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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 May 11, 2004 to June 04, 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study well documented, followed method comparable to guideline, GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Remarks:
according to U.S. FDA, OECD and JMAFF Principles of GLP
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: N,N-bis (2-hydroxyethyl)-p-phenylenediamine sulfate
- TSIN: GTS03849
- Substance type: Pure active substance
- Physical state: White powder
- Stability of test material: Stable for the treatment period, demonstrated by analysis purity of active ingredient in the test material for second and third time during the treatment period, perfumed using the HPLC method.
- Storage Conditions: At room temperature protected from light
- Solubility: Soluble in water, polar organic solvents (methanol. soluble sodium hydroxide solution & hydrochloric acid solution).

Test animals

Species:
rat
Strain:
other: Crl:CD (SD)IGS BR VAF/Plus
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina, USA
- Age at study initiation: 68 d
- Weight at study initiation: 219 - 247 g (weight of females at study assignment)
- Fasting period before study: No
- Housing: individually in stainless steel, wire-bottomed cages, except during the cohabitation period. The study rooms were maintained under conditions of positive airflow relative other places.
- All cage sizes and housing conditions were in compliance with the "Guide for the Care and Use of Laboratory Animals". Cage pan liners were changed at least three times weekly. Cages were changed approximately every other week.
- Diet: Certified Rodent Diet # 5002 (PMI Nutrition International, St. Louis, Missouri, USA) in individual feeders; ad libitum. Diet was routinely analysed by the feed supplier. No contaminants at levels exceeding the maximum concentration limits for certified feed or deviations from expected nutritional requirements were detected by these analyses.
- Water: Local water (purified by reverse osmosis) provided from an automatic watering access system; ad libitum. The processed water was analyzed twice annually for possible chemical contamination (Lancaster Laboratories, Lancaster, Pennsylvania, USA) and monthly for possible bacterial contamination (QC Laboratories, Southampton, Pennsylvania, USA).
- Acclimation period: 6 d

ENVIRONMENTAL CONDITIONS
- Temperature: 69.0-80°F (20.6-26.7°C).
- Relative humidity: 39.0-57.2%.
- Air changes: A minimum of 10 air changes/h with 100% fresh air that had been passed through 99.97% HEPA filters.
- Photoperiod: An automatically controlled 12 h fluorescent light/12 h dark cycle/d.

IN-LIFE DATES: From: May 17, 2004 To: June 04, 2004

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: aqueous 0.2% w/v erythorbic acid (prepared using erythorbic acid (D-araboascorbic acid; isoascorbic acid), a white, crystalline powder, and reverse osmosis membrane processed deionized water (R.O. deionized water))
Details on exposure:
PREPARATION OF DOSING FORMULATIONS (SOLUTIONS): Appropriate (weighed) quantity of test substance was mixed thoroughly with vehicle using magnetic stirer until the test substance was completely dissolved.
- Rate of preparation of dosing solutions: Weekly
- Storage condition of dosing solutions: Refrigerated (4-8°C)

VEHICLE
- Justification for use and choice of vehicle: Not reported
- Concentration in vehicle: aqueous 0, 0.5, 2 and 5 mg/mL
- Amount of vehicle: 10 mL/kg bw (adjusted daily on the basis of the individual body weights recorded immediately before administration of the test substance).
- Lot/batch no. ( erythrobic acid): Spectrum Chemical MFG. CORP; Lot# SW0767
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Homogeneity and concentration (all levels) analyses were performed by HPLC method (Covance Method Procedure MA-PG61-2050) in Covance Laboratories Inc. Wisconsin 53704 USA.
- For Homogeneity analysis, quadruplicate samples were taken from the top, middle and bottom of the high dose concentration (5.0 mg/mL) from the first preparation used on this study to establish homogeneity of the high dose formulation. Two samples from each quadruplicate were shipped for analysis.
- For concentration analysis, quadruplicate samples were taken from the middle of all dose formulations on all preparations performed on the study. Two samples from each quadruplicate set were shipped for analysis. For the first preparation on study, the mean of the homogeneity sample concentration values served as the concentration value for the high dose formulation.
- The mean concentration results for all samples analyzed varied from 95.0% to 104% of the respective theoretical value. For the homogeneity analyses, the overall relative standard deviation from all sampling locations for each respective concentration was within 3%.
- Stability of the dose formulations was established by Covance study# 6114-461 and this information was provided to the Study Director. No stability determination was performed during the conduct of this study.
Details on mating procedure:
- Impregnation procedure: Cohoused
- M/F ratio per cage: 1M:1F
- Length of cohabitation: 4 d
- Verification of same strain and source of both sexes: Male and female rats were of same strain and were obtained from same source.
- Proof of pregnancy: Mating was evaluated daily during the cohabitation period and confirmed by observation of spermatozoa in a smear of the vaginal contents and/or a copulatory plug observed in situ. The Day mating was confirmed was designated as Day 0 of (presumed) gestation.
Duration of treatment / exposure:
Gestation Day 6 through Day 20
Frequency of treatment:
Daily (at approx. the same time each day)
Duration of test:
21 d
Doses / concentrationsopen allclose all
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25 mated females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosages were selected on the basis of a range-finding study (Argus Protocol 916-061P). The highest dosage level selected was anticipated to induce some overt developmental and/or maternal toxicity (clinical signs or a decrease in body weight) but not death or severe suffering. The intermediate dose level was selected to produce minimal observable toxicity. The lowest dosage level was selected to not produce any evidence of either maternal or developmental toxicity. A descending sequence of dose levels was selected with the purpose of demonstrating any dosage-related response and to establish a no-observed-adverse-effect level (NOAEL).
Available additional information on metabolism and kinetics of the test substance or related materials was also considered.
- Rationale for animal assignment: Rats were assigned to individual housing on the basis of computer-generated random units. Healthy, mated female rats were assigned to four dosage groups (1 control and 3 treatment), 25/group, by using a computer-generated (weight-ordered) randomization procedure based on body weights recorded on Gestation Day 0.

Examinations

Maternal examinations:
MORTALITY: Yes
- Time schedule: At least twice each day

DETAILED CLINICAL OBSERVATIONS: Yes, animals were examined for clinical observations, general appearance, abortions and premature deliveries.
- Time schedule: Weekly during the acclimation period and on gestation Day 0, thereafter daily before and after dosage and on the day of sacrifice.
Post dosage observations were recorded at approx. hourly intervals for the first 4 h and at the end of the normal working day on the first three days of dosage administration. On subsequent days, post dosage observations were recorded approx. 2 h (±30 min) after administration.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly during the acclimation period, on Gestation Day 0 and daily during the dosage and post dosage periods.

FOOD CONSUMPTION: Yes, feed consumption values were recorded on Gestation Day 6, 9, 12, 15, 18, and 20

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21, all surviving animals were sacrificed by CO2 asphyxiation and caesarean sectioned.
- Organs examined: Gross necropsy of the thoracic, abdominal and pelvic viscera was performed.
- Gross lesions were retained in neutral buffered 10% formalin for possible future evaluation. A table of random units was used to select one control group rat from which all tissues examined at necropsy were retained, in order to provide comparative tissues for any possible histopathological evaluations of gross lesions.
- To minimize bias, Caesarean-sectioning and subsequent fetal observations were conducted without knowledge of dosage group.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number and distribution of corpora lutea: Yes
- Number and distribution of implantations: Yes
- Number of early resorptions: Yes (early resorption was defined as one in which organogenesis was not grossly evident).
- Number of late resorptions: Yes (late resorption was defined as one in which the occurrence of organogenesis was grossly evident).
- Number of live and dead fetuses: Yes (A live fetus was defined as a term fetus that responded to stimuli. Nonresponding term fetuses were considered to be dead (there were no dead fetuses). Dead fetuses and late resorptions were differentiated by the degree of autolysis present; marked to extreme autolysis indicated that the fetus was a late resorption).
- Uteri of apparently nonpregnant rats were stained with 10% ammonium sulfide to confirm the absence of implantation sites.
- Other: Placentae were examined for size, color and shape.
Fetal examinations:
- External examinations: Yes: all per litter. Each fetus was removed from the uterus, placed in an individual container and individually identified with a tag noting the study number, litter number, uterine distribution and fixative.
- Each fetus was subsequently weighed and examined for sex and gross external alterations. Live fetuses were sacrificed by an intraperitoneal injection of EUTHASOL (Diamond Animal Health, Inc., Des Moines, Iowa, USA).
- Soft tissue examinations: Yes: half per litter. Totals of 167, 175, 129 and 157 fetuses were examined for soft tissue alterations in the four respective dosage groups following a variation of the microdissection technique of Staples.
- Skeletal examinations: Yes: half per litter. Totals of 177, 189, 137 and 170 fetuses were examined for skeletal alterations and fetal ossification site averages, in the four respective dosage groups. Fetuses were eviscerated, cleared, stained with alzarin red and examined for skeletal alterations. The fetuses were initially fixed in alcohol. Skeletal preparations were retained in glycerin, with thymol added as a preservative.
- Head examinations: Yes: half per litter. The heads of the fetuses undergoing soft tissue examinations were fixed in Bouin's solution and subsequently examined by free-hand sectioning; head sections were retained in alcohol.
Statistics:
- All data were tabulated, summarized and/or statistically analyzed using the Argus Automated Data Collection and Management System, the Vivarium Temperature and Relative Humidity Monitoring System, Microsoft Excel (part of Microsoft Office 97/2000/XP), Quattro Pro 8 and/or The SAS System (version 6.12). Averages and percentages were calculated. Litter values were used where appropriate. Statistically significant probabilities were reported as either p<=0.05 or p<=0.01.
- Clinical observations and other proportional data were analyzed for the Variance Test for Homogeneity of the Binomial Distribution. Continuous data (e.g., maternal body weights, body weight changes, feed consumption values, organ weights and litter averages for percent male fetuses, percent resorbed conceptuses, fetal body weights and fetal anomaly data) were analyzed using Bartlett's Test of Homogeneity of Variances and the Analysis of Variance, where appropriate [i.e., Bartlett's Test was not significant (P>0.001)].
- If the Analysis of Variance was significant (p<=0.05), Dunnett's Test was used to identify the statistical significance of the individual groups. If the analysis of Variance was not appropriate [i.e., Bartlett's Test was significant (p<=0.001)], the Kruskal-Wallis Test was used, when less than or equal to 75% ties were present. In cases where the Kruskal-Wallis Test was statistically significant (p<=0.05), Dunn's Method of Multiple Comparisons was used to identify the statistical significance of the individual groups. If there were greater than 75% ties, Fisher's Exact Test was used to analyze the data. Count data obtained at Caesarean-sectioning of the dams were evaluated using the procedures described above for the Kruskal-Wallis Test.
Historical control data:
- Summary of following historical control data has been provided in the report:

Summary of reproductive indices, summary of maternal necropsy observations Crl:CD(SD)IGS BR rats - Day 21 C-section, summary of fetal gross external alterations Crl:CD(SD)IGS BR rats - Day 21 C-section, summary of fetal soft tissue alterations Crl:CD(SD)IGS BR rats - Day 21 C-section, summary of fetal skeletal alterations Crl:CD(SD)IGS BR rats - Day 21 C-section, summary of fetal ossification sites skeletal averages Crl:CD(SD)IGS BR rats - Day 21 C-section.

Details are provided in the study report.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical observations related to the test substance occurred. Variations like localized alopecia in limb(s) or head, chromorhinorrhea, urine-stained abdominal fur, rales, mass on the nose, incisor(s) missing/broken and scant feces were observed. However, these findings were not considered to be treatment related as they were sporadic incidences or were common in species and strain of the test animal.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One dam in the high dose group was found dead on Day 18 of gestation. No specific cause of death was determined and was therefore not considered to be treatment related.
- All other dams survived to scheduled necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Maternal body weight gains were reduced in the mid dose group and significantly reduced (p≤0.05) in the high dose group animals during the entire dosage period when compared to vehicle control group value.
Body weight gains in the low, mid and high dose groups were 107.1%, 92.6% and 90.5% of the vehicle control group value respectively.
During the dosing period significant reductions in body weight gains occurred during gestation Day 6 to 9 for high and mid dose groups. Body weight gains in the mid and high dose groups were 76.1% and 65.7% of control value respectively.
Body weights were significantly reduced on gestation Day 8 and 11 to 21 in the mid dose group (p≤0.05 or p≤0.01) and on gestation Day 8, 18, 19 and 21 in the high dose group (p≤0.05), as compared to the vehicle control group. Gravid uterine weights were reduced in the mid and high dose groups (91.3% and 91.0%, respectively, of the control value), as compared to the vehicle control value. These reductions were not dosage dependent and reflected a slightly smaller litter size that occurred spontaneously in the mid and high dose groups.
Corrected maternal body weights (gestation Day 21 body weight minus the gravid uterine weight) and corrected body weight changes (calculated as gestation Day 6 to 21 and 0 to 21) were comparable among the groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Corresponding to reductions in body weight gain, absolute (g/day) and relative (g/kg/day) feed consumption values were significantly reduced in the mid dose group (p≤0.05 or p≤0.01) and high dose group (p≤0.05) for the dosage interval of gestation Day 6 to 9 (92.2% and 92.5%, respectively, of the vehicle control group absolute value).
Absolute feed consumption values in the low, mid, and high dose groups were 101.1%, 96.6% and 97.0%, respectively, of the vehicle control group values forthe entire dosage period (calculated as gestation Day 6 to 21).
Absolute and relative feed consumption values were unaffected in the low dose group.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
No treatment related gross lesions were observed in necropsy. Isolated occurrence of urinary tract lesions was noted in one rat; this is a common finding in this species and strain and was considered unrelated to the treatment.

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
CAESAREAN-SECTIONING AND LITTER OBSERVATIONS: Pregnancy occurred in 24 (96.0%), 24 (96.0%), 20 (80.0%) and 25 (100%) rats in the control, low, mid and high dose groups respectively. One dam in the control group delivered its litter on gestation Day 21 and was sacrificed. This dam was given 15 dosages of the vehicle, and had no adverse clinical observations.
- No treatment related effects were noted in caesarean-sectioning or litter observations. The litter averages for corpora lutea, implantations, litter sizes, live fetuses, early and late resorptions, fetal body weights, percent resorbed conceptuses and percent live male fetuses were comparable among the four dosage groups and did not significantly differ.
There were no dead fetuses and no litters with all conceptuses resorbed; all placentae appeared normal. All values were within the ranges observed historically at the testing facility.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: effects obserevd in the 20 and 50 mg/kg/day dosage groups

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
External malformations:
effects observed, non-treatment-related
Skeletal malformations:
effects observed, non-treatment-related
Visceral malformations:
effects observed, non-treatment-related
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- Excluding fetuses from the dam that was found dead in the high dose group and fetuses from the dam that delivered on gestation Day 21 in the control group, fetal evaluations were based on 344, 364, 266 and 327 live, caesarean-delivered fetuses from 23, 24, 20 and 24 litters in the control, low, mid and high dose groups, respectively.
- Summary of fetal alterations: In four respective dose groups (control, low, mid and high) fetal alteration were observed in 3 (13.0%), 8 (33.3%; significant at p<=0.05), 10 (50%; significant at p<=0.01) and 5 (20.8%) litters respectively.
- The numbers of fetuses with any alteration observed were 3 (0.9%), 11 (3.0%; significant at p≤0.01), 11 (4.1%; significant at p≤0.01) and 6 (1.8%), in control, low, mid and high dose group respectively.
- The percentages of fetuses with any alteration per litter were 0.8, 3.3, 5.4 (significant at p≤0.01) and 1.8 for the four dosage groups, respectively.
- The significant increases (p≤0.05 to p≤0.01) in the number of litters with fetuses with alterations, the number of fetuses with alterations and the percentage of fetuses with alterations per litter were not considered related to the test substance, as the increases were not dosage dependent and) the increases reflected low values in the control group.
- With the exception of wavy ribs that occurred in three (significant at p≤0.01) fetuses in two high dose group litters, there were no dosage-dependent significant differences in the litter or fetal incidences of any gross external, soft tissues or skeletal alterations. The incidence of wavy ribs was not considered related to the test substance because it was within the historical control range for this test facility and the more relevant litter incidence was not significantly increased. There was a significant (p≤0.05) decrease in the number of ossified hindlimb phalanges in the high dose group; however, this was not considered toxicologically important since there was no other adverse effect on fetal weights or fetal alterations.

- There was a significant (p≤0.05) decrease in the number of ossified hindlimb phalanges in the 50 mg/kg/day dosage group; however, this was not considered toxicologically important since there was no other adverse effect on fetal weights or fetal alterations. No other gross external, soft tissue or skeletal fetal alterations (malformations or variations) were attributable to treatment with the test substance.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: no developmental toxicity observed up to the highest dose tested

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Administration of N,N-bis (2-hydroxyethyl)-p-phenylenediamine sulfate by oral gavage to mated female Crl:CD (SD)IGS BR VAF/Plus rats during gestation Day 6-20 at dose levels of 0, 5, 20 and 50 mg /kg bw/day revealed a NOAEL of 5 mg/kg bw/day for maternal toxicity and 50 mg/kg bw/day for developmental toxicity.
Executive summary:

The maternal and developmental toxicity potential of N,N-bis (2-hydroxyethyl)-p-phenylenediamine sulfate was determined following the OECD Guideline 414 (Prenatal Developmental Toxicity Study).

The purpose of this study was to evaluate the developmental toxicity (embryo-fetal toxicity and teratogenic potential) of N,N-bis (2-hydroxyethyl)-p-phenylenediamine sulfate when administered to female Crl:CD (SD)IGS BR VAF/Plus rats (presumed pregnant) by oral gavage at dose levels of 0, 5, 20 and 50 mg /kg bw/day for a period of gestation Day 6 through 20.

One hundred female Crl:CD (SD)IGS BR VAF/Plus rats of 68 d age (Source: Charles River Laboratories, Inc., Raleigh, North Carolina, USA) weighing 219 - 247 g were housed individually in stainless steel, wire-bottomed cages. The animals were maintained under standard laboratory conditions (temperature: 20.6-26.7°C, humidity: 39.0-57.2%, minimum of 10 air changes/h, 12 h light/12 h dark cycle per d) and fed on Certified Rodent Diet #5002; ad libitum. The animals were acclimatized for 6 d prior to mating.

 

After 6 d of acclimation, 140 virgin female rats were placed into cohabitation with 140 breeder male rats, one male rat per female rat for a 4 d cohabitation period. Female rats with spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug observed in situ were confirmed pregnant (The day pregnancy was confirmed was designated as gestation Day 0). Post-coitum, mated females were randomly divided into 3 different treatment groups consisting of 25 animals/dose group. A similar sized group of animals that received 0.2% w/v erythorbic acid in R.O. deionized water (vehicle) served as control. Solutions of the test substance and/or the vehicle were administered orally (gavage) once daily from Days 6 through 20 of presumed gestation. The dosage volume was 10 mL/kg, adjusted daily on the basis of the individual body weights recorded immediately before administration of the test substance.

 

Viabilities, clinical observations, body weights and feed consumption values were recorded. All surviving rats were sacrificed on gestation Day 21. The gravid uterus was excised, weighed and subsequently examined for the number and distribution of corpora lutea, implantation sites and uterine contents. A gross necropsy was performed. Fetuses were weighed and examined for gross external, soft tissue and skeletal alterations and sex. To minimize bias, Caesarean-sectioning and subsequent fetal observations were conducted without knowledge of dosage group.

One dam in the high dose group was found dead on gestation Day 18. No other mortality or any clinical necropsy observations related to the test substance occurred. Maternal body weight gains were reduced in the mid dose group and significantly reduced in the high dose group during the entire dosage period (calculated as gestation Day 6 -21). The gravid uterine weights were reduced in the mid and high dose groups, as compared to the control group value. Although not always dosage dependent, these reductions reflected a slightly smaller litter size that occurred spontaneously in the mid and high dose groups (13.3 and 13.6, respectively) compared to the 0 (Vehicle) and low dose groups (15.0 and 15.2, respectively). Corrected maternal body weights (gestation Day 21 body weight minus the gravid uterine weight) and corrected body weight changes were comparable among the groups. Corresponding to reductions in body weight gain, absolute (g/day) and relative feed consumption values (g/kg/day) were significantly reduced in the mid and high dose groups for the dosage interval of gestation Day 6 -9 (92.2% and 92.5%, respectively, of the control group absolute value).

No treatment related effects were noted in caesarean-sectioning or litter observations. The litter averages for corpora lutea, implantations, litter sizes, live fetuses, early and late resorptions, fetal body weights, percent resorbed conceptuses and percent live male fetuses were comparable among the four dosage groups and did not differ significantly.

The significant increases (p<=0.05 to p<=0.01) in the number of litters with fetuses with alterations, the number of fetuses with alterations and the percentage of fetuses with alterations per litter were not considered related to the test substance as the increases were not dosage dependent and the increases reflected low values in the control group. There was a significant (p≤0.05) decrease in the number of ossified hindlimb phalanges in the 50 mg/kg/day dosage group; however, this was not considered toxicologically important since there was no other adverse effect on fetal weights or fetal alterations. No other gross external, soft tissue or skeletal fetal alterations (malformations or variations) were attributable to treatment with the test substance. 

Based on the above observations, the maternal no-observable-adverse-effect-level (NOAEL) was 5 mg/kg/day. Dosages of 20 and 50 mg/kg/day produced reductions in body weight and feed consumption. The developmental NOAEL was determined by the study authors to be 50 mg/kg/day. No developmental toxicity was produced at dosages as high as 50 mg/kg/day, the highest dose tested.

This teratogenicity study is classified as acceptable, and satisfies the guideline requirements of the OECD 414 method.