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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
3-(2-hydroxyethyl)-p-phenylenediammonium sulphate
EC Number:
298-995-1
EC Name:
3-(2-hydroxyethyl)-p-phenylenediammonium sulphate
Cas Number:
93841-25-9
Molecular formula:
C8H12N2O.H2O4S
IUPAC Name:
3-(2-hydroxyethyl)-p-phenylenediammonium sulphate
Specific details on test material used for the study:
Batch number : 30/01
Purity : 99.9a/a%

Test animals

Species:
rat
Strain:
other: CRL : (WI) BR Wistar

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1% aqueous methylcellulose
Duration of treatment / exposure:
Animals were treated on Day 6-20 of gestation.
Frequency of treatment:
Animals were treated once daily.
Duration of test:
Animals were sacrificed on Day 21 of gestation.
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
70 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Twenty two females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
CRL:(WI) BR Wistar rats (22 sperm positive females/group) were treated daily from gestation day 6 to gestation day 20 by gavage with the test item at a dose levels of 10, 30 and 70 mg/kg bw prepared in 1% aqueous methylcellulose. These dose levels were based on a preliminary dose range finding study. The concentrations corresponded to a constant treatment volume of 5 mL/kg bw. Control animals were treated with 1% aqueous methylcellulose.

Examinations

Maternal examinations:
Cage observations were performed twice daily. Individual examination of the animals was undertaken at least once per day and condition of the animals, including behaviour changes and signs of overt toxicity were recorded. Body weight and food consumption was recorded on a regular basis. On gestation day 21 sperm positive females were asphyxated using carbondioxide and sacrificed by cervical dislocation. Gross pathology and histology were performed.
Ovaries and uterine content:
Foetal weight, placental weight and corrected body weight was determined. The number of implantation sites, corpora lutea, number and position of live foetuses, early and late embryonic death and foetal death were counted.
Fetal examinations:
Foetal examination was performed.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Clinical symptoms were not observed in the control, 10 or 30 mg/kg bw/day dose groups. Urine was coloured orange in the animals of the 70 mg/kg bw/day dose group during the treatment period. Compound related clinical signs were observed in the 70 mg/kg bw/day treated dose group for 22 animals from the second or third day of administration. Reduced activity, hunched position, salivation, piloerection, dyspnea and reduced righting reflex were noted.In three cases brownish discolouration around the mouth and nose was observed in the high dose group.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The body weight was at the control level in the 10 and 30 mg/kg bw/day dose groups. It was significantly below the control level in the 70 mg/kg bw/day dose group on day 12 of gestation. Body weight gain was statistically lower between days 18 and 21 of gestation in the 30 mg/kg bw/day group but taking into account the profile of effects at 70 mg/kg bw/day, this was considered to be of no biological significance. The body weight gain at 70 mg/kg bw/day was statistically significantly lower in the first week of the treatment period (between days 6 and 12 of gestation). This corresponded with clinical signs and food intake differences and was considered to be treatment related. A statistically significant higher body weight gain was observed in this group between days 12 and 18 of gestation, this was attributed to a compensation effect following the reduced body weight gain in the previous period. No significant differences were found between the control and test item treated groups in respect of the gravid uterine weight, corrected body weight or corrected body weight gain.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Significantly decreased food consumption which correlated with the reduction in body weight was noted on the first week of the treatment period in the 70 mg/kg bw/day dose group. Significantly decreaded food consumption was also observed in the 10 mg/kg bw/day group between days 18 and 21 of gestation but was considered as biologically negligible and not treatment related.
Details on results:
No mortality was observed. In the control and the 10 and 30 mg/kg bw/day dose groups no clinical symptoms were observed. Clinical symptoms were observed in the 70 mg/kg bw/day dose group. Animals in the 70 mg/kg bw/day group showed orange coloured urine, reduced activity, hunched position, salivation, piloerection, dyspnoea and reduced righting reflex. In three animals of the 70 mg/kg bw/day dose group, brownish discolouration around the mouth and nose was observed. Body weight and boby weight gain in this dose group was significantly reduced in the first week of the treatment. These effects corresponded with clinical signs and reduced food consumption.

Maternal developmental toxicity

Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
The early embryonic death and the post implantation loss were significantly higher in the 70 mg/kg bw/day dose group.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
clinical signs
pre and post implantation loss

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The average fetal body weight and the weight of placenta were similar to control values in each treated group.
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
The early embryonic death and the post implantation loss were significantly higher in the 70 mg/kg bw/day dose group.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
In the course of external examination two malformed fetuses were found in the 10 mg/kg bw/day group, one with hypoplastic mandible and the other with cleft palate. There were no similar findings in the higher dose groups, these observations were considered to be incidental and not related to treatment. In case of one fetus in the 70 mg/kg bw/day dose group white point-like formations were found on the placenta. Histopathological examination of this placenta showed purulent placentitis, this is ascribed to poor blood circulation associated with maternal toxicity (occasionally observed in previous studies in this laboratory). The incidence of edematous and fibrinoid degenerated placentas was low but statistically significant in the 10 and 70 mg/kg bw/day dose groups, the incidence is within the expected range and is not considered to be of any biological significance. In the 10 mg/kg bw/day dose group the number of fetuses found with abnormalities was statistically significantly higher than controls, but this was only because of the 2 incidental malformed fetuses and is not considered to be a treatment related effect. Most of the observations were compatible with historical data in the laboratory, others were clearly not dose related. There were no treatment related external findings in any treated group.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Signs of delayed calcification were observed in all experimental groups at a normal incidence. None showed any relationship with treatment. At skeletal examination the incidence of variations was significantly higher in the 10 mg/kg bw/day treated groups. The absence of pubic bones was also significantly higher in the 30 mg/kg bw/day dose group. These changes were directly associated with individual low fetal body weights and were not seen at highe dose levels. They were not considered attributable to treatment. Three skeletal malformations were found in the control group. All these fetuses had short, bent scapula. In the 10 mg/kg bw/day dose group three malformed fetuses were observed. Two malformed fetuses were found in the 30 mg/kg bw/day dose group. Both had short and bent scapula. One malformed fetus was observed in the 70 mg/kg bw/day dose group with short and bent scapula and shorter humerus on the right. The incidence of sporadic skeletal malformations in the scapula/humerus, cleft palate were compatible with historical data in the laboratory. These changes are known spontaneous findings in rats. There was no dose-relationship with no similar findings found in the two higher treatment levels, so these findings are not considered to be related to treatment. There were no skeletal malformations considered to be related to treatment.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
In the course of visceral examination three malformed fetuses were found, all in the 10 mg/kg bw/day dose group. In two cases hydronephrosis occurred as malformations (in one litter) and in the third case a multiple malformed fetus was found showing hypoplastic kidney on the right, dilated aquaeductus cerebri, hydrureter with dilated renal pelvis and an unjoined palate with only the mucouse membrane closed. The incidence of hydrureter with dilated renal pelvis was statistically significantly higher in the 10 mg/kg bw/day dose group because of one litter where its frequency was 80%. This finding was litter related and not considered to be treatment related effect. The hydronephrosis, hydrureter and dilated brain ventricle were findings compatible with historical data in the laboratory. The fetuses with multiple malformations had changes that are known spontaneous findings in rats and were only found in the low dose group with no evidence of any dose-response. All observations were incidental sporadic findings with no dose relationship. There were no treatment related visceral findings in any group.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: early embryonic death considered to be a consequence of maternal toxicity

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Gravid uterine weight, corrected body weight and corrected body weight gain was not affected by the test item. Necropsy did not reveal any treatment related effects. Early embryonic death and post implantation loss were higher in the 70 mg/kg bw/day dose group as a consequence of maternal toxicity. Foetal body weight and placental weight were similar to control values. Sporadic external, visceral and skeletal alterations were observed but these effects were not considered treatment related. No specific compound related teratogenic effects were observed in the study.

Applicant's summary and conclusion

Conclusions:
Based on the study a NOAEL for maternal and developmental toxicity of 30 mg/kg bw/day was determined. No specific compound related teratogenic effects were observed in this teratogenicity study. The NOAEL for teratogenicity was >70 mg/kg bw/day.
Executive summary:

A teratology study with the test item hydroxyethyl-p-phenylenediamine sulfate (Betoxol II) was performed with doses 10, 30 and 70 mg/kg bw/day in groups of 22 Crl(WI)BR-Wistar rats in their first pregnancy. Sperm positive females were treated orally from day 6 to and including day 20 of pregnancy. Clinical observations, body weight and food consumption measurements were made during the in-life phase. Dams were subjected to Caesarean section and gross pathology on day 21 of pregnancy. After examination of the uterine content and external examination of fetuses about half of each litter was fixed for visceral examination and the other half for skeletal examination. After fixation the head was examined and the body was dissected with emphasis on a detailed examination of blood vessels and sectioning of heart and kidneys. Examination of skeletons was undertaken and all abnormalities were recorded. Compound related clinical signs (orange coloured urine, reduced activity, hunched position, piloerection, salivation, dyspone and reduced righting reflex, brownish discolouration around the mouth and nose) were found in all animals at 70 mg/kg bw/day only. Body weight, body weight gain and food consumption were lower at 70 mg/kg bw/day only. Early embryonic death and post implantation loss were significantly higher in the 70 mg/kg bw/day dose group only. The effects at 70 mg/kg bw/day were ascribed to maternal toxicity. There was no evidence for any direct effects on the fetuses. No effects of treatment were observed in dams or fetuses at 10 or 30 mg/kg bw/day. Significant maternal toxicity was observed at 70 mg/kg bw/day characterised by reductions in body weight, body weight gain, food intake in addition to clinical signs of systemic toxicity. In the 70 mg/kg bw/day dose group a significantly increased post-implantation mortality and early embryonic loss was observed, these were attributed to the consequences of maternal toxicity. No teratogenic or embryotoxic and/or fetotoxic effects were directly attributable to treatment of pregnant rats with hydroxyethyl-p-phenylenediamine sulfate (Betoxol II). Under the conditions employed in the study, a NOAEL for maternal and developmental toxicity of 30 mg/kg bw/day was determined. The NOAEL for teratogenicity was >70 mg/kg bw/day.

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