Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005-05-24 till 2007-05-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline Study, test substance used is methylamine hydrochloride
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005-05-24 till 2007-05-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline Study, as test substance methylamine hydrochloride was used
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to
Guideline:
other: U. S. EPA Health Effects Test Guidelines OPPTS 870.3650 Combined Repeated Dose Toxicity Study With the Reproduction/Developmental Toxicity Screening Test.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD®(SD)IGS BR rats
Sex:
male/female
Details on test animals and environmental conditions:
On May 12, 2005, 52 male and 52 female Crl:CD®(SD)IGS BR rats, with an assigned birth date of March 21, 2005, were received from Charles River Laboratories, Inc., Raleigh, North Carolina for use on this study. The rats were approximately 52 days old upon arrival with body weights on the day after arrival that ranged from 195.9 - 243.9 grams (males) and 162.6 - 200.6 grams (females). Male and female rats were nonsiblings; females were nulliparous. The rats were approximately 65 days old at the start of treatment (May 25, 2005) when they weighed between 296.5 – 356.2 grams (males) and 200.4 - 250.1 grams (females).

All male rats were housed individually during non-mating periods in stainless steel, wire-mesh cages suspended above cageboards.

Cohabitation Period: All rats were housed as breeding pairs in stainless steel, wire-mesh cages suspended above cageboard. At the end of the cohabitation period, females without evidence of copulation were housed individually in polycarbonate pans.

Gestation Period (Females with evidence of copulation)
• Days 0 - 19 Gestation (G): Dams were housed individually in stainless steel, wire-mesh cages suspended above cageboards.
• Day 20G - Delivery, Day 4 Lactation (L): Females were housed individually in polycarbonate pans with bedding.
Lactation Period: Dams were housed with their litters in polycarbonate pans with bedding.

temperature of 18-26ºC.
an acceptable relative humidity of 30%-70%.
artificially illuminated (fluorescent light) on a 12-hour light/dark cycle (approximately 0600-1800 hours).
All rats were fed pelleted PMI® Nutrition International, Certified Rodent LabDiet® 5002 ad libitum, except when fasted.
Tap water from United Water Delaware was provided ad libitum.

During the pretest period, a unique number was assigned to each animal which was tattooed on the animal’s tail and included on the animal’s cage label.
Health Monitoring Program
As specified in the Haskell Laboratory animal health and environmental monitoring program, the following procedures are performed periodically to ensure that contaminant levels are below those that would be expected to impact the scientific integrity of the study:
• Water samples are analyzed for total bacterial counts, and the presence of coliforms, lead, and other contaminants.
• Samples from freshly washed cages and cage racks are analyzed to ensure adequate sanitation by the cagewashers.

Certified animal feed is used, guaranteed by the manufacturer to meet specified nutritional requirements and not to exceed stated maximum concentrations of key contaminants, including specified heavy metals, aflatoxin, chlorinated hydrocarbons, and organophosphates. The presence of these contaminants below the maximum concentration stated by the manufacturer would not be expected to impact the integrity of the study.

The animal health and environmental monitoring program is administered by the attending laboratory animal veterinarian. Evaluation of these data did not indicate any conditions that affected the validity of the study.

Upon arrival at Haskell Laboratory, the rats were quarantined for 7 days of the 13-day pretest period and were released from quarantine on test day -6.

The rats were observed daily for any apparent signs of disease or injury and weighed twice during the quarantine period. Rats were released from quarantine by the laboratory animal veterinarian on the basis of acceptable body weight gains and clinical observations.
Route of administration:
oral: gavage
Vehicle:
other: NanoPure water
Details on oral exposure:
quantity 4 mL/kg, based on the most recently recorded body weight, administered by oral intubation
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test substance methylammonium chloride, a white, crystalline powder, was supplied by Alfa Aesar, Ward Hill, Massachusetts, and was assigned Haskell Number 26809.
The bulk test substance was provided by Alfa Aesar, a Johnson Matthey Company, 30 Bond Street, Ward Hill, MA 01835. A Certificate of Analyses (COA) was provided by the supplier and indicated that the purity of the test substance was 99.46%.
To confirm that the test substance was indeed stable over the period of use for the current study, a sample was analyzed near the end of the study by Case Consulting Laboratories, Inc.; the results of these analyses confirmed the purity of the test substance to be 99.2%.

Formulations of the test substance methylammonium chloride in the vehicle (NanoPure® water) were prepared daily until stability data were available that supported weekly preparation of the formulations. After these stability data were generated, the dosing formulations were prepared weekly and stored refrigerated up to seven days. The pH values for all dose preparations were recorded in the study records. Dose formulations with pH values that were initially > 6.0 or < 4.0 were adjusted to a pH value between 4.0 and 6.0 prior to dosing.

Two sets of samples were collected from the dosing formulations prepared near the beginning of the study. Analyses addressed concentration, uniformity of mixing, and stability.
• The first sets of samples were analyzed immediately to verify mixing uniformity, concentration, and 5-hour room temperature stability of the test substance in the formulation.
• The second sets of samples were analyzed to verify stability after 7 days of refrigeration and stability after 7 days of refrigeration followed by 5 hours at room temperature.
Duration of treatment / exposure:
All animals were dosed once daily by gavage for 71 days prior to cohabitation and during the cohabitation period (up to 2 weeks).
• Male rats and female rats showing no evidence of copulation were dosed after the end of the cohabitation period until the day before sacrifice.
• Females showing evidence of copulation were dosed throughout gestation.
• Pregnant females in the process of delivery or showing signs of delivery were not dosed.
• Females were dosed after delivering litters, until day 3 postpartum.
• Females that did not deliver a litter continued to be dosed until the day before sacrifice.
Frequency of treatment:
once daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
Following a 71-day premating period, P1 males and females were cohoused for up to 2 weeks within their respective treatment groups to produce F1 litters. Dams were allowed to deliver and rear their offspring until postpartum day 4.

After being transferred into polycarbonate pans (on day 20 of gestation for mated females, or at the end of the cohabitation period for females without evidence of copulation), female rats were observed at least twice daily for signs of delivery and offspring.

The day when delivery was complete was designated day 0 postpartum. At each examination period (days 0 and 4 postpartum), offspring were individually handled and examined for abnormal behavior and appearance; any dead or abnormal pups were recorded.
1. Day 0 Postpartum: Live and dead pups in each litter were counted by sex as soon as possible after delivery was completed. Live pups in each litter were individually weighed.
2. Day 4 Postpartum: Live pups in each litter were counted by sex and individually weighed and a gross external examination was performed. All pups were euthanized by decapitation.

Rats of each sex were selected for use on study based on adequate body weight gain and freedom from any clinical signs of disease or injury. They were distributed by computerized, stratified randomization into study groups as designated in the Study Design so that there were no statistically significant differences among group body weight means within a sex (p > 0.05). The weight variation at study start (the beginning of test substance administration) did not exceed ± 20% of the mean for each sex. During pretest, on test day –1, detailed clinical observations were performed. Rats not assigned to the study were sacrificed by carbon dioxide euthanasia and discarded without evaluation.
Observations and examinations performed and frequency:
Cage-site examinations to detect moribund or dead animals and abnormal behavior and/or appearance were performed on all animals at least once daily during the quarantine/pretest period and twice daily during the dosing period.
Careful clinical observations were recorded once daily at approximately the same time each morning during dosing; detailed clinical observations were recorded once during the pretest period and weekly thereafter.
Body weights and food consumption were recorded weekly for P1 males and females (premating), on days 0, 7, 14, and 21 of gestation and on days 0 and 4 of lactation; food consumption was not measured during cohabitation or thereafter for males, or for females with no evidence of copulation.

Once during pretest (baseline), and weekly thereafter at approximately the same time of day (+ 2 hours), animals were individually handled and examined for abnormal behavior and appearance in a standardized arena. Observations included (but were not limited to) evaluation of fur, skin, eyes, mucous membranes, occurrence of secretions and excretions, autonomic nervous system activity (lacrimation, piloerection, and unusual respiratory pattern), changes in gait, posture, response to handling, presence of clonic, tonic, stereotypical, or bizarre behavior.

An abbreviated neurobehavioral evaluation consisting of a functional observational battery and motor activity was conducted in P1 rats once during pretest and again near the end of the premating period. Clinical pathology parameters were measured in P1 rats near the end of the premating period (haematology, clinical chemistry, urinalysis) and at terminal sacrifice (coagulation). F1 litter examinations (pup viability, individual pup weights, clinical observations) were performed at birth and on lactation day 4.
Sacrifice and pathology:
All P1 rats were given a gross pathological examination at terminal sacrifice (euthanized by carbon dioxide anaethesia and exsanguination on days 118-119 (males) or 98 - 112 (females)). Selected tissues and gross lesions were weighed and/or retained for microscopic examination. Uterine implantation sites and ovarian corpora lutea were counted in P1 females. Final body weights and organ weights were recorded. A histological examination of all tissues saved was conducted for all animals in the control and 1000 mg/kg/day group. Examination of tissues from the remaining groups was limited to relevant gross lesions and those tissues that demonstrated treatment-related histological effects in the 1000 mg/kg/day group.

A clinical pathology evaluation was conducted on all animals once before the cohabitation period on test days 69/70 (haematology, clinical chemistry, and urinalysis) and again on test days 118-119 and 98-112 (coagulation) for males and females, respectively. The day before collection of samples for the clinical pathology evaluation for haematology, clinical chemistry, and urinalysis, the animals were placed in metabolism cages. These animals were fasted after 3 p.m. for at least 15 hours and urine was collected from each animal. Blood samples for haematology and clinical chemistry measurements were collected from the orbital sinus of each animal while the animal was under carbon dioxide anaesthesia. Blood samples for coagulation parameters were collected at sacrifice from the abdominal vena cava of each animal while the animal was under carbon dioxide anaesthesia. Additional blood collected from the vena cava was placed in a serum tube, processed to serum, and frozen at approximately -80°C. Serum was discarded without analysis because further tests were not required to support experimental findings. Bone marrow smears were prepared at sacrifice from all surviving animals. Bone marrow smears were stained with Wright-Giemsa stain, but analysis was not necessary to support experimental findings. All blood samples were evaluated for quality by visual examination prior to analysis. Results were maintained in the study records and reported only if the sample was analyzed. Unless otherwise indicated, any historical control clinical pathology data referenced in the text is maintained in the study records.

The following tissues were weighed from all adult rats: liver, kidneys, adrenal glands, thymus, brain, spleen, heart, testes, and epididymides. Organ weight ratios (% final body weight, % brain weight) and group mean values were calculated.
All tissues were fixed in 10% neutral buffered formalin except the testes and epididymides, which were fixed in modified Davidson’s solution. Processed tissues were embedded in paraffin, sectioned approximately 5-6 microns thick, stained with hematoxylin and eosin (H&E), and examined microscopically by a veterinary pathologist.
The reproductive organs from all (12/sex/dose) control (0 mg/kg/day) and high-dose (1000 mg/kg/day) rats were processed to slides and evaluated microscopically. The remaining collected tissues were processed to slides and evaluated microscopically from randomly selected individual (5/sex/dose) control and high-dose rats. Gross observations (recorded at necropsy) were examined microscopically for all animals.
Tissue identified as having test substance-related changes, in a given sex, based on the microscopic examination of selected control and high-dose rats (5/sex/dose), were processed and examined microscopically for all rats at each dose level (12/sex/dose). In this study, the trachea and stomach were identified as target organs in both sexes. These two tissues, from all adult rats, were processed to slides and examined microscopically. In addition, the lungs from all rats and the hearts from all male rats were processed to slides and examined in order to confirm the absence of any test substance-related effects.
Other examinations:
Evidence of Copulation: once daily, each female was examined for the presence of an intravaginal copulation plug or sperm in the vaginal lavage sample, either of which was considered evidence of copulation. The presence of an intravaginal plug and/or sperm was recorded. The day evidence of copulation was observed was designated as day 0 of gestation.

Neurobehavioral Evaluation: Prior to initiation of test substance administration (test day -2 for male rats and test day -1 for female rats) and prior to the end of the premating period (test day 62 for male rats and test day 63 for female rats), an abbreviated functional observational battery (FOB) and motor activity (MA) were conducted on all animals. Body weights were collected on the day of neurobehavioral assessments. The FOB and MA assessments were conducted in 4 replicates for each evaluation. Assignment to a given replicate was counterbalanced across all groups. Replicate designation was not reported in the final report, but was recorded in the study records.
For all the following assessments, the experimenter was unaware of the group designation of the animal.
1. Abbreviated Functional Observational Battery (FOB):Assessments of responses to approach/touch, sharp auditory stimulus, and tail pinch were made while the animal was in a standard arena. Fore- and hindlimb grip strength were measured by a strain gauge device (Chatillon® Digital Force gauge). Pupillary constriction was measured immediately prior to removing the rats from the motor activity chambers (Section 2. below) because the darkened room in which the apparatus was located facilitated observing the response. The presence or absence of pupillary constriction was assessed after a beam of light was directed into each eye.
2. Motor Activity (MA): Following the evaluation of grip strength and sensory function, assessment of motor activity (MA) was conducted. Rats were individually tested in 1 of 30 identical, automated activity monitors (Coulbourn® Infrared Motor Activity System). Group and sex were counterbalanced across the monitors and time of day to the fullest extent possible. The infrared monitoring device enables measurement of 2 dependent variables: duration of movement and number of movements. A continuous movement was counted as 1 movement regardless of duration. Each test session was 60 minutes in duration, and the results were expressed for the total session, as well as for 6 successive 10-minute blocks. Presence of diarrhea and polyuria on the cageboards below the motor activity monitor were also recorded if observed following each motor activity session.
3. Test Facility Positive Control Data: Procedures and data describing the effects of trimethyltin, acrylamide, carbaryl, and d-amphetamine are presented in 5 separate reports.(2,3,4,5,6,) These positive control studies are the basis of training certification for the people making judgments in the neurobehavioral and neuropathology tests. The data also document that the equipment and procedures are capable of detecting effects that may be seen in studies of this type.
Statistics:
Mating index, Fertility index, Gestation index, Impantation Efficiency, Pups born alive, 0-4 day viability.

levene's test for homogeneity and Shapiro-Wilk test for normality
one-way analysis of variance and Dunnett's test
Kruskal-Wallis test and Dunnett's test
Cochran-Armitage test for trend
Analysis of covariance and Dunnett-Hsu
non-parametric analysis covariance
repeated measures analysis of variance followed by linear contrasts
sequential application of the Jonckheere-terpstra trend test
Clinical signs:
no effects observed
Description (incidence and severity):
No specific test substance-related deaths in males or females at any level tested, no test substance-related clinical observations at any level tested
Mortality:
no mortality observed
Description (incidence):
No specific test substance-related deaths in males or females at any level tested, no test substance-related clinical observations at any level tested
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related, adverse effects at 1000 mg/kg/day: significant reductions in body weight in P1 animals. No test substance-related effects on body weight parameters at 500 mg/kg/day or lower.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Test substance-related, adverse effects at 1000 mg/kg/day: slightly significant reduction in food consumption parameters in P1 animals. Did not persist longer than 2 weeks. No effects on maternal food consumption during gestation at 500 mg/kg/day or lower
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):
Compound-related but not adverse(females), unrelated effects (males) changes in haematologic parameters
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No adverse changes in clinical chemistry. minimal changes in Cholesterol, Glucose, Globulin, total protein, inorganic phosphorus, AP activity, tryglycerides (dose ranges: 500 - 1000 mg/kg /day). Changes mainly likely to be related but not adverse
Urinalysis findings:
no effects observed
Description (incidence and severity):
No adverse changes in urinalysis parameters, minimal changes in urine osmolarity, pH, ketones (dose ranges: 500 - 1000 mg/kg /day). Changes mainly likely to be related but not adverse
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No test substance-related effects or statistically significant differences on fore- / hinglimb grip strength. No test substance-related effects or statistically significant effects for any behavioral parameter. No effects on duration of movement.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Liver and kidney weight parameters were increased in both sexes. Liver weights were increased in males at ≥ 250 mg/kg/day and in females at ≥ 500 mg/kg/day. Kidney weights were increased in both sexes only at the highest dose (1000 mg/kg/day).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related, adverse effects at 1000 mg/kg/day: significantly reduced corpora lutea counts and subsequently lower implantation site counts and litter sizes.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related microscopic findings were observed in the trachea (squamous metaplasia) and stomach (mucoid metaplasia) of males and females given 1000 mg/kg/day of the test substance.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
There was a test substance-related and statistically significant reduction in mean corpora lutea count at 1000 mg/kg/day that resulted in a reduction in the mean number of implantation sites per litter and a subsequent significant reduction in the mean litter size at this dose level. There were no other test substance-related effects on reproductive outcome at this level nor were there any effects on reproductive outcome at 500 mg/kg/day or lower.
There was a significant reduction in mean litter size at 1000 mg/kg/day that was associated with a test substance-related reduction in the mean numbers of corpora lutea observed at this level. Otherwise, there were no additional test substance-related effects in offspring at 1000 mg/kg/day and no evidence of any test substance-related effects on offspring at 500 mg/kg/day or lower.

There were no test substance-related clinical observations in the pups at any dose level tested.

There were no test substance-related effects on mean pup weight either at birth or on day 4 of lactation at any dose level tested.
Dose descriptor:
NOEL
Effect level:
500 mg/kg bw (total dose)
Based on:
other: reductions in parental body weights and food consumption and effects on reproductive outcome (reduced corpora lutea and subsequent reductions in implantations and litter size).
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
other: Reproductive outcome
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Pathology
Critical effects observed:
not specified

A. Test Substance Stability (Appendix B): Alfa Aesar supplied Haskell Laboratory with the test substance methylammonium chloride, conducted the purity analysis for the beginning of the study, and supplied the Certificate of Analysis (COA) confirming the purity of the test substance methylammonium chloride to be 99.46%. The stability of the test substance methylamine hydrochloride was confirmed by purity analysis conducted near the end of the study by Case Consulting Laboratories, Inc., confirming the purity of the test substance to be 99.2% (average of 2 samples analyzed).

B. Test Formulation Analysis (Appendix C): The analytical method used is described in Appendix C. Data from the analysis of the samples during the study indicate that the test substance methylammonium chloride was at the targeted levels, mixed uniformly, and was stable under the conditions of the study. No test substance was found in the 0 mg/mL samples.

1. Stability of the Test Substance Methylammonium chloride in the Vehicle: The data show that the concentrations of the test substance Methylamonium chloride in the vehicle were stable for the 7 days of refrigeration followed by 5 hours at room temperature.

2. Homogeneity and Concentration Verification: The data for samples submitted show that the concentrations of the samples were at the targeted levels and were uniformly mixed.

Conclusions:
Test substance-related effects were observed at 1000 mg/kg/day and were limited to reductions in parental body weights and food consumption parameters and a significant reduction in the mean corpora lutea and associated reduction in mean implantation sites and mean litter size. There was no evidence of systemic or reproductive toxicity at 500 mg/kg/day or lower.
Executive summary:

Test substance-related effects were observed at 1000 mg/kg/day and were limited to reductions in parental body weights and food consumption parameters and a significant reduction in the mean corpora lutea and associated reduction in mean implantation sites and mean litter size. There was no evidence of systemic or reproductive toxicity at 500 mg/kg/day or lower. Under the conditions of the study, there were no test substance-related effects on any neurobehavioral parameter evaluated in either males or females at any dose level tested. Daily gavage administration of up to 1000 mg/kg/day of the test substance Methylammonium chloride to male and female rats resulted in no adverse changes in clinical pathology parameters.

Liver: Since there was no gross or microscopic finding that correlated with the increased liver weights, and there was no increase in serum liver enzymes (see Clinical Chemistry), the weight effect was considered pharmacological and not adverse. Exposure to xenobiotics commonly induces hepatic metabolic enzymes in laboratory animals. While microscopic hypertrophy of hepatocytes may occur in hepatic enzyme induction, small increases in liver weight parameters are often not associated with a morphological change.

The increased kidney weight parameters in both sexes at 1000 mg/kg/day did not correlate with any gross or microscopic renal pathology. As in liver hepatocytes, xenobiotics may induce P450 microsomal enzymes in the renal proximal tubular epithelium. Increased kidney weights often precede morphological changes (i.e., hypertrophy) in non-adverse enzyme induction.

The metaplasia consisted of the partial replacement of the normal ciliated pseudostratified columnar epithelium on the ventral aspect of the tracheal mucosa with round and slightly flattened epithelial cells resembling mucous membrane. The change was not associated with inflammation of either the trachea or the lung. The minimal to mild squamous metaplasia was probably the result of transient topical exposure of the tracheal mucosa to the test substance Methylammonium chloride following gavage administration. It was interpreted to be a non-adverse adaptive response, most likely reversible, that was due to imperfect dosing or transient esophageal reflux.

The metaplasia consisted of a partial replacement of the normal parietal cells in the gastric glands with mucous epithelial cells. The change was evident in the fundic region of the glandular mucosa adjacent to the esophageal inlet. The change was not associated with any inflammation and was considered to be non-adverse. The minimal mucoid metaplasia was probably the result of the daily gavage administration of a slightly irritating solution to the glandular mucosa adjacent to the esophageal inlet. The degree of focal metaplasia appeared to be biologically insignificant. As with the focal tracheal metaplasia, the focal gastric metaplasia was interpreted to be a non-adverse, most likely reversible, adaptive response.

---

Daily gavage administration of 0, 250, 500, and 1000 mg/kg/day of the test substance methylamine hydrochloride, to male and female rats for 98 to 119 consecutive days, resulted in increased liver and kidney weights, squamous metaplasia of the tracheal mucosa, and mucoid metaplasia of the glandular gastric mucosa. The increased liver weights in males (≥ 250 mg/kg/day) and females (≥ 500 mg/kg/day) and kidney weights in both sexes (1000 mg/kg/day) were consistent with pharmacological enzyme induction and were considered to be non-adverse. Minimal to mild squamous metaplasia of the tracheal mucosa and minimal focal mucoid metaplasia of the gastric glandular mucosa of several high-dose rats of both sexes were interpreted to be the result of topical exposure of the test substance Methylammonium chloride. The metaplastic changes in both tissues were interpreted to be adaptive and probably reversible. Therefore, both the tracheal and gastric metaplasia were considered to be non-adverse. There were no test substance-related effects on cause of death, gross pathology, or reproductive failure in adult rats. Gross examination of nursing pups that died before day 4 of lactation did not reveal any test substance-related findings. Under the conditions of this study, the no-observed-effect level (NOEL) for pathology for male and female rats was the highest dose tested, 1000 mg/kg/day.

Under the conditions of this study, the no-observed-effect level (NOEL) for systemic and reproductive toxicity was 500 mg/kg/day Methylammonium chloride based on reductions in parental body weights and food consumption and effects on reproductive outcome (reduced corpora lutea and subsequent reductions in implantations and litter size).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to
Guideline:
other: U. S. EPA Health Effects Test Guidelines OPPTS 870.3650 Combined Repeated Dose Toxicity Study With the Reproduction/Developmental Toxicity Screening Test.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Composition: Methylamine hydrochloride 99.46%
Purity: 99.46%
Physical Characteristics: White crystalline powder
Study Initiated/Completed: May 24, 2005 / (see report cover page)
Experimental Start/Termination: May 25, 2005 /
(see anatomic pathology report date on Certification page)

test substance applie to rats in NanoPure® water (4 mL/kg )

Test animals

Species:
rat
Strain:
other: Crl:CD®(SD)IGS BR rats
Sex:
male/female
Details on test animals and environmental conditions:
On May 12, 2005, 52 male and 52 female Crl:CD®(SD)IGS BR rats, with an assigned birth date of March 21, 2005, were received from Charles River Laboratories, Inc., Raleigh, North Carolina for use on this study. The rats were approximately 52 days old upon arrival with body weights on the day after arrival that ranged from 195.9 - 243.9 grams (males) and 162.6 - 200.6 grams (females). Male and female rats were nonsiblings; females were nulliparous. The rats were approximately 65 days old at the start of treatment (May 25, 2005) when they weighed between 296.5 – 356.2 grams (males) and 200.4 - 250.1 grams (females).

All male rats were housed individually during non-mating periods in stainless steel, wire-mesh cages suspended above cageboards.

Cohabitation Period: All rats were housed as breeding pairs in stainless steel, wire-mesh cages suspended above cageboard. At the end of the cohabitation period, females without evidence of copulation were housed individually in polycarbonate pans.

Gestation Period (Females with evidence of copulation)
• Days 0 - 19 Gestation (G): Dams were housed individually in stainless steel, wire-mesh cages suspended above cageboards.
• Day 20G - Delivery, Day 4 Lactation (L): Females were housed individually in polycarbonate pans with bedding.
Lactation Period: Dams were housed with their litters in polycarbonate pans with bedding.

temperature of 18-26ºC.
an acceptable relative humidity of 30%-70%.
artificially illuminated (fluorescent light) on a 12-hour light/dark cycle (approximately 0600-1800 hours).
All rats were fed pelleted PMI® Nutrition International, Certified Rodent LabDiet® 5002 ad libitum, except when fasted.
Tap water from United Water Delaware was provided ad libitum.

During the pretest period, a unique number was assigned to each animal which was tattooed on the animal’s tail and included on the animal’s cage label.
Health Monitoring Program
As specified in the Haskell Laboratory animal health and environmental monitoring program, the following procedures are performed periodically to ensure that contaminant levels are below those that would be expected to impact the scientific integrity of the study:
• Water samples are analyzed for total bacterial counts, and the presence of coliforms, lead, and other contaminants.
• Samples from freshly washed cages and cage racks are analyzed to ensure adequate sanitation by the cagewashers.

Certified animal feed is used, guaranteed by the manufacturer to meet specified nutritional requirements and not to exceed stated maximum concentrations of key contaminants, including specified heavy metals, aflatoxin, chlorinated hydrocarbons, and organophosphates. The presence of these contaminants below the maximum concentration stated by the manufacturer would not be expected to impact the integrity of the study.

The animal health and environmental monitoring program is administered by the attending laboratory animal veterinarian. Evaluation of these data did not indicate any conditions that affected the validity of the study.

Upon arrival at Haskell Laboratory, the rats were quarantined for 7 days of the 13-day pretest period and were released from quarantine on test day -6.

The rats were observed daily for any apparent signs of disease or injury and weighed twice during the quarantine period. Rats were released from quarantine by the laboratory animal veterinarian on the basis of acceptable body weight gains and clinical observations.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: NanoPure water
Details on exposure:
quantity 4 mL/kg, based on the most recently recorded body weight, administered by oral intubation
Details on mating procedure:
Breeding
1. Start of Cohabitation
Animals were cohoused after approximately 10 weeks of exposure to the test substance Methylammonium chloride. The day animals were first cohoused was designated as day 1 of cohabitation.
2. Duration of Cohabitation Period
Animals were cohoused until evidence of copulation was observed or until 2 weeks had elapsed. The cohabitation period ended in the morning of day 15 of cohabitation.
3. Evidence of Copulation
Once daily, each female was examined for the presence of an intravaginal copulation plug or sperm in the vaginal lavage sample, either of which was considered evidence of copulation. The presence of an intravaginal plug and/or sperm was recorded. The day evidence of copulation was observed was designated as day 0 of gestation.
4. Cohousing
Each female was continually housed on a 1:1 basis with a randomly selected, nonsibling male of the same dose level, in the male's cage. On the day copulation was confirmed, the female was transferred back to individual cage housing.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test substance Methylammonium chloride, a white, crystalline powder, was supplied by Alfa Aesar, Ward Hill, Massachusetts, and was assigned Haskell Number 26809.

The bulk test substance was provided by Alfa Aesar, a Johnson Matthey Company, 30 Bond Street, Ward Hill, MA 01835. A Certificate of Analyses (COA) was provided by the supplier and indicated that the purity of the test substance was 99.46%.

To confirm that the test substance Methylammonium chloride was indeed stable over the period of use for the current study, a sample was analyzed near the end of the study by Case Consulting Laboratories, Inc.; the results of these analyses confirmed the purity of the test substance to be 99.2%.

Formulations of the test substance Methylammonium chloride in the vehicle (NanoPure® water) were prepared daily until stability data were available that supported weekly preparation of the formulations. After these stability data were generated, the dosing formulations were prepared weekly and stored refrigerated up to seven days. The pH values for all dose preparations were recorded in the study records. Dose formulations with pH values that were initially > 6.0 or < 4.0 were adjusted to a pH value between 4.0 and 6.0 prior to dosing.

Two sets of samples were collected from the dosing formulations prepared near the beginning of the study. Analyses addressed concentration, uniformity of mixing, and stability.
• The first sets of samples were analyzed immediately to verify mixing uniformity, concentration, and 5-hour room temperature stability of the test substance Methylammonium chloride in the formulation.
• The second sets of samples were analyzed to verify stability after 7 days of refrigeration and stability after 7 days of refrigeration followed by 5 hours at room temperature.
Duration of treatment / exposure:
• Females showing evidence of copulation were dosed throughout gestation.
• Pregnant females in the process of delivery or showing signs of delivery were not dosed.
• Females were dosed after delivering litters, until day 3 postpartum.
• Females that did not deliver a litter continued to be dosed until the day before sacrifice.
Frequency of treatment:
once daily
Details on study schedule:
Following a 71-day premating period, P1 males and females were cohoused for up to 2 weeks within their respective treatment groups to produce F1 litters. Dams were allowed to deliver and rear their offspring until postpartum day 4.

After being transferred into polycarbonate pans (on day 20 of gestation for mated females, or at the end of the cohabitation period for females without evidence of copulation), female rats were observed at least twice daily for signs of delivery and offspring.

The day when delivery was complete was designated day 0 postpartum. At each examination period (days 0 and 4 postpartum), offspring were individually handled and examined for abnormal behavior and appearance; any dead or abnormal pups were recorded.
1. Day 0 Postpartum: Live and dead pups in each litter were counted by sex as soon as possible after delivery was completed. Live pups in each litter were individually weighed.
2. Day 4 Postpartum: Live pups in each litter were counted by sex and individually weighed and a gross external examination was performed. All pups were euthanized by decapitation.

Rats of each sex were selected for use on study based on adequate body weight gain and freedom from any clinical signs of disease or injury. They were distributed by computerized, stratified randomization into study groups as designated in the Study Design so that there were no statistically significant differences among group body weight means within a sex (p > 0.05). The weight variation at study start (the beginning of test substance administration) did not exceed ± 20% of the mean for each sex. During pretest, on test day –1, detailed clinical observations were performed. Rats not assigned to the study were sacrificed by carbon dioxide euthanasia and discarded without evaluation.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
nominal in water
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
nominal in water
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
nominal in water
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
nominal in water

Examinations

Parental animals: Observations and examinations:
Cage-site examinations to detect moribund or dead animals and abnormal behavior and/or appearance were performed on all animals at least once daily during the quarantine/pretest period and twice daily during the dosing period.
Careful clinical observations were recorded once daily at approximately the same time each morning during dosing; detailed clinical observations were recorded once during the pretest period and weekly thereafter.
Body weights and food consumption were recorded weekly for P1 males and females (premating), on days 0, 7, 14, and 21 of gestation and on days 0 and 4 of lactation; food consumption was not measured during cohabitation or thereafter for males, or for females with no evidence of copulation.

Once during pretest (baseline), and weekly thereafter at approximately the same time of day (+ 2 hours), animals were individually handled and examined for abnormal behavior and appearance in a standardized arena. Observations included (but were not limited to) evaluation of fur, skin, eyes, mucous membranes, occurrence of secretions and excretions, autonomic nervous system activity (lacrimation, piloerection, and unusual respiratory pattern), changes in gait, posture, response to handling, presence of clonic, tonic, stereotypical, or bizarre behavior.

An abbreviated neurobehavioral evaluation consisting of a functional observational battery and motor activity was conducted in P1 rats once during pretest and again near the end of the premating period. Clinical pathology parameters were measured in P1 rats near the end of the premating period (haematology, clinical chemistry, urinalysis) and at terminal sacrifice (coagulation).
Litter observations:
F1 litter examinations (pup viability, individual pup weights, clinical observations) were performed at birth and on lactation day 4.
Postmortem examinations (parental animals):
All P1 rats were given a gross pathological examination at terminal sacrifice (euthanized by carbon dioxide anesthesia and exsanguisation on days 118-119 (males) or 98 - 112 (females)). Selected tissues and gross lesions were weighed and/or retained for microscopic examination. Uterine implantation sites and ovarian corpora lutea were counted in P1 females. Final body weights and organ weights were recorded. A histological examination of all tissues saved was conducted for all animals in the control and 1000 mg/kg/day group. Examination of tissues from the remaining groups was limited to relevant gross lesions and those tissues that demonstrated treatment-related histological effects in the 1000 mg/kg/day group.

A clinical pathology evaluation was conducted on all animals once before the cohabitation period on test days 69/70 (haematology, clinical chemistry, and urinalysis) and again on test days 118-119 and 98-112 (coagulation) for males and females, respectively. The day before collection of samples for the clinical pathology evaluation for haematology, clinical chemistry, and urinalysis, the animals were placed in metabolism cages. These animals were fasted after 3 p.m. for at least 15 hours and urine was collected from each animal. Blood samples for haematology and clinical chemistry measurements were collected from the orbital sinus of each animal while the animal was under carbon dioxide anesthesia. Blood samples for coagulation parameters were collected at sacrifice from the abdominal vena cava of each animal while the animal was under carbon dioxide anesthesia. Additional blood collected from the vena cava was placed in a serum tube, processed to serum, and frozen at approximately -80°C. Serum was discarded without analysis because further tests were not required to support experimental findings. Bone marrow smears were prepared at sacrifice from all surviving animals. Bone marrow smears were stained with Wright-Giemsa stain, but analysis was not necessary to support experimental findings. All blood samples were evaluated for quality by visual examination prior to analysis. Results were maintained in the study records and reported only if the sample was analyzed. Unless otherwise indicated, any historical control clinical pathology data referenced in the text is maintained in the study records.

The following tissues were weighed from all adult rats: liver, kidneys, adrenal glands, thymus, brain, spleen, heart, testes, and epididymides. Organ weight ratios (% final body weight, % brain weight) and group mean values were calculated.
All tissues were fixed in 10% neutral buffered formalin except the testes and epididymides, which were fixed in modified Davidson’s solution. Processed tissues were embedded in paraffin, sectioned approximately 5-6 microns thick, stained with hematoxylin and eosin (H&E), and examined microscopically by a veterinary pathologist.
The reproductive organs from all (12/sex/dose) control (0 mg/kg/day) and high-dose (1000 mg/kg/day) rats were processed to slides and evaluated microscopically. The remaining collected tissues were processed to slides and evaluated microscopically from randomly selected individual (5/sex/dose) control and high-dose rats. Gross observations (recorded at necropsy) were examined microscopically for all animals.
Tissue identified as having test substance-related changes, in a given sex, based on the microscopic examination of selected control and high-dose rats (5/sex/dose), were processed and examined microscopically for all rats at each dose level (12/sex/dose). In this study, the trachea and stomach were identified as target organs in both sexes. These two tissues, from all adult rats, were processed to slides and examined microscopically. In addition, the lungs from all rats and the hearts from all male rats were processed to slides and examined in order to confirm the absence of any test substance-related effects.
Postmortem examinations (offspring):
F1 pup pathology data was limited to the gross examination of individuals that did not survive until day 4 of lactation. No organ weight data was collected and no microscopic evaluation was conducted.
Statistics:
Mating index, Fertility index, Gestation index, Impantation Efficiency, Pups born alive, 0-4 day viability.

levene's test for homogeneity and Shapiro-Wilk test for normality
one-way analysis of variance and Dunnett's test
Kruskal-Wallis test and Dunnett's test
Cochran-Armitage test for trend
Analysis of covariance and Dunnett-Hsu
non-parametric analysis covariance
repeated measures analysis of variance followed by linear contrasts
sequential application of the Jonckheere-terpstra trend test
Reproductive indices:
Mating index, Fertility index, Gestation index, Impantation Efficiency
Offspring viability indices:
Pups born alive, 0-4 day viability

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No specific test substance-related deaths in males or females at any level tested, no test substance-related clinical observations at any level tested
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related, adverse effects at 1000 mg/kg/day: significant reductions in body weight in P1 animals. No test substance-related effects on body weight parameters at 500 mg/kg/day or lower.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Test substance-related, adverse effects at 1000 mg/kg/day: significant reductions in body weight in P1 animals. No test substance-related effects on body weight parameters at 500 mg/kg/day or lower.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Liver and kidney weight parameters were increased in both sexes. Liver weights were increased in males at ≥ 250 mg/kg/day and in females at ≥ 500 mg/kg/day. Kidney weights were increased in both sexes only at the highest dose (1000 mg/kg/day).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related, adverse effects at 1000 mg/kg/day: significantly reduced corpora lutea counts and subsequently lower implantation site counts and litter sizes.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related microscopic findings were observed in the trachea (squamous metaplasia) and stomach (mucoid metaplasia) of males and females given 1000 mg/kg/day of the test substance.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Test substance intake: Methylammonium chloride was administered by gavage in water

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related & statistically significant reduction in mean corpora lutea count at 1000 mg/kg/day, results in a reduction in the mean number of implantation sites per litter and a significant reduction in the mean litter size at this dose level
Reproductive performance:
no effects observed

Effect levels (P0)

Key result
Dose descriptor:
NOEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

There was a test substance-related and statistically significant reduction in mean corpora lutea count at 1000 mg/kg/day that resulted in a reduction in the mean number of implantation sites per litter and a subsequent significant reduction in the mean litter size at this dose level. There were no other test substance-related effects on reproductive outcome at this level nor were there any effects on reproductive outcome at 500 mg/kg/day or lower.
There was a significant reduction in mean litter size at 1000 mg/kg/day that was associated with a test substance-related reduction in the mean numbers of corpora lutea observed at this level. Otherwise, there were no additional test substance-related effects in offspring at 1000 mg/kg/day and no evidence of any test substance-related effects on offspring at 500 mg/kg/day or lower.

There were no test substance-related clinical observations in the pups at any dose level tested.

There were no test substance-related effects on mean pup weight either at birth or on day 4 of lactation at any dose level tested.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: test substance-related reduction in litter size observed at 1000 mg/kg bw/day.

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
Test substance-related effects were observed at 1000 mg/kg/day and were limited to reductions in parental body weights and food consumption parameters and a significant reduction in the mean corpora lutea and associated reduction in mean implantation sites and mean litter size. There was no evidence of systemic or reproductive toxicity at 500 mg/kg/day or lower.
Executive summary:

Test substance-related effects were observed at 1000 mg/kg/day and were limited to reductions in parental body weights and food consumption parameters and a significant reduction in the mean corpora lutea and associated reduction in mean implantation sites and mean litter size. There was no evidence of systemic or reproductive toxicity at 500 mg/kg/day or lower. Under the conditions of the study, there were no test substance-related effects on any neurobehavioral parameter evaluated in either males or females at any dose level tested. Daily gavage administration of up to 1000 mg/kg/day of the test substance Methylammonium chloride to male and female rats resulted in no adverse changes in clinical pathology parameters.

Liver: Since there was no gross or microscopic finding that correlated with the increased liver weights, and there was no increase in serum liver enzymes (see Clinical Chemistry), the weight effect was considered pharmacological and not adverse. Exposure to xenobiotics commonly induces hepatic metabolic enzymes in laboratory animals. While microscopic hypertrophy of hepatocytes may occur in hepatic enzyme induction, small increases in liver weight parameters are often not associated with a morphological change.

The increased kidney weight parameters in both sexes at 1000 mg/kg/day did not correlate with any gross or microscopic renal pathology. As in liver hepatocytes, xenobiotics may induce P450 microsomal enzymes in the renal proximal tubular epithelium. Increased kidney weights often precede morphological changes (i.e., hypertrophy) in non-adverse enzyme induction.

The metaplasia consisted of the partial replacement of the normal ciliated pseudostratified columnar epithelium on the ventral aspect of the tracheal mucosa with round and slightly flattened epithelial cells resembling mucous membrane. The change was not associated with inflammation of either the trachea or the lung. The minimal to mild squamous metaplasia was probably the result of transient topical exposure of the tracheal mucosa to the test substance Methylammonium chloride following gavage administration. It was interpreted to be a non-adverse adaptive response, most likely reversible, that was due to imperfect dosing or transient esophageal reflux.

The metaplasia consisted of a partial replacement of the normal parietal cells in the gastric glands with mucous epithelial cells. The change was evident in the fundic region of the glandular mucosa adjacent to the esophageal inlet. The change was not associated with any inflammation and was considered to be non-adverse. The minimal mucoid metaplasia was probably the result of the daily gavage administration of a slightly irritating solution to the glandular mucosa adjacent to the esophageal inlet. The degree of focal metaplasia appeared to be biologically insignificant. As with the focal tracheal metaplasia, the focal gastric metaplasia was interpreted to be a non-adverse, most likely reversible, adaptive response.

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Daily gavage administration of 0, 250, 500, and 1000 mg/kg/day of the test substance Methylammonium chloride, to male and female rats for 98 to 119 consecutive days, resulted in increased liver and kidney weights, squamous metaplasia of the tracheal mucosa, and mucoid metaplasia of the glandular gastric mucosa. The increased liver weights in males (≥ 250 mg/kg/day) and females (≥ 500 mg/kg/day) and kidney weights in both sexes (1000 mg/kg/day) were consistent with pharmacological enzyme induction and were considered to be non-adverse. Minimal to mild squamous metaplasia of the tracheal mucosa and minimal focal mucoid metaplasia of the gastric glandular mucosa of several high-dose rats of both sexes were interpreted to be the result of topical exposure of the test substance Methylammonium chloride. The metaplastic changes in both tissues were interpreted to be adaptive and probably reversible. Therefore, both the tracheal and gastric metaplasia were considered to be non-adverse. There were no test substance-related effects on cause of death, gross pathology, or reproductive failure in adult rats. Gross examination of nursing pups that died before day 4 of lactation did not reveal any test substance-related findings. Under the conditions of this study, the no-observed-effect level (NOEL) for pathology for male and female rats was the highest dose tested, 1000 mg/kg/day.

Under the conditions of this study, the no-observed-effect level (NOEL) for systemic and reproductive toxicity was 500 mg/kg/day based on reductions in parental body weights and food consumption and effects on reproductive outcome (reduced corpora lutea and subsequent reductions in implantations and litter size).