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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:
January 11,2015 - February 10,2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Follows OECD and GLP guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
- Name of test material: 1,3-Benzenedimethanamine polymer with 4(1,1-dimethylethyl)phenol and formaldehyde

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (CR) (Raleigh, North Carolina)
- Age and at study initiation: Sexually mature adult weighing approximately 200-250 g
- Fasting period before study:
- Housing: After assignment, animals were housed one per cage in stainless steel cages. Cages had solid floors with corncob bedding and shredded Aspen for enrichment. Cages contained a feed crock and a pressure activated lixit valve-type watering system.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: During the acclimation period each animal was evaluated by a laboratory veterinarian to determine the general health status and acceptability for study purposes. The Toxicology and Environmental Research and Consulting Laboratory is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). The animals were housed one per cage in stainless steel cages in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle) and acclimated to the laboratory for at least four days prior to the start of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C with a range of 20°C-26°C
- Humidity (%): 50% with a range of 30-70%
- Air changes (per hr): 10-15 times/hour (average)
- Photoperiod (hrs dark / hrs light): 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)

IN-LIFE DATES: From: January 11,2015 To: February 10,2015

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: All dosing solutions were prepared by mixing the test material in PG at concentrations of 0, 1.7, 5.0, or 16.7 mg/mL and administered at a dose volume of 6 mL/kg body weight to achieve the targeted dose levels. Dose solutions were not corrected for purity. Dose volumes were adjusted daily based on individual body weights. The control rats were dosed with PG at 6 mL/kg body weight. Dose solutions were prepared periodically throughout the study.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The solubility of Mannich base PTBP-MXDA in PG was previously determined in PG up to 250 mg/mL
- Amount of vehicle (if gavage): All dosing solutions were prepared by mixing the test material in PG at concentrations of 0, 1.7, 5.0, or 16.7 mg/mL and administered at a dose volume of 6 mL/kg body weight to achieve the targeted dose levels.
- Lot/batch no. (if required): Lot #’s MKBS5987V and MKBT3258V
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of all dosing solutions from the first mix revealed acceptable mean concentrations of Mannich base PTBP-MXDA ranging from 90.6% to 104.6% of targeted concentrations. Analysis of aliquots for the low- and high-dose solutions indicated that the test material was homogeneously distributed based on relative standard deviations of ≤ 4.5%.
Details on mating procedure:
Sexually mature, adult virgin females were naturally mated with males of the same strain (one male: one female) at the supplier’s facility. Females were checked for in situ copulation plugs the following morning, and those found with such a plug were removed from the males’ cages. The day on which a vaginal plug was detected was considered GD 0. GD 0 body weights were provided by the supplier and maintained in the study record. Rats arrived in our laboratory on GD 1 or 2.
Duration of treatment / exposure:
GD 6-20
Frequency of treatment:
Daily
Duration of test:
GD 0-21
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
10 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
30 mg/kg/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels for this study were selected on the basis of the developmental toxicity probe study discussed previously. The data driving selection of the high dose were decreases in body weight gain measurements and increased morbidity. All animals in the 200 mg/kg/day group were removed from the probe study because of excessive toxicity (excessive body weight loss/decreased body weight gain and decreased feed consumption) and one animal in the 150 mg/kg/day group was removed from the study on GD 17 in a moribund condition, manifested as labored breathing with evidence of mouth breathing prior to euthanasia (gastrointestinal tract gaseous distention was observed at necropsy). At a dose level of 150 mg/kg/day in the probe study, a 79% decrease in body weight gain was observed during the GD 6-9 interval, indicating excessive maternal toxicity. At a dose level of 75 mg/kg/day in the probe study, a body weight gain decrease of 26% during the GD 9-12 interval and an overall decrease of 13% during the treatment period (GD 6-21) were observed. Therefore, the high dose of 100 mg/kg/day was expected to induce signs of maternal toxicity, including decreased body weight gain, but not death or severe suffering. the lower dose levels were selected to provide dose-response doata for any toxicity that may have been observed among the high- dose group animals.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: 0, 6, 9, 12, 15, 18, 21 (terminal)

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #21.
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
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: Yes
Statistics:
Maternal body weights, maternal body weight gains, organ weights, gravid uterine weights, fetal body weights and feed consumption were evaluated by Bartlett’s test (alpha = 0.01; Winer, 1971) for equality of variances. Based on the outcome of Bartlett's test, a parametric (Steel and Torrie, 1960) or non-parametric (Hollander and Wolfe, 1973) analysis of variance (ANOVA) was performed. If the ANOVA was significant at alpha = 0.05, analysis by Dunnett's test (alpha = 0.05; Winer, 1971) or the Wilcoxon Rank-Sum test (alpha = 0.05; Hollander and Wolfe, 1973) with Bonferroni's correction (Miller, 1966) was performed, respectively. Feed consumption values were excluded from analysis if the feed was spilled or scratched. Frequency of pre- and post-implantation loss (calculations shown below), and fetal alterations were analyzed using a censored Wilcoxon test (Haseman and Hoel, 1974) with Bonferroni’s correction applied when the incidence was greater than 5%. The number of corpora lutea, implantations and litter size was evaluated using a nonparametric ANOVA (alpha = 0.05) followed by the Wilcoxon Rank-Sum test (alpha = 0.05) with Bonferroni's correction. Pregnancy rates were analyzed using the Fisher exact probability test (alpha = 0.05; Siegel, 1956) with Bonferroni’s correction. Fetal sex ratios were analyzed using a binomial distribution test. Females lacking visible implantations at the scheduled necropsy were excluded from the appropriate analyses. Statistical outliers were identified, using a sequential method (alpha = 0.02; Grubbs, 1969) and, if excluded, were excluded for sound scientific reasons. Both Dunnett’s test and Bonferroni’s correction were applied for multiple comparisons to the control group to keep the experiment-wise alpha at 0.05. Both were reported at the experiment-wise alpha level.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Organ Weight: Relative liver weights of animals in the 100 mg/kg/day group showed a treatment-related 5.4% statistically-identified increase. This increase was consistent with the 14% relative liver weight increases seen at 150 mg/kg/day on the probe study (Johnson et al., 2015) discussed in the Previous Toxicity section of the report. There were no treatment-related differences in kidney weights for any treated group when compared to controls.
Gross Pathology: There were no treatment-related gross pathologic observations noted on any animal. Incidental observations bearing no relationship to treatment included a mammary gland mass-nodule in one animal in the 30 mg/kg/day group and an ulcer of the glandular wall of the stomach in one animal in the 10 mg/kg/day group.
Reproductive Parameters: There were no treatment-related effects on pregnancy rates, resorption rates, litter size, numbers of corpora lutea or implantations, percent preimplantation loss, percent postimplantation loss, fetal sex ratios, fetal body weights, or gravid uterine weights at any dose level

Effect levels (maternal animals)

Dose descriptor:
NOEL
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
Fetal Examination Summary:
External Examination: There were no treatment-related external alterations in any dose group. Incidental findings bearing no relationship to treatment included one fetus in the 30 mg/kg/day group (animal 5993) with multiple malformations of the face (anophthalmia, agnathia, micrognathia mandible, proboscis on the face, and astomia) and one fetus in the 10 mg/kg/day group (animal 5976) with hindlimb rotation.

Visceral Examination: There were no treatment-related visceral alterations in any dose group. Incidental findings bearing no relationship to treatment included supernumerary hepatic lobule in one fetus in each of the 10 and 30 mg/kg/day groups (animals 5970 and 6009, respectively) and three fetuses in the 100 mg/kg/day group (animals 6018 and 6033). This variation was considered unrelated to treatment as the incidence was low and similar to recent historical control values.

Craniofacial Examination: There were no treatment-related craniofacial alterations in any dose group. Incidental findings bearing no relationship to treatment included dilated cerebral ventricle in one fetus in the 10 mg/kg/day group.

Skeletal Examination: There were no treatment-related skeletal alterations in any dose group. Incidental findings bearing no relationship to treatment included delayed ossification (DO) of the parietal, DO interparietal, DO sternebrae, DO thoracic centra, irregular ossification of the sternebrae, fused sternebrae, class I wavy ribs, class II wavy ribs, and calloused ribs. In addition, there was one fetus in the 30 mg/kg/day group (animal 5993) with multiple malformations (sternoschisis, 14 ribs bilaterally (extra rib bilaterally), fused ribs bilaterally, multiple hemivertebrae, misshapen nasal bones, and misshapen head with no mandible, maxilla, or zygomatic). These observations were consistent with external malformations noted on this fetus.

Effect levels (fetuses)

Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: embryotoxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Selected Body Weight Gains (g)

 Dose level (mg/kg/day)  GD 6-9  GD 9-12
 0  12.0  19.4
 10  13.8  21.7
 30  13.7  21.9
 100  9.8  19.4

Bold text indicates a treatment related effect.

Relative Liver Weights

Dose Level (mg/kg/day)   Final Body Weight (g)  Relative Liver Wt. (g/100)
 0  392.6  3.420±0.249
 10  401.1  3.316± 0.279
 30  407.7  3.361±0.228
 100  389.3  3.604*±0.258

*Statistically different from control mean by Dunnett's Test, Alpha=0.05

Bold text indicates a treatment-related effect.

External Malformations:

 Dose Level  0 mg/kg/day  10 mg/kg/day  30 mg/kg/day  100 mg/kg/day
 Multiple malformations  F 0/299L0/24  F 0/302L 0/24  F1/307L 1/23  F 0/298L 0/24
 Rotation hindlimb  F 0/299L 0/24  F 1/302L 1/24  F 0/307 L 0/23  F 0/298L 0/24

F=Fetus L=Litter

Incidence of Visceral Alterations:

 Dose Level  0 mg/kg/day  10 mg/kg/day  30 mg/kg/day  100 mg/kg/day
 Supernumerary hepatic lobule  F 0/148 L 0/24  F 1/149 L 1/24  F 1/152 L 1/23  F 3/149 L 2/24

F= Fetus L=Litter

Craniofacial Malformations:

 Dose Level  0 mg/kg/day  10 mg/kg/day  30 mg/kg/day  100 mg/kg/day
 Dilated Cerebral ventricles  F 0/148 L 0/24  F 1/149 L 1/24  F 0/152 L 0/23  F 0/149L 0/24

F=Fetus L=Litter

Skeletal Malformations

 Dose Level  0 mg/kg/day  10 mg/kg/day  30 mg/kg/day  100 mg/kg/day
 Multiple Malformations  F 0/151 L 0/24  F 0/153 L 0/24  F 1/155 L 1/23  F 0/149 L 0/24

F= Fetus L=Litter

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the no-observed-effect level (NOEL) for maternal toxicity was 30 mg/kg/day, and the embryo/fetal NOEL was 100 mg/kg/day.
Executive summary:

The purpose of this study was to evaluate the maternal and developmental toxicity of the reaction mass of 4-tert-butylphenol and 1,3-phenylenedimethanamine and 2-({[3- (aminomethyl)benzyl]amino}methyl)-4-tert-butylphenol (hereafter referred to as Mannich Base PTBP-MXDA) in Crl:CD(SD) rats following repeated gavage administration.

Groups of 24 time-mated female Crl:CD(SD) rats were administered Mannich Base PTBP-MXDA by gavage at dose levels of 0, 10, 30, or 100 mg/kg/day on gestation day (GD) 6-20. In-life parameters evaluated for all groups included clinical observations, body weight, body weight gain, and feed consumption. On GD 21, all surviving rats were euthanized and examined for gross pathologic alterations. Liver, kidneys, and gravid uterine weights were recorded, along with the number of corpora lutea, uterine implantations, resorptions, and live/dead fetuses. All fetuses were weighed, sexed, and examined for external alterations. Approximately one half of the fetuses were examined for visceral and craniofacial alterations while skeletal examinations were conducted on the remaining fetuses.

Administration of Mannich base PTBP-MXDA via gavage at 100 mg/kg/day produced treatment-related maternal toxicity evidenced by decreased body weight gain from GD 6-9 and increased relative liver weights. There were no treatment-related effects on clinical observations, body weight, or feed consumption, and no effect on gross pathologic alterations, kidney weights, gravid uterine weights, number of corpora lutea, uterine implantations, or resorptions. There was no indication of embryo/fetal toxicity or teratogenicity at any dose level tested.

Therefore, under the conditions of this study, the no-observed-effect level (NOEL) for maternal toxicity was 30 mg/kg/day, and the embryo/fetal NOEL was 100 mg/kg/day.