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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2012-12-13 to 2013-07-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline conforming GLP study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
m-phenylenebis(methylamine)
EC Number:
216-032-5
EC Name:
m-phenylenebis(methylamine)
Cas Number:
1477-55-0
Molecular formula:
C8H12N2
IUPAC Name:
1-[3-(aminomethyl)phenyl]methanamine
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): 1,3-Benzenedimethanamine
- Physical state: clear, colorless liquid
- Storage condition of test material: at room temperature, protected from light, under nitrogen

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: 93 days old
- Weight at study initiation: 228 - 295 g
- Fasting period before study: no
- Housing: until pairing individually in clean, stainless steel wire-mesh cages suspended above cage-board
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.7 - 22.4
- Humidity (%): 38.8 - 45.8
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Dosing formulations were prepared at the test substance concentrations indicated below:

Group 1: vehicle control 0 mg/kg/day dose level, 0 mg/mL test substance concentration, pH 6.82
Group 2: 1,3-benzenedimethanamine 30 mg/kg/day dose level, 3 mg/mL test substance concentration, pH 11.19
Group 3: 1,3-benzenedimethanamine 100 mg/kg/day dose level, 10 mg/mL test substance concentration, PH 11.31
Group 4 1,3-benzenedimethanamine 300 mg/kg/day dose level, 30 mg/mL test substance concentration, pH 11.57

The appropriate amounts of vehicle and test substance were transferred to sterile septum vials. The containers were purged with nitrogen, capped with a septum, and inverted to ensure mixing. The formulations were maintained at room temperature, protected from light, until use.

VEHICLE
- Concentration in vehicle: 0, 3, 10, 30 test substance mg/mL
- Amount of vehicle (if gavage): 10 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses to demonstrate the stability (following at least 10 days of room temperature storage) of the test substance formulations were conducted. Samples for concentration analysis were collected from the dosing formulations (including the control group) prepared during the first and last week of the study. One set of samples from each collection was subjected to the appropriate analyses. The remaining set of samples was stored at room temperature as back-up. All analyses were conducted using a validated gas chromatography method with flame ionization detection.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: no data
- Length of cohabitation: no data
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
gestation days 6-19
Frequency of treatment:
once daily
Duration of test:
from 2012-12-18 to 04-04-2013
No. of animals per sex per dose:
25 bred female rats
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosage levels were selected based on the results of a previous study in which 1,3-benzenedimethanamine was administered orally to female rats at dosage levels of 0, 50, 150, 450, and 650 mg/kg/day from gestation days 6 through 19. One female each in the 450 and 650 mg/kg/day groups was found dead during the treatment period. All other animals survived until the scheduled euthanasia on gestation day 20. Test substance-related reductions in mean body weight gains were noted throughout the treatment period for both the 450 and 650 mg/kg/day groups resulting in statistically significant lower mean body weights for these groups compared to the control group beginning on gestation day 15 and generally persisting for the remainder of the study. Reductions in mean food consumption were also noted for the 450 and 650 mg/kg/day groups when compared to the control group; the changes were generally statistically significant throughout the treatment period. In addition, statistically significant reduced mean fetal weights were noted in the 450 and 650 mg/kg/day groups when compared to the control group. Based on the results of the previous study, a high dose of 300 mg/kg/day was chosen for the current study and was expected to have effects on maternal body weight gain without causing mortality.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily. Animals were onserved for signs of toxicity approximately 1 hour following dose administration.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily from gestation days 0 through 20.

BODY WEIGHT: Yes
- Time schedule for examinations: on gestation days 0 6-20 (daily)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
- Organs examined: contents of thoracic, abdominal and pelvic cavities.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter
Statistics:
All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group. Each mean was presented with the standard deviation (SD), standard error (SE), and the number of animals (N) used to calculate the mean. Data obtained from nongravid animals were excluded from statistical analyses. Where applicable, the litter was used as the experimental unit.
Maternal body weights (absolute and net), body weight changes (absolute and net), and food consumption, gravid uterine weights, numbers of corpora lutea, implantation sites, and viable fetuses, and fetal body weights (separately by sex and combined) [were subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-treated groups to the control group. Mean litter proportions (percent per litter) of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and fetal sex distribution), total fetal malformations and developmental variations (external, visceral, skeletal, and combined) and each particular external, visceral, and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test (Kruskal and Wallis, 1952) to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test (Dunn, 1964) was used to compare the test substance-treated groups to the control group.
Indices:
No data
Historical control data:
yes

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects: Moribundity, mean body weight losses, and lower mean body weight gains with corresponding reduced mean food consumption at 300 mg/kg bw/day.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: intrauterine growth and survival or embryo/fetal development

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the results of the study, a dosage level of 100 mg/kg bw/day is considered to be no-observed-adverse-effect level (NOAEL) for maternal toxicity. There were no test substance-related effects on intrauterine growth and survival of embryo/fetal development at any dosage level. Therefore, a dosage level of 300 mg/kg bw/day is considered to be the NOAEL for embryo/fetal development when 1,3-benzenedimethanamine was administered orally by gavage to bred Crl:CD(SD) rats.
Executive summary:

The objective of the study was to determine the potential of the test substance to induce developmental toxicity after maternal exposure from implantation to 1 day prior to expected parturition, to characterize maternal toxicity at the exposure levels tested, and to determine a no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity according to the OECD TG 414 and EPA OPPTS Guideline 870.3700. The test substance, 1,3-benzenedimethanamine, in the vehicle (sterile water for injection, USP) was administered orally by gavage to 3 groups of 25 bred female Crl:CD(SD) rats once daily from gestation days 6 through 19. Dosage levels were 30, 100, and 300 mg/kg bw/day administered at a dosage volume of 10 mL/kg. A concurrent control group composed of 25 bred females received the vehicle on a comparable regimen. The females were approximately 14 weeks of age at the initiation of dose administration. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. On gestation day 20, a laparohysterectomy was performed on each surviving female. The uteri, placentae, and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

Three females in the 300 mg/kg bw/day group were euthanized in extremis on gestation day 11, 12, or 15 following severe body weight losses and decreased food consumption during the 2-5 days prior to euthanasia. Clinical findings noted for these females included rales, labored respiration, gasping, pale body, decreased defecation, small feces, soft stool, and/or red, clear, brown, and/or yellow material on various body surfaces at the daily examinations and/or approximately 1 hour following dose administration beginning up to 4 days prior to euthanasia. At necropsy, these females were noted with internal findings of distended stomach and/or intestine, dark red areas on the stomach, and dark red discoloration of the lungs. All other females survived to the scheduled necropsy where no remarkable macroscopic findings were observed at any dosage level. Clinical findings of rales, gasping, labored respiration, red and/or clear material on the forelimbs, around the nose, and/or mouth, and excreta-related findings (decreased defecation, soft stool, and small feces) were noted for surviving females in the 300 mg/kg/day group primarily throughout the treatment period at the daily examinations and at approximately 1 hour following administration and were considered test substance-related and adverse. There were no test substance-related clinical findings observed at dosage levels of 30 and 100 mg/kg bw/day. A mean body weight loss or lower mean body weight gains were noted for the 300 mg/kg bw/day group throughout the treatment period, corresponded to decrements in mean food consumption, and were considered test substance-related and adverse. As a result, a lower mean body weight gain was noted for this group when the overall treatment period (gestation days 6-20) was evaluated. These effects resulted in mean body weight for the 300 mg/kg/day group that was up to 7.2 % lower than the control group throughout the study, with correspondingly lower mean net body weight and net body weight gain in this group. Mean gravid uterine weight in the 300 mg/kg bw/day group was unaffected by test substance administration. Mean body weights, body weight gains, net body weights, net body weight gains, and gravid uterine weights, and food consumption in the 30 and 100 mg/kg bw/day groups were unaffected by test substance administration. Intrauterine growth and survival and fetal developmental morphology at 30, 100, and 300 mg/kg kg/day were unaffected by test substance administration.

Moribundity, mean body weight losses, and lower mean body weight gains with corresponding reduced mean food consumption were noted at a dosage level of 300 mg/kg bw/day. Based on these results, a dosage level of 100 mg/kg bw/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity.

There were no test substance-related effects on intrauterine growth and survival or embryo/fetal development at any dosage level. Therefore, a dosage level of 300 mg/kg bw/day was considered to be the NOAEL for and embryo/fetal development when 1,3-benzenedimethanamine was administered orally by gavage to bred Crl:CD(SD) rats.