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Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Reason / purpose for cross-reference:
data waiving: supporting information
Reproductive effects observed:
not specified
Effect on fertility: via oral route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

The oral administration of 4,4'-Isopropylidenediphenol, oligmoeric reaction products with 1-chloro-2, 3-expoxypropane, reaction products with 2-methylimidazole to pregnant rats by gavage during gestation Days 3 to 19, at dose levels of 100, 300 or 750 mg/kg bw/day resulted in an adverse effect on body weight gain and food consumption at the high dose level. Although some improvement was evident, cumulative body weight gains for females treated with 750 mg/kg bw/day remained lower than controls throughout the dosing period with body weight gain adjusted for the contribution of gravid uterus also being markedly lower than controls. Taking into consideration the overall results, the No Observed Adverse Effect Level’ (NOAEL) for the pregnant female was considered to be 300 mg/kg bw/day within the confines of this type of study.

At 750 mg/kg bw/day, fetal and litter weights were marginally lower than control with skeletal examination identifying a number of treatment-related variants. These observations were, however, considered to be due to maternal toxicity which results in slight reduction in fetal growth rather than an indication of a direct effect of the test item on fetal growth and development. The ‘No Observed Adverse Effect Level’ (NOAEL) for developmental toxicity was therefore considered to be 750 mg/kg bw/day whilst the ‘No Observed Effect Level’ (NOEL) for developmental toxicity was deemed to be 100 mg/kg bw/day within the confines of this type of study.

Link to relevant study records
Reference
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
A total of ninety-six time-mated female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. Animals were delivered in two batches containing females prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation. On arrival the females weighed 192 to 283g.
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
Animals were allocated to treatment groups as follows:

Treatment Dose Level Treatment Concentration Animal Numbers
Group (mg/kg bw/day) Volume (mL/kg) (mg/mL)

Control 0 4 0 24 (1-24)
Low 100 4 25 24 (25-48)
Intermediate 300 4 75 24 (49-72)
High 750 4 187.5 24 (73-96)

The numbers in parentheses ( ) show the individual animal numbers allocated to each treatment group.

The test item was administered daily, from Day 3 to Day 19 of gestation, by gavage. Control animals were treated in an identical manner with the vehicle alone.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
See Annex 1
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
This study was designed to be compatible with the procedures indicated by the following internationally accepted guidelines and recommendations:
 US EPA Health Effects Test Guidelines OPPTS 870.3700 “Prenatal Developmental Toxicity Study” (August 1998)
 Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147 (24 November 2000)
 OECD Guidelines for Testing of Chemicals, No 414 “Prenatal Developmental Toxicity Study” (adopted 22 January 2001)
 Commission Regulation (EC) No 440/2008 of 30 May 2008 test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)
Maternal examinations:
General Observations/Measurements

Clinical Observations
Following arrival, all animals were examined for overt signs of toxicity, ill-health or behavioral changes once daily during the gestation period. Additionally, during the dosing period, observations were recorded immediately before and soon after dosing and one hour post dosing. All observations were recorded.

Body Weight
Individual body weights were recorded on Day 3 (start of treatment) and on Days 4, 5, 8, 11, 14 and 17 of gestation. Body weights were also recorded for animals at terminal kill (Day 20).

Food Consumption
Food consumption was recorded for each individual animal at Day 3, 5, 8, 11, 14, 17 and 20 of gestation.

Water Consumption
Water intake was observed daily by visual inspection of the water bottles for any overt changes.
Ovaries and uterine content:
Necropsy
All animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on Day 20 of gestation. All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded. The ovaries and uteri of pregnant females were removed, examined and the following data recorded:

i) Number of corpora lutea
ii) Number, position and type of intrauterine implantation
iii) Fetal sex
iv) External fetal appearance
v) Fetal weight
vi) Placental weight
vii) Gravid uterus weight

The uteri of any apparently non-pregnant females were immersed in 0.5% ammonium polysulphide solution to reveal evidence of implantation.

Implantation types were divided into:

Early Death: No visible distinction between placental/decidual tissue and embryonic tissue
Late Death: Separate embryonic/fetal and placental tissue visible
Dead Fetus: A fetus that had died shortly before necropsy. These were included as late deaths for reporting purposes
Fetal examinations:
All implantations and viable fetuses were numbered according to their intrauterine position as follows (as an example):

Left Horn Cervix Right Horn
L1 L2 L3 L4 L5 L6 L7 L8 R1 R2 R3 R4 R5 R6 R7 R8
V1 V2 V3 V4 V5 V6 V7 V8 V9 V10 V11 V12 V13 V14 V15 V16
V = viable fetus

The fetuses were killed by subcutaneous injection of a suitable barbiturate agent. Fetuses from each litter were divided into two groups and examined for skeletal alterations and soft tissue alterations. Alternate fetuses were identified using an indelible marker and placed in Bouin’s fixative. Fetuses were subsequently transferred to distilled water and examined for visceral anomalies under a low power binocular microscope and then stored in 10% Buffered Formalin. The remaining fetuses were identified using cardboard tags marked with chinagraph pencil and placed 70% IMS in distilled water. The fetuses were subsequently eviscerated, processed and the skeletons stained with alizarin red S before being transferred to 50% glycerol for examination of skeletal development and anomalies and storage.
Statistics:
The following parameters were analyzed statistically, where appropriate, using the test methods outlined below:

Female body weight change, food consumption and gravid uterus weight: Shapiro Wilk normality test and Bartlett’s test for homogeneity of variance and one way analysis of variance, followed by Dunnett’s multiple comparison test or, if unequal variances were observed, on alternative multiple comparison test.

All caesarean necropsy parameters and fetal parameters: Kruskal-Wallis non-parametric analysis of variance; and a subsequent pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test, where significance was seen.

Fetal evaluation parameters, including skeletal or visceral findings: Kruskal-Wallis nonparametric analysis of variance and Mann-Whitney ‘U’ test.

Probability values (p) are presented as follows:

p<0.001 ***
p<0.01 **
p<0.05 *
p≥0.05 (not significant)
Indices:
Pre and Post Implantation Loss
Percentage pre-implantation loss was calculated as:

((number of corpora lutea - number of implantations)/number of corpora lutea) x 100

Percentage post-implantation loss was calculated as:

((number of implantations - number of live fetuses)/number of implantations) x 100

Sex Ratio
Sex ratio was calculated as:

% male fetuses (sex ratio) = (Number of male fetuses/Total number of fetuses) x 100
Clinical signs:
no effects observed
Description (incidence and severity):
A summary incidence of daily clinical observations is given in Table 2. Individual data are presented in Appendix 1.

There were no clinical signs of toxicity detected.

One female treated with 750 mg/kg bw/day showed generalised fur loss towards the end of gestation. Such observations are common in this type of study and in isolation this was considered unlikely related to treatment. One female treated with 100 mg/kg bw/day showed red urine on the day of arrival. Two control females were observed with scab formation. In the absence of treatment with the test item this finding was considered be incidental.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths on the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Group mean body weights and standard deviations are given in Table 3 and presented graphically in Figure 1. Group mean body weight gains and adjusted body weights and standard deviations are given in Table 4 and Table 5 (statistically significant differences are indicated). Individual data are given in Appendix 2, Appendix 3 and Appendix 4.

At 750 mg/kg bw/day females showed statistically significant reductions (p<0.01 or p<0.001) in body weight gains for the majority of the treatment period. The mean cumulative body weight gains for these females were lower than controls throughout the dosing phase achieving statistical significance between Days 3 to 4 and 11 to 14 (p<0.01 and p<0.001, respectively) in relation to controls. Group mean body weights for these females were also lower than controls throughout the treatment period with the intergroup differences attaining statistical significance for Day 11 of gestation. Overall body weight gain, when adjusted for gravid uterus weight for females at this dose level were statistically significantly lower (p<0.001) when compared to controls.

Females treated with 300 mg/kg bw/day showed a statistically significant increase (p<0.01) in body weight gains between Days 11 and 14. These females also showed a statistically significant reduction (p<0.05) in body weight gains in relation to controls between gestation Days 17 and 20. This resulted in slightly lower cumulative body weight gains for these animals over this period but statistical significance was not attained. Overall body weight and body weight gain, when adjusted for gravid uterus weight, in 300 mg/kg bw/day females were also slightly lower than controls, but without attaining statistical significance.

Body weight development in females treated with 100 mg/kg bw/day was generally similar to controls throughout the treatment period. Minor but statistically significant increases in body weight gains observed for females receiving 300 or 100 mg/kg bw/day over Days 11 to 14 of gestation were considered to be due to biological variation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Group mean food consumptions are given in Table 6 and presented graphically in Figure 2. Individual data are given in Appendix 5.

At 750 mg/kg bw/day, food intake was statistically significantly lower than controls (p<0.01 or p<0.001) throughout the treatment period. At 300 mg/kg bw/day, food intake was lower at the start (Days 3 to 5) and at the end (Days 14 to 20) of the treatment period, attaining statistical significance only at the start. There were no effects on food consumptions for females at 100 mg/kg bw/day.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily visual inspection of water bottles did not reveal any treatment-related intergroup differences.
Gross pathological findings:
no effects observed
Description (incidence and severity):
A summary incidence of female necropsy findings is given in Table 7. Individual data are given in Appendix 6.

There were no macroscopic abnormalities detected in treated females.
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
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
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
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Skeletal Examination of fetuses/litters revealed a number of statistically significant changes in relation to controls which resulted in a statistically significant increase (p<0.001) in 750 mg/kg bw/day fetuses/litters being affected. At 750 mg/kg bw/day there were statistically significant higher incidences of incomplete ossification of the nasal (p<0.01), squamosal (p<0.01), occipital (supra-occipital) (p<0.01), interparietal (p<0.01), presphenoid (p<0.01) and the hyoid bones (p<0.01). There was a statistically significant increase in some hyoid bones not ossified (p<0.001). All the findings at this dose level were above the background control ranges.

At 300 mg/kg bw/day there were statistically significant higher incidences of incomplete ossification of the nasal (p<0.05), interparietal (p<0.01), presphenoid (p<0.05) bones and some costal cartilage not fused to the sternebra (p<0.05). With the exception of the latter, the findings were above the background control ranges.

There were no statistically significant differences detected in the type and incidence of skeletal findings in fetuses/litters from females treated with 100 mg/kg bw/day. The cervical (neural) arch showed a statistically significant higher (p<0.01) incidence in this dose group. This finding showed no dose relationship and was considered to be an incidental finding. The group mean percentage for this finding was within the background control ranges.
Visceral malformations:
effects observed, treatment-related
Description (incidence and severity):
At the visceral examination, the external observations revealed major malformations confined to one fetus from the 750 mg/kg bw/day dose group showing encephalocoele. This anomaly is known to occur spontaneously in this strain of rat, and as there were no similar observations in any other test item-treated dose groups, this finding was considered to be incidental.

In addition, one fetus from the 300 mg/kg bw/day and two fetuses from one litter from the 750 mg/kg bw/day dose groups were shown to have enlarged cardiac atrium. The same two fetuses from the 750 mg/kg bw/day dose group also had thickened ventricular wall and septum. These observations are known to occur spontaneously and showed no dose relationship. Therefore these findings in isolation were considered to be incidental and of unknown etiology.

The visceral examination at 750 or 300 mg/kg bw/day also revealed a statistically significant lower incidence of kinked ureter (p<0.01 and p<0.001, respectively) with no dose relationship, and increased renal pelvic cavitation (p<0.01 and p<0.05, respectively) showed a dose related response for this observation. At 750 mg/kg bw/day a statistically significant lower incidence of rugae showing a non-uniform patterning (p<0.05) was observed. With the exception of kinked ureter, all the other findings were within the background control ranges. There were no statistically significant visceral findings in fetuses/litters at 100 mg/kg bw/day.
Key result
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations
Abnormalities:
not specified
Localisation:
skeletal: skull
Description (incidence and severity):
At 750 or 300 mg/kg bw/day, skeletal evaluation revealed statistically significant higher incidences of incomplete ossification of the nasal, squamosal, occipital (supra-occipital), interparietal, presphenoid and the hyoid bones. There were some incidences of hyoid bones not ossified at 750 mg/kg bw/day. Although group mean values were outside the background control ranges, these observations are likely due to the slightly slower fetal growth rate resulting from maternal toxicity rather than an indication of a direct effect of the test item on fetal development. Any other skeletal findings at all dose levels were considered to be within normal biological variation.
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
750 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
yes
Relevant for humans:
no
Conclusions:
The oral administration of 4,4'-Isopropylidenediphenol, oligmoeric reaction products with 1-chloro-2, 3-expoxypropane, reaction products with 2-methylimidazole to pregnant rats by gavage during gestation Days 3 to 19, at dose levels of 100, 300 or 750 mg/kg bw/day resulted in an adverse effect on body weight gain and food consumption at the high dose level. Although some improvement was evident, cumulative body weight gains for females treated with 750 mg/kg bw/day remained lower than controls throughout the dosing period with body weight gain adjusted for the contribution of gravid uterus also being markedly lower than controls. Taking into consideration the overall results, the No Observed Adverse Effect Level’ (NOAEL) for the pregnant female was considered to be 300 mg/kg bw/day within the confines of this type of study.

At 750 mg/kg bw/day, fetal and litter weights were marginally lower than control with skeletal examination identifying a number of treatment-related variants. These observations were, however, considered to be due to maternal toxicity which results in slight reduction in fetal growth rather than an indication of a direct effect of the test item on fetal growth and development. The ‘No Observed Adverse Effect Level’ (NOAEL) for developmental toxicity was therefore considered to be 750 mg/kg bw/day whilst the ‘No Observed Effect Level’ (NOEL) for developmental toxicity was deemed to be 100 mg/kg bw/day within the confines of this type of study.
Executive summary:

Introduction

The study was performed to investigate the effects of the test item on embryonic and fetal development following repeated administration by gavage to the pregnant female during gestation including the period of organogenesis.

The study was designed to comply with the following guidelines:

 US EPA Health Effects Test Guideline OPPTS 870.3700, ‘Prenatal Developmental Toxicity Study’ (August 1998)

 Japanese Ministry of Agriculture, Forestry and Fisheries Testing guidelines for Toxicology studies, 12 NohSan No 8147, (24 November 2000)

 OECD Guidelines for Testing of Chemicals, No 414, ‘Prenatal Developmental Toxicity Study’ (adopted 22 January 2001)

 Commission Regulation (EC) No 440/2008 of 30 May 2008 test methods pursuant to Regulations (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

Methods

The test item was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, between Days 3 and 19 of gestation inclusive at dose levels 100, 300, and 750 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle only (Arachis oil) to serve as a control.

Clinical signs, body weight change, food and water consumptions were monitored during the study.

All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

Results

Mortality

There were no unscheduled deaths during the study.

Clinical Observations

There were no clinical signs of toxicological significance throughout the study.

Body Weight

At 750 mg/kg bw/day females showed a statistically significant reduction for the body weight gains for the majority of the treatment period. Overall body weight gain for these females was lower than control females and overall body weight gains when adjusted for gravid uterus weight for females treated with 750 mg/kg bw/day was also lower when compared to controls.

Females treated with 300 mg/kg bw/day showed statistically significant increase in body weight gains between Days 11 and 14 and statistically significant reduction between Days 17 and 20. Overall body weight gain and body weight gain when adjusted for gravid uterus weight in 300 mg/kg bw/day females was slightly lower than controls.

Females treated with 100 mg/kg bw/day were generally similar to controls.

Food Consumption

There was a statistically significant reduction in dietary intake for females treated with 750 mg/kg bw/day throughout the treatment period. Food consumptions for females treated with 300 or 100 mg/kg bw/day showed generally similar dietary intake to controls.

Water Consumption

Daily visual inspection of water bottles did not reveal any treatment-related intergroup differences.

Post Mortem Studies

There were no macroscopic abnormalities detected in treated females.

Litter Data and Litter Placental and Fetal Weights

There was no effect of maternal treatment on litter data as assessed by numbers of implantations, in-utero offspring survival (as assessed by the mean numbers of early or late resorptions), live litter size, sex ratio and pre and post-implantation losses at 100, 300 or 750 mg/kg bw/day.

There were no effects of maternal treatment on mean placental weights or litter weight at 100, 300 or 750 mg/kg bw/day. However, the mean foetal weights from 750 mg/kg bw/day litters showed a statistically significant reduction in body weight in relation to controls. This finding is considered to be associated to maternal toxicity rather than a direct effect of treatment on fetal growth and development.

Fetal Examination

There were no findings apparent for foetuses from treated females at external examination on Day 20 of gestation of toxicological importance.

At 750 mg/kg bw/day there were statistically significant higher incidences of incomplete ossification of the nasal, squamosal, occipital (supra-occipital), interparietal, presphenoid and the hyoid bones. There were some incidences of hyoid bones not ossified. The majority of these values were above the background control ranges. The visceral examination for 750 mg/kg bw/day litters indicated there was a statistically significant lower incidence of kinked ureter, increased renal pelvic cavitation and the rugae showing a non-uniform patterning. These visceral findings showed a reduced incidence of structures completing ossification. The group mean values for these findings were within the background control ranges, with the exception of kinked ureters which values were outside the background control ranges, and therefore considered of no toxicological significance.

At 300 mg/kg bw/day there were statistically significant higher incidences of incomplete ossification of the nasal, interparietal, presphenoid bones and also there was some costal cartilage not fused to the sternebra. There was also a statistically significant lower incidence of kinked ureter and increased renal pelvic cavitation.

These observations are likely due to a slightly slower fetal growth rate resulting from maternal toxicity rather than an indication of a direct effect of the test item on fetal development. Any other skeletal and visceral findings at all dose levels were considered to be within normal biological variation.

Conclusion

The oral administration of 4,4'-Isopropylidenediphenol, oligmoeric reaction products with 1-chloro-2, 3-expoxypropane, reaction products with 2-methylimidazole to pregnant rats by gavage during gestation Days 3 to 19, at dose levels of 100, 300 or 750 mg/kg bw/day resulted in an adverse effect on body weight gain and food consumption at the high dose level. Although some improvement was evident, cumulative body weight gains for females treated with 750 mg/kg bw/day remained lower than controls throughout the dosing period with body weight gain adjusted for the contribution of gravid uterus also being markedly lower than controls. Taking into consideration the overall results, the No Observed Adverse Effect Level’ (NOAEL) for the pregnant female was considered to be 300 mg/kg bw/day within the confines of this type of study.

At 750 mg/kg bw/day, fetal and litter weights were marginally lower than control with skeletal examination identifying a number of treatment-related variants. These observations were, however, considered to be due to maternal toxicity which results in slight reduction in fetal growth rather than an indication of a direct effect of the test item on fetal growth and development. The ‘No Observed Adverse Effect Level’ (NOAEL) for developmental toxicity was therefore considered to be 750 mg/kg bw/day whilst the ‘No Observed Effect Level’ (NOEL) for developmental toxicity was deemed to be 100 mg/kg bw/day within the confines of this type of study.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Dose descriptor:
NOAEL
750 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Justification for classification or non-classification

Additional information

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