology:
Ovarian tissues for quantitative assessment were processed at PAI Durham. All other tissues were processed at the Testing Facility. Tissues enlisted above from F1 animals in Cohort 1A in the control and high-dose groups and from all animals found dead, as well as nasal section level two and gross lesions from all animals in all groups, and reproductive organs (ovaries, oviducts, uterus, cervix, and vagina of females; testes, epididymides, prostate gland, seminal vesicles, coagulating glands, and levator ani/bulbocavernosus [LABC] muscle group of males) of all animals suspected of reduced fertility (e.g., those that failed to mate, conceive, sire or deliver healthy offspring, or for which estrous cyclicity or sperm number, motility or morphology were affected) were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. Processing of the testes, epididymides, and ovaries were performed as noted below.
Sections of 2–4 microns of the testis (transverse) and epididymis (longitudinal) were stained with PAS and hematoxylin staining in addition to the routine hematoxylin and eosin (H&E) staining. Testes and epididymides from males that were found dead were stained with H&E only. The following regions of the epididymis were embedded in paraffin: caput, corpus, and cauda; the vas deferens was examined when possible.
Five (5) sections were taken approximately 100 µm apart from the inner third of each ovary from all F1 Cohort 1A females at the scheduled termination. In addition, a single section was taken from all F1 Cohort 1A females for a qualitative bilateral evaluation of each ovary.
The coagulating glands, ovaries, pituitary gland, prostate gland, seminal vesicles, testes with epididymides, uterus with cervix and vagina, and gross lesions from Cohort 1B animals were processed to the block stage (in paraffin).

Histopathology (Cohort 1A)
Pathological evaluation was performed by a board-certified veterinary pathologist. Tissues enlisted above for microscopic examination were evaluated from all animals in the control and high dose groups and from all animals found dead. Nasal section level two and gross lesions were examined from all animals in all groups.
In addition, reproductive organs of all animals suspected of reduced fertility, e.g., those that failed to mate, conceive, sire or deliver healthy offspring, or for which estrous cyclicity or sperm number, motility or morphology were affected, were subjected to a histopathologic evaluation.
Histopathological examination of the testis included a qualitative assessment of the stages of spermatogenesis (Russell et al., 1990). For males that survived to the scheduled necropsy, microscopic evaluation included a qualitative assessment of the relationships between spermatogonia, spermatocytes, spermatids, and spermatozoa seen in cross-sections of the seminiferous tubules. The progression of these cellular associations defines the cycle of spermatogenesis. In addition, sections of both testes were examined for the presence of degenerative changes (e.g., vacuolation of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis). When possible, sections of the rete testis were examined in the F1 Cohort 1A males.
For all F1 Cohort 1A females in the control and high-dose groups at scheduled termination, a quantitative histopathologic evaluation of multiple sections was conducted. This examination included enumeration of the total number of primordial follicles primordial follicles (Bolon et al., 1997; Bucci et al., 1997; Picut et al., 2008).
For the F1 Cohort 1A females, a tiered approach was used for the evaluation of ovarian follicle counts. A quantitative evaluation of primordial and primary follicles (combined) and corpora lutea were performed on 1 of 5 sections from each of the control and high exposure group animals, as a screening measure for potential adverse effects. If any 1 of the mean counts of the high-exposure level group differed by more than 10% of the corresponding count of the control group, then the quantitative evaluation was extended to include all 5 sections of ovary per animal, as a more definitive measure for potential adverse effects. If the results were equivocal, the quantitative evaluation was performed on the low- and mid-exposure groups for F1 females in Cohort 1A.

Statistics:

Each mean was presented with the standard deviation (S.D.) and the number of animals or cages (N) used to calculate the mean. Where applicable, the litter was used as the experimental unit. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means, standard deviations, and coefficients of variation on the summary and individual tables may differ slightly. Therefore, the use of reported individual values to calculate subsequent parameters or means will, in some instances, yield minor variations from those listed in the report data tables. Data obtained from nongravid animals were excluded from statistical analyses following the mating period. Statistical analyses were not performed on F0 female weekly food or body weight data after 1 or more animals had entered the gestation phase or remained in the lactation phase.
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 by sex.

Clinical signs:

no effects observed

Description (incidence and severity):

No test substance-related clinical findings were noted for F0 animals in the 4, 12, and 30 ppm groups at the detailed clinical observations and at 0.5-3 hours postexposure. Clinical observations noted in the test substance-exposed groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not exposure-related.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

In the 30 ppm group, 1 male (Animal No. 5997) was found dead on Study Day 46 and 1 female (Animal No. 6100) was found dead on Study Day 133. Although both animals had test substance-related microscopic lesions in the nasal cavity that were similar to those described in other animals in this group examined at scheduled necropsy, a specific cause of death was not determined based on microscopic examination. These deaths were deemed unlikely to be test substance related based on the lack of any other evidence of systemic toxicity at this dosage level.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

weekly bodyweights: No test substance related effects on mean body weights, body weight gains, and cumulative body weight gains were noted in the 4, 12, and 30 ppm groups. The values in the test substance exposed groups were generally comparable to the control group values for the pre-mating period (females) or the entire generation (males).
Sporadic statistically significant values were observed in a non-toxicologically relevant manner, or in a non exposure related manner and were considered incidental.

Body weight during Gestation: Mean maternal body weights, body weight gains, and cumulative body weight gains were unaffected by test substance exposure during gestation. Differences between the control, 4, 12, and 30 ppm groups were slight and not statistically significant.

Body weights during Lactation: Mean maternal body weights, body weight gains, and cumulative body weight gains were unaffected by test substance exposure during lactation. Sporadic statistically significant values were observed in a non-toxicologically relevant manner, or in a non exposure related manner and were considered incidental.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

weekly Food consumption, evaluated as g/animal/day, and food efficiency in the 4, 12, and 30 ppm groups was unaffected by test substance exposure. The values in the test substance exposed groups were generally comparable to the control group values for the pre-mating period (females) or the entire generation (males). Sporadic statistically significant values were observed in a non-toxicologically relevant manner, or in a non exposure related manner and were considered incidental.

Food consumption during gestation: Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, and food efficiency were unaffected by test substance exposure during gestation. Differences between the control, 4, 12, and 30 ppm groups were slight and not statistically significant.

Food consumption during lactation: Mean maternal food consumption, evaluated as g/animal/day, and food efficiency were unaffected by test substance exposure during lactation. Differences between the control, 4, 12, and 30 ppm groups were slight and not statistically significant. Sporadic statistically significant values were observed transiently, in a non-toxicologically relevant manner, or in a non exposure related manner and were considered incidental.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Haematology: There were no test substance-related effects on hematology parameters. A statistically significantly lower mean % basophil value was noted for males in the 30 ppm group; however, the magnitude of the change was minimal. Other differences from the control group were slight and not statistically significant.
Coagulation: There were no test substance-related effects on coagulation parameters. Differences from the control group were slight and not statistically significant.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on serum chemistry parameters. A statistically significantly lower mean urea nitrogen value was noted for females in the 30 ppm group; however, this was attributed to the lower values for 2 females. Other differences from the control group were slight and not statistically significant.

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 and given the lack of any correlating findings, and because an increase in thyroid hormones is generally not considered toxicologically relevant, these changes were considered likely to be unrelated to test substance administration.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on urinalysis parameters. Differences from the control group were slight and not statistically significant.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

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 of the F0 generation. 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.
Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of dimethylamine. One female in the 30 ppm group (Animal No. 6036) had an incidental mammary adenocarcinoma, which correlated grossly with the subcutaneous mass. This tumor was deemed incidental because of the single occurrence and known spontaneous occurrence in young Sprague Dawley rats (Kuzutani et al., 2012; Oishi et al., 1995).
In the F0 generation, there were nine pairs of males and females with suspected reduced fertility. The suspected reduced fertility was not associated with dimethylamine-related histologic findings. Three pairs were in Group 2 (4 ppm), one pair in Group 3 (12 ppm), and five pairs in Group 4 (30 ppm). Of these, 3 females (Animal No. 6088 in Group 2; 6087 in Group 4; and 6131 in Group 4) had histologic findings in the ovary consistent with early reproductive senescence (increased numbers of atretic follicles, decreased numbers of corpora lutea), which can be occasionally seen in this age of rat (Vidal, 2017). The cause of the suspected reduced fertility in the other animals was not determined by light microscopic evaluation.
F0 generation females with suspected reduced fertility had quantitative counts of primordial/small growing ovarian follicles performed. Primordial/small growing ovarian follicular counts of these animals were similar to values obtained from the F1 Cohort 1A control females (155.95 ± 53.8). Dimethylamine exposure did not have any effect on the number of primordial/small growing ovarian follicles in these F0 animals.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

not further specified

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No test substance related effects were observed on F0 sperm parameters (mean testicular and epididymal sperm numbers and sperm production rate, motility, progressive motility, and morphology) in males at any dosage concentration. Differences from the control group were slight and were not statistically significant.

Reproductive performance:

no effects observed

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 (Female Nos. 6036, 6060, 6084, 6087, and 6131 and Male Nos. 5955, 5957, 5968, 5969, and 6016) did not produce a litter; differences from the control group were not statistically significant. There were no macroscopic or microscopic lesions to account for reduced fertility in any of the listed animals, with the exception of 2 females in the 30 ppm group that exhibited abnormal estrous cycles (≥ 6 consecutive days of estrus, Nos. 6087 and 6131) and had histologic findings in the ovary consistent with early reproductive senescence (increased numbers of atretic follicles, decreased numbers of corpora lutea), which can be occasionally seen in this age of rat (Vidal, 2017); these observations were hence considered incidental and unrelated to dimethylamine administration. Male No. 5968, which did not sire a litter with the first female (No. 6084), was also paired with a second female (No. 6062) and successfully sired a litter.
In addition, 3 and 1 mating pairs in the 4 and 12 ppm groups also did not sire a litter, but these findings were considered unrelated to dimethylamine exposure, based on the lack of a dose response. Based on the lower male and female fertility in the 30 ppm group, F1 animals assigned to Cohort 1B for follow-up reproductive assessments were bred to obtain reproductive performance data for the F1 generation.
There were no effects of test substance exposure on reproductive performance in the 4 and 12 ppm groups and the mean numbers of days between pairing and coitus in the test substance exposed groups were comparable to the control group value. The mean lengths of estrous cycles in these groups were also comparable to the control group value. None of these differences were statistically significant.

Gestation length / parturition:
No test substance related effects were noted on mean gestation lengths or the process of parturition at any dosage concentration. Mean F0 gestation lengths in the test substance exposed groups were comparable to the control group value. Differences were slight and were not statistically significant. The mean gestation lengths in the 4, 12, and 30 ppm groups were 22.0, 22.0, and 22.0 days, respectively, compared to mean gestation lengths of 22.1 days in the concurrent control group and 21.9 days in the Charles River Ashland historical control data. No signs of dystocia were noted at any dosage/exposure level.

Dose descriptor:

NOAEC

Remarks:

systemic toxicity

Effect level:

30 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Absence of any systemically adverse effects in either generation (F0 and F1 males and females) during the study

Dose descriptor:

LOAEC

Remarks:

local effects

Effect level:

4 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Dose descriptor:

NOAEC

Remarks:

reproductive toxicity

Effect level:

30 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on the lack of any evidence of reproductive toxicity at any exposure level following the evaluation of reproductive performance, sperm measurements, and estrous cyclicity

Clinical signs:

not specified

Description (incidence and severity):

please refer to observations described in the fields for F1 (Cohort 1A).

Dermal irritation (if dermal study):

not examined

Mortality:

not specified

Description (incidence):

please refer to observations described in the fields for F1 (Cohort 1A).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Maternal Body Weights during Gestation (Cohort 1B). Mean maternal body weights, body weight gains, and cumulative body weight gains were unaffected by test substance exposure during gestation. Differences between the control, 4, 12, and 30 ppm groups were slight and not statistically significant.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Maternal Food Consumption and Food Efficiency during Gestation (Cohort 1B): Mean maternal food consumption, evaluated as g/animal/day, and food efficiency were unaffected by test substance exposure during gestation. Differences between the control, 4, 12, and 30 ppm groups were slight and not statistically significant.

Food efficiency:

not specified

Description (incidence and severity):

please refer to observations described in the fields for F1 (Cohort 1A).

Water consumption and compound intake (if drinking water study):

not specified

Description (incidence and severity):

please refer to observations described in the fields for F1 (Cohort 1A).

Ophthalmological findings:

not specified

Description (incidence and severity):

please refer to observations described in the fields for F1 (Cohort 1A).

Haematological findings:

not specified

Description (incidence and severity):

please refer to observations described in the fields for F1 (Cohort 1A).

Clinical biochemistry findings:

not specified

Description (incidence and severity):

please refer to observations described in the fields for F1 (Cohort 1A).

Endocrine findings:

no effects observed

Description (incidence and severity):

please refer to observations described in the fields for F1 (Cohort 1A).

Urinalysis findings:

not specified

Description (incidence and severity):

please refer to observations described in the fields for F1 (Cohort 1A).

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Cohort 1A: No dimethylamine-related organ weight changes were noted. Statistically significantly higher mean left cauda epididymal weights were noted at 12 and 30 ppm; however, mean values were within the historical control data range, and a significant increase in weight was not observed in the contralateral right cauda epididymis. In addition, there was no effect on cauda epididymal weights for animals in Cohort 1B (see Section 8.4.16.2). Therefore, this finding was not considered to be related to dimethylamine administration. There were isolated organ weight values that were statistically different from their respective controls. There were, however, no patterns, trends, or correlating data to suggest these values were toxicologically relevant. Thus, the organ weight differences observed were considered incidental and unrelated to administration of dimethylamine.
Cohort 1B: No dimethylamine-related organ weight changes were noted in the males of Cohort 1B. Organ weights were not collected from females as they were in gestation.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Cohort 1A: No dimethylamine-related gross findings were noted. The gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of dimethylamine.
Cohort 1B: No dimethylamine-related gross findings were noted. The gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration of dimethylamine.

Neuropathological findings:

not specified

Description (incidence and severity):

please refer to observations described in the fields for F1 (Cohort 1A).

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Dimethylamine-related microscopic findings are summarized in Text Table 33 (please refer to field 'Any other information on results (incl. tables).
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.
Dimethylamine-related lesions included minimal to moderate hyperplasia of the respiratory and transitional epithelium, minimal to mild mixed cell inflammation, and minimal vacuolar degeneration of the respiratory epithelium, and minimal to mild vacuolar degeneration of the transitional epithelium.
Histologically, the epithelial hyperplasia and inflammation were as described for the F0 generation animals (See Section 8.2.14.). The vacuolar degeneration was characterized by a single to few, round to irregular, variably sized cytoplasmic vacuoles and disorganized epithelium.
Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to dimethylamine exposure. There were no dimethylamine-related effects on primordial/small growing ovarian follicle counts between control and 30 ppm F1 females from Cohort 1A. Any variation in counts between those groups was due to normal biological variability.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

please refer to observations described in the fields for F1 (Cohort 1A).

Other effects:

no effects observed

Description (incidence and severity):

please refer to observations described in the fields for F1 (Cohort 1A).

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

Reproductive Performance (Cohort 1B): No test substance related effects on F1 reproductive performance were observed at any exposure concentration. No statistically significant differences were noted between the control and test substance exposed groups. 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.

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

30 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: lack of any evidence of reproductive toxicity at any exposure level following the evaluation of reproductive performance, sperm measurements, and estrous cyclicity in the F0 and Final Report Laboratory Project ID 00235551 Page 69 F1 generations

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

30 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

other: absence of any systemically adverse effects in either generation (F0 and F1 males and females) during the study

Dose descriptor:

LOAEL

Remarks:

local effects

Effect level:

4 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: observation of locally adverse histologic lesions at all exposure levels

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

F1 Generation Following Weaning: No test substance related clinical findings were noted during the generation at the detailed clinical observations or at 0.5-3 hours postexposure observations. Findings noted in the test substance exposed groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not exposure related.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

F1 Generation Following Weaning: One F1 generation Cohort 1B male (Animal No. 6077-05) at 12 ppm was found dead on PND 63. No remarkable clinical or macroscopic findings were noted for this male. No other mortality or moribundity was noted at the higher exposure level 30 ppm; therefore, this single death was considered incidental and not attributed to test substance exposure.
All other F1 parental animals in the control, 4, 12, and 30 ppm groups survived to the scheduled necropsy.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

F1 Generation Following Weaning: No test substance related effects on mean body weights, body weight gains, and cumulative body weight gains were noted in the 4, 12, and 30 ppm groups. The values in the test substance exposed groups were generally comparable to the control group values for the premating period (females) or the entire generation (males). Statistically significant differences from the control group were discordant in direction of change, transient, and/or did not occur in a dose related manner.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

F1 Generation Following Weaning: Food consumption, evaluated as g/animal/day, and food efficiency in the 4, 12, and 30 ppm groups were unaffected by test substance exposure. The values in the test substance exposed groups were generally comparable to the control group values for the pre-mating period (females) or the entire generation (males). Statistically significant differences from the control group were discordant in direction of change, transient, and/or did not occur in a dose related manner.

Food efficiency:

no effects observed

Description (incidence and severity):

F1 Generation following Weaning: Food consumption, evaluated as g/animal/day, and food efficiency in the 4, 12, and 30 ppm groups were unaffected by test substance exposure. The values in the test substance exposed groups were generally comparable to the control group values for the pre-mating period (females) or the entire generation (males). Statistically significant differences from the control group were discordant in direction of change, transient, and/or did not occur in a dose related manner.

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Cohort 1A: There were no test substance-related effects on hematology parameters. Statistically significantly higher mean red cell distribution width values were noted for females in the 12 and 30 ppm groups; however, the magnitudes of change were small, and the mean values were comparable to males in the control, 4, 12 and 30 ppm groups. The statistically significant differences were ascribed to the atypically low control group value for females, rather than an effect of dimethylamine exposure. All other differences from the control group were slight and not statistically significant.
There were no test substance-related effects on coagulation parameters. Differences from the control group were slight and not statistically significant.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Cohort 1A: There were no test substance-related effects on serum chemistry parameters. Differences from the control group were slight and not statistically significant.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Cohort 1A: There were no test substance-related effects on urinalysis parameters. Differences from the control group were slight and not statistically significant.

Sexual maturation:

no effects observed

Description (incidence and severity):

Balanopreputial separation: Mean ages of attainment of balanopreputial separation and mean body weights at the age of attainment were unaffected by test substance exposure. The mean ages of attainment of balanopreputial separation were 45.0, 45.5, and 44.8 days in the 4, 12, and 30 ppm groups, respectively, when compared to 45.6 in the control group. Mean body weights at the age of attainment were 242.7 g, 247.9 g, and 242.5 g in the same respective groups compared to 255.1 g in the control group. None of the differences from the control group were statistically significant.
Vaginal Patency: Mean ages of attainment of vaginal patency and mean body weights at the age of attainment were unaffected by test substance exposure. The mean ages of attainment of vaginal patency were 34.1, 34.5, and 34.4 days in the 4, 12, and 30 ppm groups, respectively, when compared to 34.1 days in the control group. Mean body weights at the age of attainment were 128.1 g, 129.9 g, and 129.8 g in the same respective groups compared to 132.9 g in the control group. None of the differences from the control group were statistically significant.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

F1 Generation before weaning: The anogenital distances (absolute, relative to pup body weight, and relative to the cube root of pup body weight) in the 4, 12, and 30 ppm groups were comparable to the control group values. Differences from the control group were slight and not statistically significant.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Areolae/nipple anlagen in the F1 male pups was unaffected by parental exposure to the test substance when evaluated on PND 12. The test substance treated group values were not statistically different from the control group values.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Terminal body weights for males were comparable across all groups. No test substance related effects on organ weights (absolute, relative to final body weight, and relative to brain weight) were observed for F1 pups on PND 28 at any exposure concentration when the test substance exposed groups were compared to the control group. A statistically significantly higher mean thymus weight relative to final body weight was noted for males in the 30 ppm group; however, mean absolute and relative to brain weight thymus weights were not affected.
Terminal body weights for females in the 4, 12 and 30 ppm groups were 5.2 %, 9.4 % and 7.5 % lower respectively than the control group; differences were statistically significant at 12 ppm only. Although non-dose responsive, these differences were considered to be a consequence of the lower mean body weight gains noted across all test substance groups during PND 21-28 and were considered test substance related but nonadverse based on the transient nature of the change. A statistically significantly lower mean brain weight was noted for females in the 4 ppm group; however, this did not occur in a dose-related manner. Other differences from the control group were slight and not statistically significant.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Unscheduled Deaths
Twenty (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 from PND 0 through the selection of the F1 generation. No internal findings that could be attributed to parental test substance exposure were noted at the necropsies of pups that were found dead or euthanized in extremis. Aside from the presence or absence of milk in the stomach, no internal findings were noted for pups in the test substance-exposed groups. Pup No. 6041-05 in the control group had malformations of hemimelia (both hindlimbs, consisting of short femurs), claw absent (all digits of both hindlimbs, consisting of absent phalanges), and bent tail (distal).

PND 4 Culled Pups
No internal findings that could be attributed to parental exposure with the test substance were noted at the necropsy of culled pups euthanized on PND 4. No internal findings were noted.

PND 28 Nonselected Pups
No internal findings that could be attributed to parental exposure to the test substance were noted at the necropsy of pups euthanized on PND 28. Pup No. 6035-09 in the 12 ppm group had a kidney cyst and dilated renal pelvis and Pup No. 6111-02 in the 30 ppm group had a kidney cyst; however, these findings were noted for single animals in each group. No other internal findings were noted. No other internal findings were noted.

PND 28 Pups Selected for Hormone Analysis
At the PND 28 necropsy of F1 weanlings selected for organ weights, no test substance-related internal findings were observed at any exposure concentration. Internal findings in the test substance-exposed groups were limited to a small eye (left), liver (all lobes), spleen and thymus for Pup No. 6032-06 in the 12 ppm group; however, these findings were noted for a single pup and were not noted in an exposure-related manner. Pup No. 6065-13 in the control group had multiple cysts on the kidney. No other internal findings were noted.

Histopathological findings:

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.
Dimethylamine-related lesions included minimal to moderate hyperplasia of the respiratory and transitional epithelium, minimal to mild mixed cell inflammation, and minimal vacuolar degeneration of the respiratory epithelium, and minimal to mild vacuolar degeneration of the transitional epithelium.
Histologically, the epithelial hyperplasia and inflammation were as described for the F0 generation animals (See Section 8.2.14.). The vacuolar degeneration was characterized by a single to few, round to irregular, variably sized cytoplasmic vacuoles and disorganized epithelium.
Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rats, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to dimethylamine exposure.
There were no dimethylamine-related effects on primordial/small growing ovarian follicle counts between control and 30 ppm F1 females from Cohort 1A. Any variation in counts between those groups was due to normal biological variability.

Other effects:

no effects observed

Description (incidence and severity):

Gestation Day 15 Laparohysterectomy:
Mean numbers of corpora lutea, implantation sites, 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. No statistically significant differences from the control group were noted. Intrauterine parameters evaluated included mean litter proportions of postimplantation loss and mean numbers and litter proportions of viable embryos.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

F1 Gerneation prior to weaning:
PND 0 Litter Data and Postnatal Survival:
The mean number of pups born, live litter size, percentage of males per litter at birth, and postnatal survival between birth and PND 0 (relative to number born), PND 0–1, 1–4 (pre selection), 4 (post-selection)–7, 7–14, 14–21, 21-28, and from birth to PND 4 (pre-selection) and PND 4 (post-selection)–28 were unaffected by the test substance at all dosage concentrations. Differences from the control group were slight, were not statistically significant, and/or did not occur in an exposure related manner.
Observations:
The general physical condition (defined as the occurrence and severity of clinical findings) of all F1 pups in this study was unaffected by test substance exposure. 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.
Offspring Body Weights:
Mean male and female pup body weights and body weight changes in the 4, 12, and 30 ppm groups were unaffected by parental test substance exposure generally throughout the postnatal period. 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.

Thyroid Hormone Analysis
PND 4 Culled Pups
No test substance-related effects were noted for culled pups on PND 4 at any exposure level. Differences from the control group were slight and not statistically significant.
PND 28 Pups Selected for Hormone Analysis
No test substance-related effects were noted for pups on PND 28 at any exposure level. A statistically significantly higher mean TSH value was noted for females in the 12 ppm group; however, this did not occur in a dose-related manner. Other differences from the control group were slight and not statistically significant.

Estrous Cycle data (Cohort 1A):
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.

Sperm Evaluations (Cohort 1A):
No test substance related effects were observed on F1 sperm parameters (mean testicular and epididymal sperm numbers and sperm production rate, motility, progressive motility, and morphology) in males at any exposure concentration. Differences from the control group were slight and were not statistically significant.

Thyroid Hormone Analysis (Cohort 1A):
There were no test substance-related effects on thyroid hormones in F1 Cohort1A pups on PND 91. Differences from the control group were slight and not statistically significant.

Dose descriptor:

NOAEC

Remarks:

for neonatal and developmental toxicity

Generation:

F1

Effect level:

30 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Absence of any effects during the preweaning period

Dose descriptor:

LOAEC

Remarks:

local effects

Generation:

F1

Effect level:

4 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Dose descriptor:

NOAEC

Remarks:

reproductive toxicity

Generation:

F1

Effect level:

30 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on the lack of any evidence of reproductive toxicity at any exposure level following the evaluation of reproductive performance, sperm measurements, and estrous cyclicity

Reproductive effects observed:

no

Reproductive performance (Fo-Generation):

F₀male and female reproductive parameters are presented in the following table.

Results of F₀Reproductive Performance

Parameter	Dosage Level (mg/kg/day)				CRL HC^a Mean (Range)
	0	4	12	30
Male Mating Index (%)	100.0	100.0	100.0	100.0	96.2 (86.7-100.0)
Female Mating Index (%)	100.0	100.0	100.0	100.0	96.7 (86.7-100.0)
Male Fertility Index (%)	100.0	87.5	95.8	82.6	89.0 (70.0-100.0)
Female Fertility Index (%)	100.0	87.5	95.8	79.2	89.3 (70.0-100.0)
Male Copulation Index (%)	100.0	87.5	95.8	82.6	93.2 (70.0-100.0)
Female Conception Index (%)	100.0	87.5	95.8	79.2	93.1 (70.0-100.0)
Estrous Cycle Length (days)	4.3	4.3	4.2	4.5	4.6 (3.9-7.6)
Pre-Coital Interval (days)	2.5	2.3	2.8	2.1	3.0 (1.8-4.7)
^a Charles River Ashland historical control data.

Dimethylamine-related microscopic findings are summarized in the following Table:
Summary of Microscopic Findings – F₀Generation

	Males				Females
Group	1	2	3	4	1	2	3	4
Target Exposure Concentration (ppm)	0	4	12	30	0	4	12	30
No. Animals Examined	24	24	24	23	24	24	24	23
Nasal Level II (No. Examined)	24	24	24	23	24	24	24	23
Hyperplasia, Respiratory Epithelium	(0)^a	(6)	(24)	(20)	(0)	(5)	(12)	(21)
Minimal	0	6	15	9	0	4	10	13
Mild	0	0	9	8	0	1	2	7
Moderate	0	0	0	3	0	0	0	1
Hyperplasia, Transitional Epithelium	(0)	(14)	(23)	(22)	(0)	(15)	(15)	(23)
Minimal	0	11	13	7	0	14	12	9
Mild	0	3	6	7	0	1	3	12
Moderate	0	0	4	8	0	0	0	2
Inflammation, mixed cell	(2)	(11)	(16)	(22)	(4)	(16)	(16)	(23)
Minimal	2	11	16	22	4	15	16	23
Mild	0	0	0	0	0	1	0	0
^a  Numbers in parentheses represent the number of animals with the finding.

Text Table - Ovarian Primordial/Small Growing Ovarian Follicle Counts – F0 Generation

Animal No.	Group	Target Exposure Concentration (ppm)	Total Primordial/Small Growing Ovarian Follicles
6088	2	4	246
6055	2	4	112
6082	2	4	61
6108	3	12	187
6084	4	30	102
6060	4	30	169
6087	4	30	186
6131	4	30	154
6036	4	30	162

Reproductive Performance Cohort 1B

F₁male and female reproductive parameters are presented in the following table:
Results of F₁Reproductive Performance

Parameter	Dosage Level (mg/kg/day)				CRL HC^a Mean (Range)
	0	4	12	30
Male Mating Index (%)	100.0	95.0	100.0	100.0	96.2 (86.7-100.0)
Female Mating Index (%)	100.0	95.2	100.0	100.0	96.7 (86.7-100.0)
Male Fertility Index (%)	100.0	90.0	95.5	94.7	89.0 (70.0-100.0)
Female Fertility Index (%)	100.0	90.5	95.7	94.7	89.3 (70.0-100.0)
Male Copulation Index (%)	100.0	94.7	95.5	94.7	93.2 (70.0-100.0)
Female Conception Index (%)	100.0	95.0	95.7	94.7	93.1 (70.0-100.0)
Estrous Cycle Length (days)	4.0	4.1	4.1	4.2	4.6 (3.9-7.6)
Pre-Coital Interval (days)	2.4	3.8*	2.7	2.7	3.0 (1.8-4.7)
^a Charles River Ashland historical control data * Significantly different from the control group at 0.05

Histopathology (Cohort 1A)

Dimethylamine-related microscopic findings are summarized in the following table:

Summary of Microscopic Findings – F₁Generation Cohort 1A

	Males				Females
Group	1	2	3	4	1	2	3	4
Target Exposure Concentration (ppm)	0	4	12	30	0	4	12	30
No. Animals Examined	20	20	20	19	20	20	18	19
Nasal Level II (No. Examined)	20	20	20	19	20	20	18	19
Hyperplasia, Respiratory Epithelium	(0)^a	(19)	(18)	(18)	(0)	(8)	(16)	(17)
Minimal	0	9	1	3	0	8	9	4
Mild	0	10	15	9	0	0	5	12
Moderate	0	0	2	6	0	0	2	1
Hyperplasia, Transitional Epithelium	(0)	(20)	(19)	(19)	(0)	(14)	(18)	(17)
Minimal	0	8	1	2	0	13	7	4
Mild	0	11	16	8	0	1	8	9
Moderate	0	1	2	9	0	0	3	4
Inflammation, mixed cell	(1)	(18)	(19)	(18)	(0)	(13)	(17)	(17)
Minimal	1	16	19	17	0	13	17	17
Mild	0	2	0	1	0	0	0	0
Vacuolar degeneration, Respiratory Epithelium	(0)	(6)	(4)	(9)	(0)	(3)	(6)	(12)
Minimal	0	6	4	9	0	3	6	12
Vacuolar degeneration, Transitional Epithelium	(0)	(16)	(17)	(18)	(0)	(5)	(11)	(19)
Minimal	0	16	17	17	0	5	11	19
Mild	0	0	0	1	0	0	0	0
^a  Numbers in parentheses represent the number of animals with the finding.

 

Text Table - Ovarian Primordial/Small Growing Ovarian Follicle Counts – F1 Generation Cohort 1A

	females
Group	1	4
Target Exposure Concentration (ppm)	0	30
No. Animals per Group	20	19
Primordial/Small Growing Ovarian Follicles
Mean	155.950	147.368
Standard Deviation	53.834	51.234

DISCUSSION

The objective of this study was to evaluate the potential adverse effects of the test substance on reproduction in an extended one-generation study. This included evaluation of life stages not covered by other types of toxicity studies and test for effects that may occur as a result of pre- and postnatal chemical exposure.

Dose levels for the current study (4, 12 and 30 ppm) were selected based on a previous rat study (Wang, 2020, 00235550) where dimethylamine was administered via whole-body inhalation to F₀male and female rats at 8, 25, and 75 ppm during the premating, mating, gestation, and lactation periods. F₁male and female rats on the same study were exposed to the same concentrations from weaning (PND 28) through PND 40. While systemic toxicity was limited to lower body weights and body weight gains in the F₁generation (only at the 75 ppm level), dose limiting locally adverse histopathological lesions were noted for both F₀and F₁animals on this study and the 75 ppm exposure level was considered excessive for an extended one-generation reproductive study.

In the current study, at 30 ppm, the highest exposure level tested, lower F₀ male and female fertility and copulation/conception indices were observed in comparison with the control group. Five (5) of 24 mating pairs in the 30 ppm group did not produce a litter as opposed to the control group where 100 % of the mating pairs produced a litter. 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 and there were no effects on F₀ reproductive performance at 4 and 12 ppm. Based on these findings, F₁ animals assigned to Cohort 1B for follow-up reproductive assessments were bred to obtain reproductive performance data for the F₁ generation. All procedures for F₁ animals assigned to Cohort 1B breeding were generally the same as those for the F₀ generation, with the exception that F₁females were euthanized in mid-gestation, 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. Mean mating, fertility and conception/copulation indices were comparable between the control and test substance‑exposed groups, as were the mean numbers of days between pairing and coitus. Mean estrous cycle lengths were also comparable to the control group value. Thus, based on the lack of any effects on reproductive performance of the F₁generation, it was concluded that the lower male and female fertility, copulation and conception indices noted for animals in the 30 ppm group in the F₀generation were likely incidental and hence unrelated to test substance exposure.

Higher T4 levels noted for F₀ males and females 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, only 5 and 6 (of 10) samples were available for analysis (due to insufficient serum quantity) for both male and female control groups which may have contributed to the noted differences. Lastly, there were no noted differences in thyroid organ weights or corresponding changes in thyroid histopathology and given the lack of any correlating findings, these changes were considered likely to be unrelated to test substance administration.

Dimethylamine exposure was associated with microscopic findings 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.

With the exception of the abovementioned effects, there were no other changes noted on this study. Parental and neonatal survival, and all other systemic, reproductive and terminal endpoints in the F₀ and F₁ generations were unaffected by dimethylamine administration via whole body inhalation to Crl:CD(SD) rats at 4, 12 and 30ppm.

Conclusions:

Dimethylamine exposure was associated with locally adverse histologic lesions at all exposure levels. Thus, an exposure level of 4 ppm was considered to be the lowest-observed-adverse-effect level (LOAEL) for locally adverse effects for F0 and F1males and females when dimethylamine was administered via whole body inhalation.
In the absence of any systemically adverse effects in either generation (F0 and F1males and females) during the study, an exposure level of 30 ppm, the highest exposure level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for F0 and F1male and female systemic toxicity when dimethylamine was administered via whole body inhalation to Crl:CD(SD) rats.
Based on the lack of any effects F1offspring during the preweaning period, an exposure level of 30 ppm was considered to be the NOAEL for F1 neonatal and developmental toxicity. Based on the lack of any evidence of reproductive toxicity at any exposure level following the evaluation of reproductive performance, sperm measurements, and estrous cyclicity in the F0 and F1 generations, the NOAEL for F0 and F1 reproductive toxicity was also considered to be 30 ppm, the highest exposure level evaluated.

Executive summary:

The objective of this study was to evaluate the potential adverse effects of the test substance dimethylamine on reproduction in an extended one-generation study. This included evaluation of life stages not covered by other types of toxicity studies and tested for effects that may occur as a result of pre- and postnatal chemical exposure.

The study design was as follows: Group 1 - 0 ppm DMA (Filtered Air), Group 2 - 4 ppm DMA, Group 3 - 12 ppm Dimethylamine, Group 4 - 30 ppm DMA (Each of the four groups contained 24 Males and Females).

Animals in the parental (F₀) generation were exposed via whole-body inhalation for 6 hours daily for 70 consecutive days prior to mating and continuing through the day prior to euthanasia. Maternal exposure was suspended from Gestation Day 20 through Lactation Day 4 to prevent confounding effects on parturition and maternal nursing and nesting behavior. The offspring in the F₁ generation were potentially exposed in utero and through nursing during lactation. The offspring selected to constitute the F₁ generation were exposed beginning at weaning (PND 28) and continuing until the day prior to euthanasia. Due to observation of lower mean fertility and copulation/conception indices for F₀ males and females (see below), F₁ animals assigned to Cohort 1B for follow-up reproductive assessments were bred to obtain reproductive performance data for the F₁ generation. All procedures for F₁ animals assigned to Cohort 1B breeding were generally the same as those for the F₀ generation, with the exception that F₁ females were euthanized in mid-gestation, 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. F₁ males were euthanized following examination of females with evidence of mating.

The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, food consumption, estrous cycles, reproductive performance, parturition, litter viability and survival, pre- and postweaning developmental landmarks, thyroid hormones, clinical pathology, gross necropsy findings, sperm parameters, organ weights, and histopathologic examinations.

Target exposure concentrations were 4, 12 and 30 ppm. Overall mean analyzed exposure concentrations were 4.3, 11.9 and 30.7 ppm for the F₁ generation and 4.2, 11.9 and 31.1 ppm for the F₁ generation.

F₀Generation: There were no test substance-related effects on survival at any exposure level. One male and one female each in the 30 ppm group were found dead on Study Day 46 and 113, respectively. Although both animals had test substance-related microscopic lesions in the nasal cavity that were similar to those described in other animals in this group examined at scheduled necropsy, a specific cause of death was not determined based on microscopic examination. These deaths were deemed unlikely to be test substance related based on the lack of any other evidence of systemic toxicity at this dosage level. All other F₀ animals survived to the scheduled necropsies, and there were no test substance-related clinical observations, or any effects on mean body weights, body weight gains, food consumption or food efficiency during the study.

In the F₀generation, lower male and female fertility and copulation/conception indices were observed in the 30 ppm group, when compared to the control group. Five (5) of 24 mating pairs in the 30 ppm group did not produce a litter as opposed to the control group where 100 % of the mating pairs produced a litter. There were, however, 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 also comparable across all groups. There were no effects on F₀ reproductive performance at 4 and 12 ppm.

Increased T4 levels were noted for males and females in the 12 and 30 ppm groups; differences from the control group were statistically significant. However, due to lack of other correlating changes in the levels of TSH, thyroid organ weight, or thyroid histopathology in the F₀ generation, and the lack of any effects on thyroid endpoints in the F₁ generation, these changes were considered likely to be incidental and not related to test substance administration.

There were no test substance-related gross macroscopic findings or any changes in clinical pathology parameters (hematology, coagulation, serum chemistry, and urinalysis) noted for F₀ animals at any exposure level.

Dimethylamine exposure was associated with microscopic findings in the nasal cavity (level II) of F₀ animals at ≥ 4 ppm. 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 all exposure levels based on the presence of inflammation and epithelial hyperplasia.

F₁Generation: F₁ postnatal survival was unaffected by test substance exposure at all exposure levels. In addition, pup birth weights and the general physical condition of F₁ pups during the pre-weaning period were unaffected by test substance exposure. Mean pup body weights and body weight gains were comparable across all groups, including in the period immediately following re-initiation of maternal test substance exposures on PND 5. There were also no substance-related effects on serum levels of thyroid hormones for F₁ pups (on PND 4 and 28) or any effects on preweaning developmental landmarks (anogenital distance, areolae/nipple retention) at any exposure level in the F₁ generation.

Following weaning, there were no test substance-related effects on survival in the F₁ generation at any exposure level. One male in the 12 ppm group was found dead on PND 63; there were no remarkable clinical or macroscopic findings noted for this male and given the lack of mortality at the 30 ppm, this death was considered incidental and unrelated to test substance exposure. All other F₁ animals survived to the scheduled necropsies, and there were no test substance-related clinical observations, or any effects on mean body weights, body weight gains, food consumption or food efficiency during the generation. There were also no substance-related effects on post weaning developmental landmarks (vaginal patency, onset of estrus or balanopreputial separation), serum levels of thyroid hormones, gross macroscopic findings, estrous cyclicity, sperm parameters, or clinical pathology parameters at any exposure level for F₁ males and females (Cohort 1A, PND 90).

Microscopic findings related to test substance exposure in F₁ Cohort 1A animals were similar to the F0 generation. In the nasal cavity (level II) of animals exposed to ≥ 4 ppm, 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 was noted in males and females. 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. Similar to the F₀ generation, these findings were considered locally adverse at all exposure levels based on the presence of inflammation and epithelial hyperplasia. There were no test substance-related effects on primordial/small growing follicles in F₁ Cohort 1A females on PND 90.

Due to observation of lower mean fertility and copulation/conception indices for F₀ males and females, F₁ animals assigned to Cohort 1B for follow-up reproductive assessments were bred to obtain reproductive performance data for the F₁ generation. There were no test substance related effects on reproductive performance. Male and female mating, fertility, and copulation and conception indices, estrous cyclicity and the number of days between pairing and coitus were comparable across all exposure levels. Mean numbers of corpora lutea, implantation sites and viable embryos were similar across all exposure levels. Based on the lack of any effects on reproductive performance of the F₁ generation, it was concluded that the lower male and female fertility and copulation/conception indices noted for animals in the 30 ppm group in the F₀ generation were spurious and unrelated to test substance exposures.

In conclusion, dimethylamine exposure was associated with locally adverse histologic lesions at all exposure levels. Thus, an exposure level of 4 ppm was considered to be the lowest-observed-adverse-effect level (LOAEL) for locally adverse effects for F₀ and F₁ males and females when dimethylamine was administered via whole body inhalation.

In the absence of any systemically adverse effects in either generation (F₀ and F₁males and females) during the study, an exposure level of 30 ppm, the highest exposure level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for F₀ and F₁male and female systemic toxicity when dimethylamine was administered via whole body inhalation to Crl:CD(SD) rats.

Based on the lack of any effects F₁ offspring during the preweaning period, an exposure level of 30 ppm was considered to be the NOAEL for F₁ neonatal and developmental toxicity. Based on the lack of any evidence of reproductive toxicity at any exposure level following the evaluation of reproductive performance, sperm measurements, and estrous cyclicity in the F₀ and F₁ generations, the NOAEL for F₀ and F₁reproductive toxicity was also considered to be 30 ppm, the highest exposure level evaluated.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Remarks:

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (subacute exposure)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

23-Jan-2019 - 04-Aug-2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29 July 2016

Deviations:

yes

Remarks:

several slight deviations in Atmosphere Generation & Exposure, Husbandry, In-life Observations, Measurements & Evaluations, Laboratory Evaluations & Postmortem and Pathology (all are considered not to have had impact the overall integrity of the study);

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Airgas Specialty Products

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

OTHER SPECIFICS
- The Sponsor and/or Supplier provided to the Testing Facility documentation of the identity, strength, purity, composition, and stability for the test substance. A Certificate of Conformance was provided to the Testing Facility.

Species:

rat

Strain:

Crj: CD(SD)

Remarks:

Crl:CD(SD)

Details on species / strain selection:

Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) approximately 11 wks
- Weight at study initiation: between 220 and 423 g at the initiation of exposure
- Fasting period before study: not applicable
- Housing: Animals were housed in an animal colony room during nonexposure periods. In detail, on arrival (F0) or following selection (F1), animals were group housed (up to 3 animals of the same sex) until cohabitation or euthanasia. At initiation of cohabitation, F0 males were assigned to individual home cages and then F0 animals were paired for mating in the home cage of the male. Following the breeding period, F0 animals were individually housed. Animals were housed in solid-bottom cages containing appropriate bedding equipped with an automatic watering valve throughout the study. Animals were separated during designated procedures/activities. Each cage was clearly labeled with a color-coded cage card indicating study, group, animal, cage number(s), dosage level, and sex. Cages were arranged on the racks in group order. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals. The animal facilities at Charles River Ashland are accredited by AAALAC International
- Diet: PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 was provided ad libitum throughout the study, except during designated procedures. Food was withheld during the animal exposure periods.
- Water: Municipal tap water after treatment by reverse osmosis was freely available to each animal via an automatic watering system, except during inhalation exposure procedures. Water was withheld during the animal exposure periods.
- Acclimation period: at least 7 days prior to initiation of exposure
- Animal identification: Upon receipt, each animal was identified using a subcutaneously implanted electronic identification chip (BMDS system). Offspring were identified by tattoo markings applied to the digits after parturition and by microchip at weaning.
- Animal Enrichment: Animals were socially housed for psychological/environmental enrichment and were provided with environmental enrichment as appropriate to aid in maintaining the animals’ oral health.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 68 °F to 78 °F (20 °C to 26 °C)
- Humidity (%): 30 % to 70 %
- Air changes (per hr): Ten or greater air changes per hour with 100 % fresh air (no air recirculation) were maintained in the animal rooms
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark

IN-LIFE DATES: From: 24 Jan 2019 (Animal Arrival) To: 13 May 2019

Route of administration:

inhalation: gas

Type of inhalation exposure:

whole body

Vehicle:

other: Humidified, filtered air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 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 group. The chambers were operated under dynamic conditions, at a slight negative pressure, and with a minimum of 12 air changes per hour.
- Method of holding animals in test chamber: Animals were individually housed in standard exposure batteries of appropriate size for the whole-body exposure chamber in use.
- Source and rate of air: 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. All whole-body chamber exhaust passed through the facility exhaust system, which consists of redundant exhaust blowers preceded by activated-charcoal and HEPA-filtration units
Test substance atmospheres were generated by releasing neat test substance gas from the cylinder. The cylinder was heated to maintain pressure within the cylinder. The test substance gas was directed to a manifold. Test substance gas was directed to the chamber inlet, where it mixed with dilution supply air to achieve the desired atmosphere concentration.
- Method of conditioning air: not specified
- System of generating particulates/aerosols: not applicable
- Temperature, humidity, pressure in air chamber: The mean temperature and relative humidity of the exposure atmospheres were to be 69 °F to 75 °F (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.
- Air flow rate: between 453 and 464 Liters/min
- Air change rate: not specified
- Method of particle size determination: not applicable
- Treatment of exhaust air: All whole-body chamber exhaust passed through the facility exhaust system, which consists of redundant exhaust blowers preceded by activated-charcoal and HEPA-filtration units.

TEST ATMOSPHERE
- Brief description of analytical method used: Analyzed exposure concentrations were determined at approximately 60-minute intervals using a gas chromatograph
- Samples taken from breathing zone: yes (An external multi-position valve permitted sequential sampling from the exposure room and each exposure chamber. Gas sampling injection onto the chromatography column occurred via an internal gas sampling valve with a sample loop, the chromatograph was displayed, and the area under the sample peak was calculated and stored. An ln quadratic equation based on the gas chromatograph calibration curve(s) was used to calculate the measured concentration in ppm.)

VEHICLE (if applicable)
- Justification for use and choice of vehicle: air
- Composition of vehicle: not specified
- Type and concentration of dispersant aid (if powder): not applicable
- Concentration of test material in vehicle: not specified
- Lot/batch no. of vehicle (if required): not required
- Purity of vehicle: no details given
For exposure of the control group (Group 1, 0 ppm), humidified supply air was delivered to the chamber from the facility air supply source. To provide a comparable oxygen level in the control chamber to that as within the test substance chambers, compressed nitrogen was delivered to the inlet of the control exposure chamber at a flow rate similar to that used in the high-exposure group, where it mixed with supply air.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyzed exposure concentrations were determined at approximately 60-minute intervals using a gas chromatograph.
Samples were collected from the approximate animal-breathing zone of the exposure chamber. An external multi-position valve permitted sequential sampling from the exposure room and each exposure chamber. Gas sampling injection onto the chromatography column occurred via an internal gas sampling valve with a sample loop, the chromatograph was displayed, and the area under the sample peak was calculated and stored. An ln quadratic equation based on the gas chromatograph calibration curve(s) was used to calculate the measured concentration in ppm.

Duration of treatment / exposure:

Males: 14 days prior to mating and continuing throughout mating for a total of 28 days
Females: 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

Frequency of treatment:

6 hours/day

Dose / conc.:

0 ppm

Remarks:

Control (filtered air)

Dose / conc.:

8 ppm

Dose / conc.:

25 ppm

Dose / conc.:

75 ppm

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Justification of Route and Exposure Levels
The route of administration was whole-body inhalation exposure because inhalation would be a likely route of unintended human exposure.
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.

- Rationale for animal assignment: Animals were assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights. Males and females were randomized separately. Animals with physical abnormalities, at extremes of body weight range, or not exhibiting normal, 4- to 5-day estrous cycles were not assigned to groups.
- Fasting period before blood sampling for clinical biochemistry: Animals were fasted overnight prior to blood collection
- Other: Animals were housed in an animal colony room during nonexposure periods. Prior to each exposure, the animals were transferred to exposure caging, transported to the exposure room, exposed for the requisite duration, transported back to the colony room, and returned to their home cages. Food and water were withheld during the animal exposure periods.
Prior to exposure, females were evaluated for estrous cyclicity during the pretest period and any females that failed to exhibit normal 4-5 day estrous cycles (e.g., EDDDE), during the pretest period, were excluded from the study; therefore, the extra females were included to yield at least 10 females per group. Given the possibility of nongravid animals, unexpected deaths, total litter losses, or test substance-related moribundity and/or mortality, this was an appropriate number of animals to obtain a sample size of 8 at termination.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, once in the morning and once in the afternoon. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were removed from the cage, and a detailed clinical observation was performed once daily throughout the study. During the exposure period, these observations were performed prior to exposure. On exposure days, clinical observations were also recorded 0.5–3 hours following completion of exposure.
During social housing, some observations (e.g., fecal observations) may not have been attributable to an individual animal.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually weekly throughout the study and prior to the scheduled necropsy. Once evidence of mating was observed, female body weights were recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and Lactation Days 1, 4, 7, 10, 13, 17, 21, 25, and 28. A fasted weight was recorded on the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption was quantitatively measured weekly until cohabitation. Once evidence of mating was observed, female food consumption was recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and Lactation Days 1, 4, 7, 10, 13, 17, 21, 25, and 28.

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples for hematology were collected from the jugular vein on study day 28 (males) or lactation day 29 (females). K2EDTA was used for the anticoagulant on samples collected for hematology. Sodium citrate was used for samples collected for clotting determinations
- Anaesthetic used for blood collection: Yes (animals anesthetized by inhalation of isoflurane)
- Animals fasted: Yes
- How many animals: 5 animals/sex/group
- Parameters were examined: Differential leukocyte count (-Percent and absolute; -Neutrophil (NEU); -Lymphocyte (LYMPH), -Monocyte (MONO), -Eosinophil (EOS), -Basophil (BASO), -Large unstained cell (LUC)), Erythrocyte count (RBC); Hemoglobin (HGB); Hemoglobin distribution width (HDW), Hematocrit (HCT), Mean corpuscular hemoglobin (MCH), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (Platelet), Red cell distribution width (RDW), Reticulocyte count, Percent (RETIC), Absolute (RETIC Absolute), Total leukocyte count (WBC), Platelet estimatea, Red cell morphology (RBC Morphology)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples for hematology and serum chemistry were collected from the jugular vein. Blood samples for coagulation parameters were collected by necropsy on study day 28 (males) or lactation day 29 (females). Samples for serum chemistry were collected without anticoagulants
- Animals fasted: Yes
- How many animals: 5 animals/sex/group
- Parameters were examined:
Coagulation: Blood samples were processed for plasma, and the plasma was analyzed for: Activated partial thromboplastin time (APTT), Fibrinogen, Prothrombin time (PT).
Serum chemistry: Blood samples were processed for serum, and the serum was analyzed for: Alanine aminotransferase (ALT), Albumin, Albumin/globulin ratio (A/G Ratio) [by calculation], Alkaline phosphatase (ALP), Aspartate aminotransferase (AST), Bile Acids, Calcium, Chloride, Creatinine, Gamma glutamyltransferase (GGT), Globulin [by calculation], Glucose, Phosphorus, Potassium, Sodium, Sorbitol dehydrogenase (SDH), Total bilirubin (Total BILI), Total cholesterol (Cholesterol), Total protein, Triglycerides (Triglyceride), Urea nitrogen, Appearance
Thyroid Hormone Analysis:
Sample Collection: Blood samples for thyroid hormone analyses were collected from the jugular vein into tubes without anticoagulants.
Sample Processing: Blood samples were maintained at room temperature and allowed to clot. Serum was isolated in a refrigerated centrifuge and stored in a freezer set to maintain a target of -70 °C.
Sample Analysis: Blood samples were analyzed for Thyroxine (Total T4). Samples to be analyzed for T4 were transferred to the Charles River Ashland Bioanalytical Chemistry Department; analyses were performed using a validated UHPLC/MS/MS assay.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: FOB assessments were recorded for 5 animals/sex/group during the last week of exposure (Study Day 27) or on Lactation Day 28
- Dose groups that were examined: all dose groups
- Battery of functions tested: sensory activity / grip strength / motor activity / other:
Home-Cage Observations: Posture, Convulsions/tremors, Feces consistency, Biting, Palpebral (eyelid) closure.
Handling Observations: Ease of removal from cage, Lacrimation/chromodacryorrhea, Piloerection, Palpebral closure, Eye prominence, Red/crusty deposits, Ease of handling animal in hand, Salivation, Fur appearance, Respiratory rate/character, Mucous membranes/eye/skin color, Muscle tone.
Open Field Observations: Mobility, Rearing, Convulsions/tremors, Grooming, Bizarre/stereotypic behavior, Time to first step (seconds), Gait, Arousal, Urination/defecation, Gait score, Backing.
Sensory Observations: Approach response, Startle response, Pupil response, Forelimb extension, Air righting reflex, Touch response, Tail pinch response, Eyeblink response, Hindlimb extension, Olfactory orientation.
Neuromuscular Observations: Hindlimb extensor strength, Hindlimb foot splay, Grip strength-hind and forelimb, Rotarod performance.
Physiological Observations: Catalepsy, Body temperature, Body weight

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): No

LUNG BURDEN: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
Males were subjected to necropsy on day 28; females were subjected to necropsy on Lactation day 29. On these days organ weights were determined and tissues were collected and subsequently subject to histological and histopathogical examinations.
Animals were subjected to a complete necropsy examination, which included examination of the external surface, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera. The numbers of implantation sites and former implantation sites were recorded for females that delivered or had macroscopic evidence of implantation. The number of unaccounted-for sites was calculated for each female by subtracting the number of pups born from the number of former implantation sites observed. Uteri of females without macroscopic evidence of implantation were opened and placed in 10 % ammonium sulfide solution for detection of early implantation loss.

HISTOPATHOLOGY: Yes
Representative samples of the tissues were collected from all animals and preserved in 10 % neutral buffered formalin, unless otherwise indicated.
The following tissues were collected: Adrenal glands (2), Aorta, Bone with marrow (sternebrae), Brain, Coagulating glands (2), Eyes with optic nerve (2), Gastrointestinal tract, Esophagus, Stomach, Duodenum, Peyer’s Patches, Jejunum, Ileum, Cecum, Colon, Rectum, Heart, Kidneys (2), Liver (sections of 2 lobes), Lymph node, Axillary (2), Mandibular (2), Mesenteric, Lungs (including bronchi, fixed by inflation with fixative), Nasal cavities with turbinates, Ovaries and oviducts (2), Pancreas, Peripheral nerve (sciatic)c, Pituitary gland, Prostate gland, Salivary gland (mandibular [2]), Seminal vesicles (2), Skeletal muscle (quadriceps), Skin with mammary gland, Spinal cord (cervical), Spleen, Testes with epididymides (2) and vas deferens, Thymus gland, Thyroids (with parathyroids, if present [2]), Trachea, Urinary bladder, Uterus with cervix and vagina, All gross lesions.

Organ Weights
The organs were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together, unless otherwise indicated. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated.
The following organs were weighted: 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).

Statistics:

Each mean was presented with the standard deviation (S.D.) and the number of animals or cages (N) used to calculate the mean.
All statistical tests for the following parameters were performed using WTDMS™. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1 % and 5 %, comparing each test substance-treated group to the control group by sex.
Parental body weights and body weight changes, parental food consumption, abs. & rel. organ weights, clinical pathology values, thyroid hormone values, and FOB data values were subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunnett's test was used to compare the test substance-treated groups to the control group. FOB parameters that yielded scalar or descriptive data were analyzed using Fisher’s Exact Test.
The locomotor activity ambulatory & total counts data were statistically analyzed using SAS Software for Windows, Release 9.4 (SAS Institute Inc., Cary, NC) or later. All statistical tests were conducted at the 5 % significance level. All pairwise comparisons were performed two-sided.

Clinical signs:

no effects observed

Description (incidence and severity):

All F0 males and females in the control, 8, 25, and 75 ppm groups survived to the scheduled necropsies. There were no test substance-related clinical observations noted at the weekly examinations or 0.5–3 hours following the completion of exposure. Findings noted in the test substance-exposed groups, including hair loss, scabbing or red material on various body surfaces, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not exposure-related.

Mortality:

no mortality observed

Description (incidence):

All F0 males and females in the control, 8, 25, and 75 ppm groups survived to the scheduled necropsies.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Males, Females premating and Females during gestation: Mean body weights and body weight gains in the 8, 25, and 75 ppm group males were unaffected by test substance exposure throughout the study. None of the differences from the control group were statistically significant.
Females during lactation: Mean body weights and body weight gains in the 8, 25, and 75 ppm groups were unaffected by test substance administration during lactation. The only statistically significant difference from the control group was a lower mean body weight gain in the 75 ppm group females during Lactation Days 1–4 (when females were not being exposed to the test substance). When the overall lactation exposure period (Lactation Days 4-28) was evaluated, body weight change in the 75 mg/kg/day group was comparable to the control group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Males: Mean food consumption, evaluated as g/animal/day, in the 8, 25, and 75 ppm group males was similar to that in the control group throughout the study. No statistically significant differences were observed.
Females premating: Mean food consumption, evaluated as g/animal/day, in the 8, 25, and 75 ppm group females was unaffected by test substance administration during the premating period. None of the differences from the control group were statistically significant.
Females during Gestation: Mean maternal food consumption, evaluated as g/animal/day, in the 8, 25, and 75 ppm groups was unaffected by test substance administration during gestation. The only statistically significant differences from the control group were higher mean food consumption (31.5 % compared to control) in the 25 ppm group during Gestation Days 17–20, resulting in higher mean food consumption when the entire gestation exposure period (Gestation Days 0–20) was evaluated. However, this effect is not dose dependent and not considered to be of toxicological significance.
Females during Lactation: Mean maternal food consumption, evaluated as g/animal/day, in the 8, 25, and 75 ppm groups was unaffected by test substance administration.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on hematology parameters. The only statistically significant difference from the control group values (lower [2.9 %] mean corpuscular hemoglobin concentration in the 25 ppm group females) did not occur in an exposure-related manner and was of minimal magnitude. Therefore, this finding was considered to be the result of normal biological variation and not considered to be of toxicological significance.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on serum chemistry. The only statistically significant difference from the control group was a slightly higher sodium level [1.4 %] in the 25 ppm group males. There was no exposure-response relationship, and the change was of minimal magnitude. This difference from the control group was considered to be the result of normal biological variation and not considered to be of toxicological significance.
There were no test substance-related effects on coagulation parameters. The only statistically significant difference from the control group values (faster [14.7 %] activated prothrombin time in the 75 ppm group females) was of minimal magnitude. Therefore, this finding was considered to be the result of normal biological variation and not considered to be of toxicological significance.

Endocrine findings:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on thyroid hormone values in the F0 males at any dosage level. Differences from the control group were considered to be the result of normal biological variation and were not considered to be of toxicological significance.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional Observational Battery:
Home cage parameters; Handling parameters, Open field parameters, Sensory Observations, Neuromuscular parameters and Physiological parameters were unaffected by test substance administration. There were no statistically significant differences for the test substance-treated groups when compared to the control group during Study Day 27 (males) or on Lactation Day 28 (females).

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test substance-related alterations in organ weights.
Some organ weight differences in the 75 ppm F0 groups were statistically significant when compared to the control group. These included the left epididymis and testes (males, relative to final body weight). The changes were minimal (left testes, 15 % higher and left epididymis, 11 % higher) and in the epididymis and testes were unilateral and had no microscopic correlates.
Therefore, these organ weight differences were considered incidental.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Review of the gross necropsy observations revealed no observations that were considered to be associated with exposure to the test substance.
The mean numbers of unaccounted-for sites and implantation sites in the 8, 25, and 75 ppm groups were similar to the control group values.

Neuropathological findings:

not specified

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 characterized 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.
There were no other test substance-related histologic changes. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than exposure to the test substance. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Motor Activity:
Motor activity patterns (total activity as well as ambulatory activity counts) in F0 animals were unaffected by test substance exposure at all concentrations when evaluated on Study Day 27 (males) or on Lactation Day 28 (females). Values obtained from the 6 subintervals evaluated (0–10, 11–20, 21–30, 31–40, 41–50 and 51–60 minutes) and the overall 60-minute test session values were comparable to the concurrent control values and the Charles River Ashland historical control data. Differences from the control group were slight, not statistically significant, within the Charles River Ashland historical control data ranges, and/or did not occur in an exposure-related manner. No remarkable shifts in the pattern of habituation occurred in any of the test substance-exposed groups when the F0 animals were evaluated.

Dose descriptor:

NOAEL

Effect level:

75 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: systemic toxicity

Dose descriptor:

LOAEL

Effect level:

8 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on test substance-related adverse port of entry findings, consisting of respiratory epithelial hyperplasia and mixed cell type inflammation, and/or ulceration noted in all exposure levels.

DISCUSSION

The objective of this study was to provide preliminary information on the potential adverse effects of the test substance, Dimethylamine (DMA) on male and female reproduction within the scope of a screening study.

All F0 animals survived to the scheduled necropsies. There were no test substance-related systemic and reproductive toxicity noted at 8, 25, and 75 ppm in the F0 generation.

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. Although no systemic adverse effects were noted at any exposure level, local effects that were restricted to Level II of the nasal cavity, not associated with clinical signs, were considered adverse at 75 ppm due to the known corrosive nature of DMA gas.

Conclusions:

In conclusion, no systemic toxicity was noted in the F0 generation at exposure up to 75 ppm (the highest level tested). Therefore, the no-observed-adverse-effect level for F0 male and female systemic was 75 ppm. Based on test substance-related adverse port of entry findings, consisting of respiratory epithelial hyperplasia and mixed cell type inflammation, and/or ulceration noted in all exposure levels the LOAEL for port of entry effects for F0 generation was 8 ppm.

Executive summary:

The objective of this study was to provide preliminary information on the potential adverse effects of the test substance, Dimethylamine (DMA), on male and female reproduction within the scope of an OECD 422 study. The study design was as follows: Group 1 - Treatment: Filtered Air - Exposure Level: 0 ppm - Number of males: 10 - Number of females: 10; Group 2 - Treatment: Dimethylamine - Exposure Level: 8 ppm - Number of males: 10 - Number of females: 10; Group 3 - Treatment: Dimethylamine - Exposure Level: 25 ppm - Number of males: 10 - Number of females: 10; Group 4 - Treatment: Dimethylamine - Exposure Level: 75 ppm - Number of males: 10 - Number of females: 10

Animals were exposed via whole-body inhalation for 6 hours daily. 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. Focussing on the repeated dose part of the study, the following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, food consumption, neurobehavior, thyroid hormones, clinical pathology, gross necropsy findings, organ weights, and histopathologic examinations. Analyzed F0 mean exposure concentrations were 0.1, 9.2, 26.9, and 75.9 ppm in the control, 8, 25, and 75 ppm groups, respectively. On Study Days 3 and 4, non-zero concentrations of DMA were noted in the control chamber. Because no test substance was being directed to the control system, and the chambers were under ambient conditions on these 2 exposure days, there should have been no DMA in the chamber. Therefore, although the reason of non-zero values in the control chamber could not be determined, carryover within the gas chromatogram/sampling setup was suspected to be the cause. The non-zero values were included in the daily average (and study mean as 0.1 ppm) for the control group. No detection of test substance was noted for the rest of the exposure period.

All F0 animals survived to the scheduled necropsies. There were no test substance-related clinical observations noted at the daily examinations or 0.5–3 hours following the completion of exposure at 8, 25, and 75 ppm. No test substance-related effects were noted on mean body weights, body weight changes, or food consumption throughout the F0 generation at any exposure level. No test substance-related effects were noted on the functional observational battery or motor activity in the F0 males on Study Day 27 or F0 females on Lactation Day 28. Hematology, coagulation, and serum chemistry parameters in the 8, 25, and 75 ppm group F0 males and females and mean T4 levels in the F0 males were unaffected by test substance exposure. There were no test substance-related macroscopic findings or effects on organ weights noted at the scheduled F0 necropsies at any exposure level. 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. Although no systemic adverse effects were noted at any exposure level, 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, local effects that were restricted to Level II of the nasal cavity, not associated with clinical signs, were considered adverse at 75 ppm.

In conclusion, no systemic toxicity was noted in the F0 generation at exposure up to 75 ppm (the highest level tested). Therefore, the no-observed-adverse-effect level for F0 male and female systemic and reproductive toxicity was 75 ppm. Based on test substance-related adverse port of entry findings, consisting of respiratory epithelial hyperplasia and mixed cell type inflammation, and/or ulceration noted in all exposure levels the LOAEL for port of entry effects for F0 generation was 8 ppm.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29 July 2016

Deviations:

yes

Remarks:

several slight deviations in Atmosphere Generation & Exposure, Husbandry, In-life Observations, Measurements & Evaluations, Laboratory Evaluations & Postmortem and Pathology (all are considered not to have had impact the overall integrity of the study);

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dimethylamine

EC Number:

204-697-4

EC Name:

Dimethylamine

Cas Number:

124-40-3

Molecular formula:

C2H7N

IUPAC Name:

N-methylmethanamine

Test material form:

gas

Details on test material:

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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Airgas Specialty Products

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

OTHER SPECIFICS
- The Sponsor and/or Supplier provided to the Testing Facility documentation of the identity, strength, purity, composition, and stability for the test substance. A Certificate of Conformance was provided to the Testing Facility.

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Remarks:

Crl:CD(SD)

Details on species / strain selection:

Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) approximately 11 wks
- Weight at study initiation: between 220 and 423 g at the initiation of exposure
- Fasting period before study: not applicable
- Housing: Animals were housed in an animal colony room during nonexposure periods. In detail, on arrival (F0) or following selection (F1), animals were group housed (up to 3 animals of the same sex) until cohabitation or euthanasia. At initiation of cohabitation, F0 males were assigned to individual home cages and then F0 animals were paired for mating in the home cage of the male. Following the breeding period, F0 animals were individually housed. Animals were housed in solid-bottom cages containing appropriate bedding equipped with an automatic watering valve throughout the study. Animals were separated during designated procedures/activities. Each cage was clearly labeled with a color-coded cage card indicating study, group, animal, cage number(s), dosage level, and sex. Cages were arranged on the racks in group order. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals. The animal facilities at Charles River Ashland are accredited by AAALAC International
- Diet: PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 was provided ad libitum throughout the study, except during designated procedures. Food was withheld during the animal exposure periods.
- Water: Municipal tap water after treatment by reverse osmosis was freely available to each animal via an automatic watering system, except during inhalation exposure procedures. Water was withheld during the animal exposure periods.
- Acclimation period: at least 7 days prior to initiation of exposure
- Animal identification: Upon receipt, each animal was identified using a subcutaneously implanted electronic identification chip (BMDS system). Offspring were identified by tattoo markings applied to the digits after parturition and by microchip at weaning.
- Animal Enrichment: Animals were socially housed for psychological/environmental enrichment and were provided with environmental enrichment as appropriate to aid in maintaining the animals’ oral health.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 68 °F to 78 °F (20 °C to 26 °C)
- Humidity (%): 30 % to 70 %
- Air changes (per hr): Ten or greater air changes per hour with 100 % fresh air (no air recirculation) were maintained in the animal rooms
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark

IN-LIFE DATES: From: 24 Jan 2019 (Animal Arrival) To: 13 May 2019

Administration / exposure

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 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 group. The chambers were operated under dynamic conditions, at a slight negative pressure, and with a minimum of 12 air changes per hour.
- Method of holding animals in test chamber: Animals were individually housed in standard exposure batteries of appropriate size for the whole-body exposure chamber in use.
- Source and rate of air: 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. All whole-body chamber exhaust passed through the facility exhaust system, which consists of redundant exhaust blowers preceded by activated-charcoal and HEPA-filtration units
Test substance atmospheres were generated by releasing neat test substance gas from the cylinder. The cylinder was heated to maintain pressure within the cylinder. The test substance gas was directed to a manifold. Test substance gas was directed to the chamber inlet, where it mixed with dilution supply air to achieve the desired atmosphere concentration.
- Method of conditioning air: not specified
- System of generating particulates/aerosols: not applicable
- Temperature, humidity, pressure in air chamber: The mean temperature and relative humidity of the exposure atmospheres were to be 69 °F to 75 °F (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.
- Air flow rate: between 453 and 464 Liters/min
- Air change rate: not specified
- Method of particle size determination: not applicable
- Treatment of exhaust air: All whole-body chamber exhaust passed through the facility exhaust system, which consists of redundant exhaust blowers preceded by activated-charcoal and HEPA-filtration units.

TEST ATMOSPHERE
- Brief description of analytical method used: Analyzed exposure concentrations were determined at approximately 60-minute intervals using a gas chromatograph
- Samples taken from breathing zone: yes (An external multi-position valve permitted sequential sampling from the exposure room and each exposure chamber. Gas sampling injection onto the chromatography column occurred via an internal gas sampling valve with a sample loop, the chromatograph was displayed, and the area under the sample peak was calculated and stored. An ln quadratic equation based on the gas chromatograph calibration curve(s) was used to calculate the measured concentration in ppm.)

VEHICLE (if applicable)
- Justification for use and choice of vehicle: air
- Composition of vehicle: not specified
- Type and concentration of dispersant aid (if powder): not applicable
- Concentration of test material in vehicle: not specified
- Lot/batch no. of vehicle (if required): not required
- Purity of vehicle: no details given
For exposure of the control group (Group 1, 0 ppm), humidified supply air was delivered to the chamber from the facility air supply source. To provide a comparable oxygen level in the control chamber to that as within the test substance chambers, compressed nitrogen was delivered to the inlet of the control exposure chamber at a flow rate similar to that used in the high-exposure group, where it mixed with supply air.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: for a maximum of 14 days
- Proof of pregnancy: vaginal copulatory plug or the presence of sperm in a vaginal lavage referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually
- Any other deviations from standard protocol: none in the context of mating
Vaginal lavages were performed daily during the mating period until evidence of mating was observed. If evidence of mating was not apparent after 14 days, the animals were separated, with no further opportunity for mating. Animals cohabited over a 12-hour dark cycle were considered to have been paired for 1 day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyzed exposure concentrations were determined at approximately 60-minute intervals using a gas chromatograph.
Samples were collected from the approximate animal-breathing zone of the exposure chamber. An external multi-position valve permitted sequential sampling from the exposure room and each exposure chamber. Gas sampling injection onto the chromatography column occurred via an internal gas sampling valve with a sample loop, the chromatograph was displayed, and the area under the sample peak was calculated and stored. An ln quadratic equation based on the gas chromatograph calibration curve(s) was used to calculate the measured concentration in ppm.

Duration of treatment / exposure:

Males: 14 days prior to mating and continuing throughout mating for a total of 28 days
Females: 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
F1 pups: directly exposed from Postnatal Day (PND) 28–40

Frequency of treatment:

6 hours/day

Details on study schedule:

Parturition: The day parturition was initiated was designated Lactation Day 0 (Postnatal Day [PND] 0 for pups). During the period of expected parturition, females were observed twice daily for initiation and completion of parturition and for dystocia or other difficulties. All females were allowed to deliver naturally. Beginning on the day parturition was initiated, the numbers of stillborn and live pups were recorded. Individual gestation length was calculated using the date delivery was first observed.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Remarks:

Control (filtered air)

Doses / concentrations 2

Dose / conc.:

8 ppm

Doses / concentrations 3

Dose / conc.:

25 ppm

Doses / concentrations 4

Dose / conc.:

75 ppm

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Justification of Route and Exposure Levels
The route of administration was whole-body inhalation exposure because inhalation would be a likely route of unintended human exposure.
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.

- Rationale for animal assignment: Animals were assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights. Males and females were randomized separately. Animals with physical abnormalities, at extremes of body weight range, or not exhibiting normal, 4- to 5-day estrous cycles were not assigned to groups.
- Fasting period before blood sampling for clinical biochemistry: Animals were fasted overnight prior to blood collection
- Other: Animals were housed in an animal colony room during nonexposure periods. Prior to each exposure, the animals were transferred to exposure caging, transported to the exposure room, exposed for the requisite duration, transported back to the colony room, and returned to their home cages. Food and water were withheld during the animal exposure periods.
Prior to exposure, females were evaluated for estrous cyclicity during the pretest period and any females that failed to exhibit normal 4-5 day estrous cycles (e.g., EDDDE), during the pretest period, were excluded from the study; therefore, the extra females were included to yield at least 10 females per group. Given the possibility of nongravid animals, unexpected deaths, total litter losses, or test substance-related moribundity and/or mortality, this was an appropriate number of animals to obtain a sample size of 8 at termination.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, once in the morning and once in the afternoon. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were removed from the cage, and a detailed clinical observation was performed once daily throughout the study. During the exposure period, these observations were performed prior to exposure. On exposure days, clinical observations were also recorded 0.5–3 hours following completion of exposure.
During social housing, some observations (e.g., fecal observations) may not have been attributable to an individual animal.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually weekly throughout the study and prior to the scheduled necropsy. Once evidence of mating was observed, female body weights were recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and Lactation Days 1, 4, 7, 10, 13, 17, 21, 25, and 28. A fasted weight was recorded on the day of necropsy.

FOOD CONSUMPTION:
Food consumption was quantitatively measured weekly until cohabitation. Once evidence of mating was observed, female food consumption was recorded on Gestation Days 0, 4, 7, 11, 14, 17, and 20 and Lactation Days 1, 4, 7, 10, 13, 17, 21, 25, and 28.

WATER CONSUMPTION: No

OTHER:
Sample Collection
Animals were fasted overnight prior to blood collection. Blood samples for hematology and serum chemistry were collected from the jugular vein. Blood samples for coagulation parameters were collected by necropsy personnel from the inferior vena cava of animals anesthetized by inhalation of isoflurane and euthanized by exsanguination.
K2EDTA was used for the anticoagulant on samples collected for hematology. Sodium citrate was used for samples collected for clotting determinations. Samples for serum chemistry were collected without anticoagulants.

Hematology
Blood samples were analyzed for the parameters: Differential leukocyte count (-Percent and absolute; -Neutrophil (NEU); -Lymphocyte (LYMPH), -Monocyte (MONO), -Eosinophil (EOS), -Basophil (BASO), -Large unstained cell (LUC)), Erythrocyte count (RBC); Hemoglobin (HGB); Hemoglobin distribution width (HDW), Hematocrit (HCT), Mean corpuscular hemoglobin (MCH), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (Platelet), Red cell distribution width (RDW), Reticulocyte count, Percent (RETIC), Absolute (RETIC Absolute), Total leukocyte count (WBC), Platelet estimatea, Red cell morphology (RBC Morphology).

Coagulation
Blood samples were processed for plasma, and the plasma was analyzed for: Activated partial thromboplastin time (APTT), Fibrinogen, Prothrombin time (PT).

Serum Chemistry
Blood samples were processed for serum, and the serum was analyzed for: Alanine aminotransferase (ALT), Albumin, Albumin/globulin ratio (A/G Ratio) [by calculation], Alkaline phosphatase (ALP), Aspartate aminotransferase (AST), Bile Acids, Calcium, Chloride, Creatinine, Gamma glutamyltransferase (GGT), Globulin [by calculation], Glucose, Phosphorus, Potassium, Sodium, Sorbitol dehydrogenase (SDH), Total bilirubin (Total BILI), Total cholesterol (Cholesterol), Total protein, Triglycerides (Triglyceride), Urea nitrogen, Appearance

Thyroid Hormone Analysis
Sample Collection: Blood samples for thyroid hormone analyses were collected from the jugular vein into tubes without anticoagulants.
Sample Processing: Blood samples were maintained at room temperature and allowed to clot. Serum was isolated in a refrigerated centrifuge and stored in a freezer set to maintain a target of -70 °C.
Sample Analysis: Blood samples were analyzed for Thyroxine (Total T4). Samples to be analyzed for T4 were transferred to the Charles River Ashland Bioanalytical Chemistry Department; analyses were performed using a validated UHPLC/MS/MS assay.

Oestrous cyclicity (parental animals):

For all females, vaginal lavages were performed daily for 2 weeks prior to randomization and continuing until evidence of mating was observed or until the end of the mating period. The slides were microscopically examined to determine the stage of the estrous cycle. The average cycle length was calculated for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [D] from estrus [E] or proestrus [P] for 14 consecutive days before cohabitation and until the detection of evidence of mating). Estrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in a subsequent cycle. The cycle during which evidence of mating was observed for a given animal was not included in the individual mean estrous cycle length calculation. Vaginal lavages were also performed on the day of necropsy to determine the stage of the estrous cycle.

Sperm parameters (parental animals):

not specified

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.
Following completion of delivery, all pups will be individually identified by tattoo markings applied on the digits. To reduce variability among the litters, eight pups from each litter, of equal sex distribution (if possible), will be randomly selected on PND 4. Culled pups will be used for thyroid hormone assessments. Culled pups not selected for blood collection will be euthanized by an intraperitoneal injection of sodium pentobarbital and discarded appropriately.

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. Particular attention should be paid to the external reproductive genitals which should be examined for signs of altered development; gross evaluation of external genitalia

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

LItter observation after birth until PND28
Moribundity:
Litters were observed for general health/mortality and moribundity twice daily, once in the morning and once in the afternoon. A daily record of litter size was maintained. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

Observations
Clinical observations were performed on PND 1, 4, 7, 10, 13, 17, 21, 25, and 28.

Sex Determination
Pups were individually sexed on PND 0, 4, 13, and 28.

Body Weights
Pups were weighed individually on PND 1, 4, 7, 10, 13, 17, 21, 25, and 28.

Preweaning Developmental Landmarks
Anogenital Distance: The anogenital distance of all pups was measured on PND 1. Anogenital distance was defined as the distance from the caudal margin of the anus to the caudal margin of the genital tubercle.
Assessment of Areolas/Nipple Anlagen: On PND 13, all male pups were evaluated for the presence of nipples/areolae. The number of nipples was recorded.

Thyroid Hormone Analysis
Sample Collection: Blood samples for thyroid hormone analyses were collected via cardiac puncture (PND 4) or vena cava (PND 28) from animals anesthetized with isoflurane into tubes without anticoagulants. Samples were collected on PND 4 (at least 2/litter) and on PND 28 (1/sex/litter).
Sample Processing: Blood samples were maintained at room temperature and allowed to clot. Serum was isolated in a refrigerated centrifuge and stored in a freezer set to maintain a target of -70 °C.
Sample Analysis: Blood samples were analyzed for Thyroxine (Total T4).
Samples to be analyzed for T4 were transferred to the Charles River Ashland Bioanalytical Chemistry Department; analyses were performed using a validated UHPLC/MS/MS assay.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals on Study Day 28
- Maternal animals: All surviving animals on Lactation Day 29

Unscheduled Deaths: No animals died during the course of the study; therefore, no animals were examined.
Scheduled Euthanasia: All animals, including females that failed to deliver, were anesthetized by inhalation of isoflurane followed by euthanasia by exsanguination.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
Males were subjected to necropsy on day 28; females were subjected to necropsy on Lactation day 29. On these days organ weights were determined and tissues were collected and subsequently subject to histological and histopathogical examinations.
Animals were subjected to a complete necropsy examination, which included examination of the external surface, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera. The numbers of implantation sites and former implantation sites were recorded for females that delivered or had macroscopic evidence of implantation. The number of unaccounted-for sites was calculated for each female by subtracting the number of pups born from the number of former implantation sites observed. Uteri of females without macroscopic evidence of implantation were opened and placed in 10 % ammonium sulfide solution for detection of early implantation loss.

HISTOPATHOLOGY / ORGAN WEIGHTS
Representative samples of the tissues were collected from all animals and preserved in 10 % neutral buffered formalin, unless otherwise indicated.
The following tissues were collected: Adrenal glands (2), Aorta, Bone with marrow (sternebrae), Brain, Coagulating glands (2), Eyes with optic nerve (2), Gastrointestinal tract, Esophagus, Stomach, Duodenum, Peyer’s Patches, Jejunum, Ileum, Cecum, Colon, Rectum, Heart, Kidneys (2), Liver (sections of 2 lobes), Lymph node, Axillary (2), Mandibular (2), Mesenteric, Lungs (including bronchi, fixed by inflation with fixative), Nasal cavities with turbinates, Ovaries and oviducts (2), Pancreas, Peripheral nerve (sciatic)c, Pituitary gland, Prostate gland, Salivary gland (mandibular [2]), Seminal vesicles (2), Skeletal muscle (quadriceps), Skin with mammary gland, Spinal cord (cervical), Spleen, Testes with epididymides (2) and vas deferens, Thymus gland, Thyroids (with parathyroids, if present [2]), Trachea, Urinary bladder, Uterus with cervix and vagina, All gross lesions.

Organ Weights
The organs were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together, unless otherwise indicated. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated.
The following organs were weighted: 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).

Histology
Tissue trimming was performed at the Testing Facility. Tissues from all animals were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. In addition, PAS staining was used for the testes and epididymides.

Histopathology
Pathological evaluation was performed by a board-certified veterinary pathologist. Tissues for microscopic examination were evaluated from all animals.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at PND40 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: all F1 animals at PND28

Unscheduled Deaths
A necropsy was conducted for animals that died on study, and specified tissues were saved. Intact offspring that were found dead during PND 0–4 were necropsied using a fresh dissection technique, which included examination of the heart and major vessels. Findings were recorded as developmental variations or malformations, as appropriate. A gross necropsy was performed on any pup found dead after PND 4.

Scheduled Euthanasia
On PND 28, animals were euthanized by carbon dioxide inhalation (animals not selected for blood collection) or were anesthetized by inhalation of isoflurane and euthanized by exsanguination (animals selected for blood collection).

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
On PND 28, 1 pup/sex/litter was 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
At the time of necropsy, the following tissues and organs will be collected and placed in 10% neutral-buffered formalin (except as noted):

Organ Weights
The organs were weighed at necropsy from 1 pup/sex/litter at the scheduled euthanasia. Paired organs were weighed together, unless otherwise indicated. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated.
The following organs were weighted: 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, Thyroid (with parathyroids).

Tissue Collection and Preservation
Representative samples of the Thyroid (with parathyroids), the Nasal cavities with turbinates and all gross lesions (all groups) were collected from 1 pup/sex/litter at the scheduled euthanasia and preserved in 10 % neutral buffered formalin.

Histology
Tissue trimming was performed at the Testing Facility. Tissues (Lungs (including bronchi, fixed by inflation with fixative), Nasal cavities with turbinates and all gross lesions (all groups)) from all animals were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin.

Histopathology
Pathological evaluation was performed by a board-certified veterinary pathologist. Tissues identified above for microscopic examination were evaluated from all animals.

Statistics:

Each mean was presented with the standard deviation (S.D.) and the number of animals or cages (N) used to calculate the mean. Where applicable, the litter was used as the experimental unit.
All statistical tests for the following parameters were performed using WTDMS™. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex.
Parental mating, fertility, copulation, and conception indices were analyzed using the Chi-square test with Yates’ correction factor. Parental and offspring body weights and body weight changes, parental food consumption, estrous cycle lengths, precoital intervals, gestation lengths, former implantation sites, unaccounted-for sites, live litter size on PND0, numbers of pups born, abs. & rel. organ weights, clinical pathology values, thyroid hormone values, anogenital distance (abs. & rel. to the cube root of bw), no. of nipples/areolae, and FOB data values were subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunnett's test was used to compare the test substance-treated groups to the control group. FOB parameters that yielded scalar or descriptive data were analyzed using Fisher’s Exact Test. Mean litter proportions of postnatal survival and pup sexes at birth (% of males/litter) were subjected to the Kruskal-Wallis nonparametric ANOVA to determine intergroup differences. If the nonparametric ANOVA revealed significant (p < 0.05) intergroup variance, Dunn’s test was used to compare the test substance-treated groups to the control group.
The locomotor activity ambulatory & total counts data were statistically analyzed using SAS Software for Windows, Release 9.4 (SAS Institute Inc., Cary, NC) or later. All statistical tests were conducted at the 5% significance level. All pairwise comparisons were performed two-sided.

Reproductive indices:

Male copulation, female conception, male, and female mating and fertility indices of the treated groups are compared to the control group using the Chi-square test with Yates’ correction factor.

MALE (FEMALE) MATING INDEX (%) = (NO. OF MALES (FEMALES) WITH EVIDENCE OF MATING (OR CONFIRMED PREGNANCY)/ TOTAL NO. OF MALES (FEMALES) USED FOR MATING) * 100

MALE FERTILITY INDEX (%) = (NO. OF MALES SIRING A LITTER/ TOTAL NO. OF MALES USED FOR MATING) * 100

FEMALE FERTILITY INDEX (%) = (NO. OF FEMALES WITH CONFIRMED PREGNANCY / TOTAL NO. OF FEMALES USED FOR MATING) * 100

MALE COPULATION INDEX (%) = (NO. OF MALES SIRING A LITTER / NO. OF MALES WITH EVIDENCE OF MATING (OR FEMALES CONFIRMED PREGNANT)) * 100

FEMALE CONCEPTION INDEX (%) = (NO. OF FEMALES WITH CONFIRMED PREGNANCY / NO. OF FEMALES WITH EVIDENCE OF MATING (OR CONFIRMED PREGNANCY)) * 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

All F0 males and females in the control, 8, 25, and 75 ppm groups survived to the scheduled necropsies. There were no test substance-related clinical observations noted at the weekly examinations or 0.5–3 hours following the completion of exposure. Findings noted in the test substance-exposed groups, including hair loss, scabbing or red material on various body surfaces, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not exposure-related.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

All F0 males and females in the control, 8, 25, and 75 ppm groups survived to the scheduled necropsies.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Males, Females premating and Females during gestation: Mean body weights and body weight gains in the 8, 25, and 75 ppm group males were unaffected by test substance exposure throughout the study. None of the differences from the control group were statistically significant.
Females during lactation: Mean body weights and body weight gains in the 8, 25, and 75 ppm groups were unaffected by test substance administration during lactation. The only statistically significant difference from the control group was a lower mean body weight gain in the 75 ppm group females during Lactation Days 1–4 (when females were not being exposed to the test substance). When the overall lactation exposure period (Lactation Days 4-28) was evaluated, body weight change in the 75 mg/kg/day group was comparable to the control group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Males: Mean food consumption, evaluated as g/animal/day, in the 8, 25, and 75 ppm group males was similar to that in the control group throughout the study. No statistically significant differences were observed.
Females premating: Mean food consumption, evaluated as g/animal/day, in the 8, 25, and 75 ppm group females was unaffected by test substance administration during the premating period. None of the differences from the control group were statistically significant.
Females during Gestation: Mean maternal food consumption, evaluated as g/animal/day, in the 8, 25, and 75 ppm groups was unaffected by test substance administration during gestation. The only statistically significant differences from the control group were higher mean food consumption (31.5 % compared to control) in the 25 ppm group during Gestation Days 17–20, resulting in higher mean food consumption when the entire gestation exposure period (Gestation Days 0–20) was evaluated. However, this effect is not dose dependent and not considered to be of toxicological significance.
Females during Lactation: Mean maternal food consumption, evaluated as g/animal/day, in the 8, 25, and 75 ppm groups was unaffected by test substance administration.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Haematology:
There were no test substance-related effects on hematology parameters. The only statistically significant difference from the control group values (lower [2.9 %] mean corpuscular hemoglobin concentration in the 25 ppm group females) did not occur in an exposure-related manner and was of minimal magnitude. Therefore, this finding was considered to be the result of normal biological variation and not considered to be of toxicological significance.

Coagulation:
There were no test substance-related effects on coagulation parameters. The only statistically significant difference from the control group values (faster [14.7 %] activated prothrombin time in the 75 ppm group females) was of minimal magnitude. Therefore, this finding was considered to be the result of normal biological variation and not considered to be of toxicological significance.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on serum chemistry. The only statistically significant difference from the control group was a slightly higher sodium level [1.4 %] in the 25 ppm group males. There was no exposure-response relationship, and the change was of minimal magnitude. This difference from the control group was considered to be the result of normal biological variation and not considered to be of toxicological significance.

Endocrine findings:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on thyroid hormone values in the F0 males at any dosage level. Differences from the control group were considered to be the result of normal biological variation and were not considered to be of toxicological significance.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional Observational Battery:
Home cage parameters; Handling parameters, Open field parameters, Sensory Observations, Neuromuscular parameters and Physiological parameters were unaffected by test substance administration. There were no statistically significant differences for the test substance-treated groups when compared to the control group during Study Day 27 (males) or on Lactation Day 28 (females).

Immunological findings:

not examined

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 characterized 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.
There were no other test substance-related histologic changes. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than exposure to the test substance. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Description (incidence and severity):

Motor Activity:
Motor activity patterns (total activity as well as ambulatory activity counts) in F0 animals were unaffected by test substance exposure at all concentrations when evaluated on Study Day 27 (males) or on Lactation Day 28 (females). Values obtained from the 6 subintervals evaluated (0–10, 11–20, 21–30, 31–40, 41–50 and 51–60 minutes) and the overall 60-minute test session values were comparable to the concurrent control values and the Charles River Ashland historical control data. Differences from the control group were slight, not statistically significant, within the Charles River Ashland historical control data ranges, and/or did not occur in an exposure-related manner. No remarkable shifts in the pattern of habituation occurred in any of the test substance-exposed groups when the F0 animals were evaluated.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No test substance-related effects on reproductive performance were observed at any exposure level. No statistically significant differences were noted between the control and test substance-exposed groups. Two males each in the control and 8 ppm groups, respectively, did not sire a litter. Two females each in these same groups were determined to be nongravid. The mean numbers of days between pairing and coitus in the test substance-exposed groups were similar to the control group value. The mean lengths of estrous cycles in these groups were also similar to the control group value. None of these differences were statistically significant.

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

No test substance-related effects on reproductive performance were observed at any exposure level. No statistically significant differences were noted between the control and test substance-exposed groups. Two males each in the control and 8 ppm groups, respectively, did not sire a litter. Two females each in these same groups were determined to be nongravid. The mean numbers of days between pairing and coitus in the test substance-exposed groups were similar to the control group value. The mean lengths of estrous cycles in these groups were also similar to the control group value. None of these differences were statistically significant.

Details on results (P0)

Gestation length and Parturition:
Mean gestation lengths in the 8, 25, and 75 ppm groups were similar to those in the control group. No statistically significant differences were noted. No signs of dystocia were noted in these groups.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

75 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: systemic and reproductive toxicity

2

Dose descriptor:

LOAEL

Effect level:

8 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on test substance-related adverse port of entry findings, consisting of respiratory epithelial hyperplasia and mixed cell type inflammation, and/or ulceration noted in all exposure levels.

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test substance-related clinical observations noted at the daily examinations or 0.5–3 hours following the completion of exposure. Findings noted in the test substance-exposed groups, including scabbing or red material on various body surfaces, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not exposure-related.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

All F1 males and females in the control, 8, 25, and 75 ppm groups survived to the scheduled necropsies.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean F1 body weight gains in the 75 ppm group males and females were occasionally lower than the control group during the exposure period, resulting in slight lower mean body weight gains
when the entire exposure period (PND 28–40) was evaluated. The difference was statistically significant for males on PND 35–36. As a result, mean absolute body weights were up to 5.0 % and 6.3 % lower for these males and females, respectively, compared to the control group during the exposure; the differences were not statistically significant.
Mean F1 body weight and body weight gains in the 8 and 25 ppm group males and females were generally comparable to the control group throughout the exposure period (PND 28–40). The only statistically significant differences from the control group were higher mean body weight gains in the 8 and 25 ppm group males during PND 30–31. There was no exposure-response relationship.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

The anogenital distances (absolute, relative to pup body weight, and relative to the cube root of pup body weight) in the 8, 25, and 75 ppm groups were similar to the control group values.
Differences from the control group were slight and not statistically significant.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Areolae/nipple anlagen in the F1 male pups was unaffected by parental administration of the test substance when evaluated on PND 13. The test substance-treated group values were not statistically different from the control group values.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test substance-related alterations in organ weights. Some organ weight differences in the 75 ppm F1 group males were statistically significant when compared to the control group. These included the kidney weights (males, absolute and relative to brain weight). The changes were minimal (11 % higher). Therefore, these organ weight differences were considered incidental.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Review of the gross necropsy observations revealed no observations that were considered to be associated with exposure to the test substance.

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.

Other effects:

no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

1

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

75 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: systemic and neonatal toxicity

2

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

8 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on adverse port of entry findings in respiratory epithelial

Results: F2 generation

General toxicity (F2)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

DISCUSSION

The objective of this study was to provide preliminary information on the potential adverse effects of the test substance, Dimethylamine (DMA) on male and female reproduction within the scope of a screening study. This encompassed gonadal function, mating behavior, conception, parturition, and lactation of the F0 generation, and the development of F1 offspring from conception through day 40 of postnatal life. In addition, the F1 pups were directly exposed to DMA for 2 weeks following weaning.

All F0 animals survived to the scheduled necropsies. There were no test substance-related systemic and reproductive toxicity noted at 8, 25, and 75 ppm in the F0 generation.

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. Although no systemic adverse effects were noted at any exposure level, local effects that were restricted to Level II of the nasal cavity, not associated with clinical signs, were considered adverse at 75 ppm due to the known corrosive nature of DMA gas.

There were no test substance-related findings at any exposure level in F1 pups during pre-weaning period (PND 0 -28). However, after the initiation of exposure on the F1 generation animals (PND 28-40), slightly lower mean body weight gains were noted in the 75 ppm group F1 males and females occasionally during the exposure period (PND 28–40; statistically significant in males on PND 35-36), resulting in mean absolute body weights that were up to 5.0 % and 6.3 % lower for these males and females, respectively, compared to the control group during the exposure period; the difference was not statistically significant.

Transitional epithelial degeneration and/or ulceration, respiratory epithelial degeneration, and/or mixed cell inflammation were noted in all F1 test substance-exposure groups on PND 40. At 8 and 25 ppm, findings were generally limited to nasal cavity Level II and there was no ulceration or associated clinical signs; therefore, the changes were considered nonadverse. In contrast to the F0 generation, F1 generation initiated exposure at a much younger age and changes in the F1 animals at 75 ppm were consistently observed at multiple levels, and ulceration was prominent at Level II; the F1 animals also had body weight effects. 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, the nasal cavity microscopic findings in these groups were considered adverse.

Applicant's summary and conclusion

Conclusions:

In conclusion, no systemic and reproductive toxicity was noted in the F0 generation at exposure up to 75 ppm (the highest level tested). Therefore, the no-observed-adverse-effect level for F0 male and female systemic and reproductive toxicity was 75 ppm. Based on test substance-related adverse port of entry findings, consisting of respiratory epithelial hyperplasia and mixed cell type inflammation, and/or ulceration noted in all exposure levels the LOAEL for port of entry effects for F0 generation was 8 ppm.
No systemic and neonatal toxicity were noted in the F1 generation animals, therefore the NOAEL for systemic and neonatal toxicity for F1 generation was 75 ppm. Similar to F0 generation, adverse port of entry findings in respiratory epithelial were noted in F1 animals; therefore, the LOAEL for port of entry effects for F1 animals was 8 ppm.

Executive summary:

The objective of this study was to provide preliminary information on the potential adverse effects of the test substance, Dimethylamine (DMA), on male and female reproduction within the scope of an OECD 422 study. This encompassed gonadal function, mating behavior, conception, parturition, and lactation of the F0 generation, and the development of F1 offspring from conception through day 40 of postnatal life. In addition, the F1 pups were directly exposed to DMA for 2 weeks following weaning.
The study design was as follows:
Group 1 - Treatment: Filtered Air - Exposure Level: 0 ppm - Number of males: 10 - Number of females: 10
Group 2 - Treatment: Dimethylamine - Exposure Level: 8 ppm - Number of males: 10 - Number of females: 10
Group 3 - Treatment: Dimethylamine - Exposure Level: 25 ppm - Number of males: 10 - Number of females: 10
Group 4 - Treatment: Dimethylamine - Exposure Level: 75 ppm - Number of males: 10 - Number of females: 10

Animals were exposed via whole-body inhalation for 6 hours daily. 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.
The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, food consumption, estrous cycles, reproductive performance, parturition, litter viability and survival, anogenital distance, areolae/nipple anlagen, neurobehavior, thyroid hormones, clinical pathology, gross necropsy findings, organ weights, and histopathologic examinations.
Analyzed F0 mean exposure concentrations were 0.1, 9.2, 26.9, and 75.9 ppm in the control, 8, 25, and 75 ppm groups, respectively. Analyzed F1 mean exposure concentrations were 0.0, 7.6, 25.2, and 73.9 ppm in the same respective groups during PND 28-40. On Study Days 3 and 4, non-zero concentrations of DMA were noted in the control chamber. Because no test substance was being directed to the control system, and the chambers were under ambient conditions on these 2 exposure days, there should have been no DMA in the chamber. Therefore, although the reason of non-zero values in the control chamber could not be determined, carryover within the gas chromatogram/sampling setup was suspected to be the cause. The non-zero values were included in the daily average (and study mean as 0.1 ppm) for the control group. No detection of test substance was noted for the rest of the exposure period.
All F0 animals survived to the scheduled necropsies. There were no test substance-related clinical observations noted at the daily examinations or 0.5–3 hours following the completion of exposure at 8, 25, and 75 ppm.
No test substance-related effects were noted on mean body weights, body weight changes, or food consumption throughout the F0 generation at any exposure level.
No test substance-related effects on F0 reproductive performance (male and female mating and fertility, male copulation, and female conception indices) were observed at any exposure level.
The mean numbers of days between pairing and coitus and estrous cycle lengths in the test substance-exposed groups were similar to the control group value. There were no test substance-related effects on mean gestation lengths or the process of parturition at any exposure level.
No test substance-related effects were noted on the functional observational battery or motor activity in the F0 males on Study Day 27 or F0 females on Lactation Day 28.
Hematology, coagulation, and serum chemistry parameters in the 8, 25, and 75 ppm group F0 males and females and mean T4 levels in the F0 males were unaffected by test substance exposure.
There were no test substance-related macroscopic findings or effects on organ weights noted at the scheduled F0 necropsies at any exposure level.
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. Although no systemic adverse effects were noted at any exposure level, 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, local effects that were restricted to Level II of the nasal cavity, not associated with clinical signs, were considered adverse at 75 ppm.
The mean number of F1 pups born, live litter size on PND 0, postnatal survival, percentage of males at birth, general physical condition of the pups, anogenital distance, and areola/nipple anlagen in the 8, 25, and 75 ppm groups were unaffected by parental exposure to the test substance. Lower mean body weight gain was noted in the 75 ppm group females at the end of the preweaning period (PND 25–29). No other effects on F1 pup body weights or body weight gains were noted at any exposure level during the preweaning period.
There were no test substance-related macroscopic findings at any exposure level in F1 pups that were found dead or at the scheduled necropsy and no effects on F1 organ weights on PND 28.
All F1 animals selected for direct exposure survived to the scheduled necropsy. There were no test substance-related clinical observations noted at the daily examinations or 0.5–3 hours following the completion of exposure at 8, 25, and 75 ppm.
Slightly lower mean body weight gains were noted in the 75 ppm group F1 males and females occasionally during the exposure period (PND 28–40), resulting in mean absolute body weights that were up to 5.0% and 6.3% lower for these males and females, respectively, compared to the control group during the exposure period; due to the low magnitude of the changes, the lower body weight gains were considered nonadverse. No test substance-related effects on mean body weights or body weight gains were noted in the F1 pups at 8 and 25 ppm.
There were no test substance-related macroscopic findings or effects on organ weights on PND 40 at any exposure level. Transitional epithelial degeneration and/or ulceration, respiratory epithelial degeneration, and/or mixed cell inflammation were noted in all F1 test substance-exposure groups on PND 40. At 8 and 25 ppm, findings were generally limited to nasal cavity Level II and there was no ulceration or associated clinical signs; therefore, the changes were considered nonadverse. At 75 ppm, changes were consistently observed at multiple levels, ulceration was prominent at Level II. 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, the nasal cavity microscopic findings in these groups were considered adverse.
In conclusion, no systemic and reproductive toxicity was noted in the F0 generation at exposure up to 75 ppm (the highest level tested). Therefore, the no-observed-adverse-effect level for F0 male and female systemic and reproductive toxicity was 75 ppm. Based on test substance-related adverse port of entry findings, consisting of respiratory epithelial hyperplasia and mixed cell type inflammation, and/or ulceration noted in all exposure levels the LOAEL for port of entry effects for F0 generation was 8 ppm.
No systemic and neonatal toxicity were noted in the F1 generation animals, therefore the NOAEL for systemic and neonatal toxicity for F1 generation was 75 ppm. Similar to F0 generation, adverse port of entry findings in respiratory epithelial were noted in F1 animals; therefore, the LOAEL for port of entry effects for F1 animals was 8 ppm.