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Registration Dossier
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EC number: 915-589-8 | CAS number: -
Toxicological information
Developmental toxicity / teratogenicity
Administrative data
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 08 Oct 2018 - 18 Feb 2019
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 020
- Report date:
- 2020
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 414 (Prenatal Developmental Toxicity Study)
- Version / remarks:
- adopted in 2018
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.31 (Prenatal Developmental Toxicity Study)
- Version / remarks:
- adopted 2008
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
- Version / remarks:
- adopted 1998
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Health and Youth Inspectorate, Ministry of Health, Welfare and Sport, Utrecht, The Netherlands
- Limit test:
- no
Test material
- Reference substance name:
- Diisopropylbiphenyl and triisopropylbiphenyl
- EC Number:
- 915-589-8
- Molecular formula:
- not applicable for UVCB substance
- IUPAC Name:
- Diisopropylbiphenyl and triisopropylbiphenyl
- Test material form:
- liquid
Constituent 1
Test animals
- Species:
- rat
- Strain:
- other: Han Wistar
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories Deutschland, Sulzfeld, Germany
- Age at study initiation: 11-15 weeks
- Weight at study initiation: 175 - 261 g
- Fasting period before study: no
- Housing: individually in Macrolon plastic cages (MIII type, height 18 cm) containing appropriate bedding (Lignocel S 8-15)
- Diet: pelleted rodent diet SM R/M-Z (SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: municipal tap water in bottles, ad libitum
- Acclimation period: at least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 22
- Humidity (%): 41 - 55
- Air changes (per hr): at least 10; fresh ait (no re-circulation)
- Photoperiod: 12 hrs dark / 12 hrs light):
IN-LIFE DATES: From: 02 Dec 2018 To: 20 Dec 2018
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared as daily portions maximally 8 days in advance and stored in the refrigerator protected from light. On each day of dosing, the dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing. Also when formulations were prepared on the day of dosing, formulations were stirred at room temperature for at least 30 minutes before dosing.
Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle and the test item. No correction was made for the purity/composition of the test item. Any residual volumes were discarded.
VEHICLE
- Justification for use and choice of vehicle: Trial preparations were performed at the Test Facility to select the suitable vehicle. Corn oil was identified as a suitable vehicle.
- Concentration in vehicle: 12.5, 37.5 and 112.5 mg/mL
- Amount of vehicle: 4 mL/kg bw
- Supplier and lot/batch no.: Sigma-Aldrich, batch nos. MKC F8882 and MKCG 3257 - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Stability analyses performed before study start in conjunction with the method development and validation study demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
During the study, dose formulation samples were collected for analysis during week 1 and week 2 of dosing. At both occasions samples for determination of achieved concentration were taken from all dose groups and samples for determination of homogeneity were take from the low and high dose group. Analyses were performed using a validated analytical procedure.
Results: The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). No test item was detected in the vehicle control (Group 1) formulations. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%). - Details on mating procedure:
- Time-mated animals were received from the breeder.
- Duration of treatment / exposure:
- Day 6 -20 of gestation
- Frequency of treatment:
- daily, 7 days/week
- Duration of test:
- 15 days
Doses / concentrationsopen allclose all
- Dose / conc.:
- 50 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 150 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 450 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- 22 females
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: The dose levels were selected based on the results of the dose range finder study.
In this dose range finding study in the pregnant rat, and in an attempt to produce graded responses to the test item. In this dose range finder groups of 6 presumed pregnant female rats were treated at 35, 150 and 600 mg/kg bw/day once daily from day 6 to 20 post coitum inclusive. Animals in the control group (n = 6) received the vehicle, corn oil, only. In this study no mortality occurred. Clinical signs noted were piloerection at 600 mg/kg bw/day. No body weight gain was observed at 600 mg/kg bw/day from post-coitum Day 6 to 9, followed by a growth rate that was comparable to the concurrent control group (similar trend at 150 mg/kgbw/day). Food consumption was decreased at 600 mg/kgbw/day between post-coitum Days 6-9 and 9-12, which returned to control levels afterwards. Enlarged livers were noted in 3/6 females at 600 mg/kg bw/day with concurrent moderately to severely increased liver weights at 150 and 600 mg/kg bw/day. Although not statistically significant, thyroid weights were slightly increased at 150 and 600 mg/kg bw/day (not dose-dependent). Number of corpora lutea, implantation sites, viable and dead fetuses, late resorptions, pre- and post-implantation loss and sex ratio were unaffected up to 600 mg/kg bw/day. At 600 mg/kg bw/day, 1/6 females had early resorptions only. Lower fetal body weights were noted at 600 mg/kg bw/day (same trend at 150 mg/kg bw/day). External examination of the fetuses did not reveal any abnormalities up to 600 mg/kg bw/day.
Examinations
- Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, in the morning and at the end of the working day
- Cage side observations were included.
CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily, beginning on Day 2 post-coitum and lasting until the day prior to necropsy
BODY WEIGHT: Yes
- Time schedule for examinations: on Days 2, 6, 9, 12, 15, 18 and 21 post-coitum
FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Time schedule: measured for Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum
WATER CONSUMPTION: No
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation Day 21
All animals (including animals found dead or sacrificed before planned necropsy and females with early delivery) were subjected to an external, thoracic and abdominal examination, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). The thyroid gland, uterus and liver were weighed at necropsy for all scheduled euthanasia animals.
THYROID HORMONES:
Blood of F0-animals (except for animals which were found dead and females that delivered their offspring early) were collected on the day of scheduled necropsy bewteen 07:00 and 09:00 a.m. from the jugular vein. Animals were not fasted overnight. Blood samples at a target volume of 1 mL were collected into tubes without anticoagulant. Blood samples were processed for serum, and serum was analyzed for triiodothyronine (T3), thyroxine (T4) and thyroid-stimulating hormone (TSH).
HISTOPATHOLOGY:
Thyroid gland of all animals of Groups 1 and 4 were embedded in paraffin, sectioned at a thickness of 2-4 µm, mounted on glass slides, and stained with hematoxylin and eosin. As treatment-related changes in the thyroid gland were expected after the evaluation of Group 1 and 4 animals, histological examination of the thyroid gland was extended to Group 2 and 3 animals.
ORGAN WEIGHTS:
At scheduled necropsy the following organ weights were recorded : thyroid gland (paired organ weight), liver, uterus - 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
- Number and distribution of live and dead fetuses: Yes
- Sex of each fetus based on anogenital distance: Yes - Fetal examinations:
- - External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter] - Statistics:
- please refer to "Any other information and methods"
- Indices:
- Body Weight Gains: Calculated against the body weight on Day 6 post-coitum
Corrected Body Weight Gains: Body weight on Day 21 post-coitum minus the body weight on Day 6 post-coitum and the weight of gravid uterus.
Relative Food Consumption: Calculated against the body weight for scheduled intervals.
Organ Weight Relative to Body Weight: Calculated against the body weight on Day 21 post-coitum.
Pre-implantaion loss (%) = ((no. of corpora lutea - no. of implantation sites)/no. of corpora lutea) x 100
Post-implantaion loss (%) = ((no. of implantation sites - no. of live fetuses)/no. of implantation sites) x 100
The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison, calculating the number of affected fetuses as a mean litter proportion on a total group basis, where:
Viable fetuses affected/litter (%) = ((no. of viable fetuses affected/litter)/number of viable fetuses/litter)) x 100 - Historical control data:
- Historical control data from studies performed during 2014 to 2017 (37 studies) are appended to the report.
Results and discussion
Results: maternal animals
General toxicity (maternal animals)
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Piloerection was observed in 8/22 high dose females on two or more consecutive days, mainly at the end of the treatment period, and in a single mid dose female on the last day of treatment.
Salivation (slight) seen after dosing among individual mid and high dose females, mainly in the second part of the treatment period, was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign is considered to be a physiological response related to the taste of the test item rather than a sign of systemic toxicity.
Other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment. - Dermal irritation (if dermal study):
- not examined
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- One high dose female was found dead in her cage prior to dosing on Day 9 of treatment (Day 14 post-coitum). Blood was noted in her cage and on her vagina (externally). No clinical signs of toxicity were noted for this animal on the days preceding her death. For this female, 4% body weight loss was recorded between post-coitum Day 6-9, which recovered between Day 9-12 (13% body weight gain in this period). Food consumption for this animal was also decreased between Day 6-9, but was considered within normal range between Day 9-12. No macroscopic abnormalities were noted at necropsy. This female was pregnant and had 15 implantations in uterus (5 early resorptions and 10 normal developing implantations). Given the low incidence and in the absence of signs of ill health at the time of her death, this mortality was considered not test item-related.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- In approximately half of the high dose females body weight loss (up to 6%) was observed between post-coitum Day 6-9, resulting in a mean body weight gain of zero. This was followed by statistically significantly reduced body weight gain throughout the treatment period, with 5% lower body weights at the end of the treatment period compared to concurrent controls (not statistically significant). A trend towards a reduced body weight and body weight gain was also observed in the mid dose between post-coitum Day 6-15, reaching statistical significance for body weight gain only (post-coitum Day 9 and 15). For details see Attachment "Summary of female body weights and body weight gains" under Attached Background Material.
Body weight gain corrected for gravid uterus was also lower at 450 mg/kg bw/day. Mean corrected weight gain in this group was 20.3 g (9.4%) versus 28.8 g (13.1%) in the control group. A similar trend was observed at 150 mg/kg bw/day (20.7 g; 9.5%), although not reaching statistical significance. (for details see Attachment "Summary of corrected body weight gains" under Attached Background Material).
No toxicologically relevant changes in body weight were noted in the low dose group. - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- A dose-related reduction in food consumption before and after allowance for body weight was observed at 150 and 450 mg/kg bw/day between post-coitum Day 6-12. Absolute and relative food consumption were decreased up to 21 and 15%, respectively at 150 mg/kg bw/day, and up to 37 and 33%, respectively at 450 mg/kg bw/day. Thereafter, food consumption returned to normal values and remained within the normal range of biological variation over the rest of the treatment period.
No toxicologically relevant changes in food consumption before or after allowance for body weight were noted in the low dose group. For details see Table 1. - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- effects observed, treatment-related
- Description (incidence and severity):
- The following statistically significant changes distinguished treated from control animals (relative changes in mean values as compared to the concurrent control group are indicated between parentheses):
- increased alanine transaminase (ALAT) values at 150 and 450 mg/kg bw/day (1.32 and 1.79x of control, respectively)
- increased aspartate aminotransferase (ASAT) values at 450 mg/kg bw/day (1.20x of control)
- decreased total protein values at 450 mg/kg bw/day (0.82x of control)
- decreased total bilirubin values at 150 and 450 mg/kg bw/day (0.74 and 0.74x of control, respectively)
- increased urea at 50, 150 and 450 mg/kg bw/day (1.20, 1.27 and 1.37x of control, respectively)
- decreased creatinine at 450 mg/kg bw/day (0.91x of control)
- increased chloride at 450 mg/kg bw/day (1.02x of control)
- decreased calcium at 450 mg/kg bw/day (0.95x of control)
Although statistically significant, these alterations in clinical biochemistry parameters were considered not toxicologically relevant due to the minimal magnitude of the change (calcium, chloride and total protein values), the absence of a dose response (bilirubin), or the absence of biological relevance (decreased creatinine). Urea values achieving a level of statistical significance when compared to controls, were considered to have arisen as a result of slightly low control values and were considered to be of no toxicological significance. Given the limited number of parameters investigated in this type of study (i.e. limited collection of tissues and no histopathological evaluation), further evaluation of these findings was considered not possible.
A statistical significant increase in serum levels of TSH was observed for F0 females at 450 mg/kg bw/day (6.2 x of control). A similar trend was observed at 150 mg/kg bw/day (2.4 x of control), although the value did not reach statistical significance. Total T3 and T4 were reduced at 150 and 450 mg/kg bw/day, reaching statistical significance for total T4 at 150 mg/kg bw/day only (0.73x of control). Although mean T4 values remained within the historical control range*, most individual T4 values were below detection limit (1.00 µg/dL) and therefore actual mean T4 values at 450 mg/kg bw/day are lower than indicated. Mean T3 and mean TSH values were respectively below and above the limits of the historical control range*, although also for T3 all samples (except for one at both 150 and 450 mg/kg bw/day), were below the detection limit (40 ng/dL), which means that the actual mean T3 values at 150 and 450 mg/kg bw/day are lower than the indicated means (see Table 2).
The observed decrease in total T3/T4 together with the increase in TSH might be related to the increased liver weights observed at 150 and 450 mg/kg bw/day and the non-adverse follicular cell hypertrophy in the thyroid glands that was noted at increased incidence and severity at 450 mg/kg bw/day. Possible adversity of the effects on thyroid hormones could not be assessed within this type of screening study and was therefore not taken into account when determining the parental NOAEL.
* Historical control data for thyroid hormone analyses in pregnant Wister Han rats (period 2018-2019):
TSH mean = 0.440, P5–P95 = 0.195–0.819 (n=30)
Total T3 mean = 66.1, P5–P95 = 51.0–84.5 (n=28)
Total T4 mean = 2.08, P5–P95 = 1.47–3.03 (n=29) - Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- There was a test item-related increase in liver weights at all doses. Absolute liver weights and liver/body weight ratios were increased with statistical significance for females treated at 50, 150 and 450 mg/kg bw/day (1.16, 1.33 and 1.74x of control for absolute liver weights and 1.18, 1.36 and 1.84x of control, for relative liver weights), see Table 3.
No changes in thyroid or uterus weights were noted. - Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Enlarged livers were observed in 8/22 females treated at 450 mg/kg bw/day versus 0/22 in the concurrent control group. This correlated with the dose dependent increase in liver weights at 450 mg/kg bw/day and increased liver enzymes (ALAT and ASAT) observed at 150 and 450 mg/kg bw/day. Based on the incidence observed, this was considered test item-related.
As no histopathology of the livers was performed, taking into account the magnitude of the effect on the liver weights and the increased liver enzymes at 150 and 450 mg/kg, adversity of these findings could not be excluded. In the absence of increased liver enzymes at 50 mg/kg and as the increase in liver weights was less than 20 percent, the liver findings at 50 mg/kg were considered non-adverse.
Watery fluid in the uterus was found in one pregnant high dose female. At the incidence observed this finding was considered not related to treatment. For one pregnant female each at 50 and at 150 mg/kg bw/day, the uterine content was hemorrhagic/contained clotted blood. There was no indication for early delivery in these females and this finding was therefore considered not toxicologically relevant.
Other findings that were noted among control and/or treated animals were considered to be of no toxicological significance, since they remained within the range of biological variation for rats of this age and strain. - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Test item-related thyroid gland findings were noted in females treated at 450 mg/kg bw/day. Increased incidence and severity of follicular cell hypertrophy was present in the thyroid glands at increased incidence in females treated at 450 mg/kg bw /day up to slight degree (see Table 4).
Based on the low severity, which was within background severity for this finding, and the lack of organ weight changes in the thyroid gland this was considered non-adverse. - Histopathological findings: neoplastic:
- not examined
- Other effects:
- not examined
Maternal developmental toxicity
- Number of abortions:
- no effects observed
- Description (incidence and severity):
- A summary of maternal performance including numbers of pregnant and non-pregnant animals is given in Table 5. No effects of treatment were noted up to and including 450 mg/kg bw/day.
- Pre- and post-implantation loss:
- effects observed, treatment-related
- Description (incidence and severity):
- At 450 mg/kg bw/day, the mean % of total resorptions and the mean % post-implantation loss per litter were increased (11.2% vs concurrent control value of 3.9% for both). This value was outside the historical control range. The increase in post-implantation loss at 450 mg/kg bw/day was mainly attributed to one female, which had total post-implantation loss (11 early resorptions, 11 corpora lutea). However, post-implantation loss was still somewhat increased after excluding values of this female from calculations (6.3% vs concurrent control value of 3.9%, respectively).
- Total litter losses by resorption:
- effects observed, treatment-related
- Description (incidence and severity):
- At 450 mg/kg bw/day one female had resorptions only. All other pregnant females had litters with viable fetuses.
- Early or late resorptions:
- effects observed, treatment-related
- Description (incidence and severity):
- At 450 mg/kg bw/day, the mean % of early resorptions per litter was increased (10.6% vs concurrent control value of 3.9%), although not reaching statistical significance. This value was outside the historical control range. The increase in early resorptions was mainly attributed to one female, which had total post-implantation loss (11 early resorptions, 11 corpora lutea). However, percent of early resorptions was still somewhat increased after excluding values of this female from calculations (5.6% vs concurrent control value of 3.9%, respectively).
- Dead fetuses:
- no effects observed
- Description (incidence and severity):
- No dead fetuses were noted in any dose group.
- Changes in pregnancy duration:
- effects observed, treatment-related
- Description (incidence and severity):
- One high dose female was suspected of delivering her litter early on Day 20 post-coitum although no pups were found. This suspicion is based on an observation taken from the study day book and the marked body weight loss observed for this female between Day 18 and Day 21 post-coitum (i.e. 52 g). Pups missing were most likely cannibalised. From post-coitum Day 18-20, this female was noted with piloerection and at necropsy, pale discolouration of her whole body was noted. At necropsy, one late resorption and 10 early resorptions were recorded; however, since this female was suspected to have had an early delivery, the early resorptions could have been empty implantation sites.
As an early delivery at Day 20 post-coitum is rare for rats a relation with the test item could not be excluded. - Changes in number of pregnant:
- no effects observed
- Description (incidence and severity):
- Four females were not pregnant: two females in the control group, one female at 150 mg/kg bw/day and one female at 450 mg/kg bw/day. These cases of non-pregnancy were considered unrelated to the test item as it occurred in the same incidence in the treated as in the vehicle control group.
- Other effects:
- not examined
Effect levels (maternal animals)
open allclose all
- Key result
- Dose descriptor:
- NOAEL
- Remarks:
- general toxicity
- Effect level:
- 50 mg/kg bw/day
- Based on:
- test mat.
- Basis for effect level:
- body weight and weight gain
- food consumption and compound intake
- organ weights and organ / body weight ratios
- Key result
- Dose descriptor:
- NOAEL
- Remarks:
- developmental toxicity
- Effect level:
- 150 mg/kg bw/day
- Based on:
- test mat.
- Basis for effect level:
- early or late resorptions
- pre and post implantation loss
Results (fetuses)
- Fetal body weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Mean male and female fetal body weights were significantly reduced at 450 mg/kg bw/day (relative difference to concurrent controls: -7 and -6%, respectively), as well as mean combined fetal body weights (relative difference to concurrent controls -6%). Values were below the lower limit of the historical control range*. Even though the difference was only 6%, fetal body weights have a small variation in general and as mean values at 450 mg/kg bw/day were below the lower limit of the historical control range, these findings were considered toxicologically relevant.
Mean female and combined (male and female) fetal weights were slightly higher (relative difference to concurrent controls: 4% for both) at 50 mg/kg bw/day. Taking into consideration the direction (increase) of the effect and since values were within the historical control range of the Test Facility, this finding was considered not toxicologically relevant (for details see Table 6).
* HCD of fetal examinations are presented in Attachment "Historical Control Data of Fetal Examinations" under Attached Background Information. - Reduction in number of live offspring:
- no effects observed
- Description (incidence and severity):
- Mean litter sizes as well number of live offspring were considered unaffected by treatment up to 450 mg/kg bw/day (for details see Table 6).
- Changes in sex ratio:
- no effects observed
- Description (incidence and severity):
- The male:female ratio was unaffected by treatment up to 450 mg/kg bw/day.
- Changes in litter size and weights:
- no effects observed
- Description (incidence and severity):
- Mean litter sizes were considered unaffected by treatment up to 450 mg/kg bw/day.
- Changes in postnatal survival:
- not examined
- External malformations:
- no effects observed
- Description (incidence and severity):
- There were no treatment-related effects on external morphology following treatment up to 450 mg/kg bw/day.
The only fetus with an external finding was one mid dose fetus. This fetus had exencephaly and open eyes. Skeletal examination substantiated the skull finding and revealed small mandibles and anomalies of the sternum, vertebrae and ribs. Due to the single occurrence in the mid dose group, these malformations were considered to be of spontaneous origin.
External variations were not seen in any group.
For a summary of fetal malformations and variations (absolute numbers and percentages) see Attachment "Summary of fetuses and litters with malformations and variations" under Attached Background Material. - Skeletal malformations:
- effects observed, treatment-related
- Description (incidence and severity):
- The number of fetuses with bent ribs was statistically significant higher at 450 mg/kg bw/day. Mean litter incidences of this variation were 10.9%, 8.9%, 15.7% and 29.8% per litter in the control, 50, 150 and 450 mg/kg bw/day groups, respectively and the high dose incidence was above the historical control maximum value of 27.4% per litter. Bent ribs represent a common finding in prenatal toxicity studies with a large variety of compounds (but are rarely observed in untreated controls) and the underlying cause is most likely an interference with the chondrification and ossification process of the ribs. Various biologic activities may interfere with these processes and maternal toxicity seems to be an important causative factor for bent ribs. Regardless of the cause, as it has been demonstrated previously that bent ribs are completely reversible, this finding was considered not adverse.
Several ossification parameters indicated reduced ossification at 450 mg/kg bw/day compared to the control group. Increased incidences were observed for unossified metacarpals and metatarsals (18.7% versus 12.0% per litter), reduced ossification of the skull (11.9% versus 5.8 % per litter) and reduced ossification of vertebral centra (4.3% versus 0.0% per litter). None of these increases was statistically significant and only the incidence of unossified metacarpals and metatarsals was above the historical control value (17.6% per litter)*. The delayed skeletal ossification appears to be associated with impaired fetal growth as shown by the body weights of fetuses with unossified metacarpals and metatarsals in both the control and 450 mg/kg bw/day group. Mean weights of both affected control (4.7 g) and 450 mg/kg (4.6 g) fetuses were below their group mean values (5.2 and 4.9 g, respectively).
Noteworthy in the 150 mg/kg bw/day group, is the variation of unossified metacarpals and metatarsals that occurred at a statistically significant lower incidence compared to control Group. Mean litter incidences of this finding were 12.0%, 5.6%, 2.3% and 18.7% per litter in the control, 50, 150 and 450 mg/kg bw/day groups, respectively. Because there was no dose-response for this finding, the low value at the mid-dose level was considered a chance finding.
The other variations that were noted occurred in the absence of a dose-related incidence trend, infrequently and/or at frequencies that were within the range of available historical control data*. Therefore, they were considered not treatment-related.
Three skeletally malformed fetuses were observed in this study. Besides the mid dose fetus with exencephaly, small mandibles and anomalies of the sternum, vertebrae and ribs that was described above, another mid dose fetus and one low dose fetus had a vertebral anomaly with or without associated rib anomaly. Due to the low incidence and absence of a dose-relationship, these malformations were considered not treatment-related.
For a summary of fetal malformations and variations (absolute numbers and percentages) see Attachment "Summary of fetuses and litters with malformations and variations" under Attached Background Material.
* HCD of fetal examinations are presented in Attachment "Historical Control Data of Fetal Examinations" under Attached Background Information. - Visceral malformations:
- no effects observed
- Description (incidence and severity):
- There were no treatment-related effects on visceral morphology following treatment up to 450 mg/kg bw/day.
The only visceral malformation was observed at cephalic examination of one high dose fetus. This fetus had a small right eye and as it occurred singly and was seen previously among historical control fetuses, it was considered not to be treatment-related.
Only two visceral variations (small supernumerary liver lobes and convoluted ureter) were observed in this study and at the low incidence these occurred, they were not considered to be related to treatment.
For a summary of fetal malformations and variations (absolute numbers and percentages) see Attachment "Summary of fetuses and litters with malformations and variations" under Attached Background Material. - Other effects:
- no effects observed
- Description (incidence and severity):
- The anogenital distance before and after correction for body weight was unaffected by treatment up to 450 mg/kg bw/day (see Table 7).
Effect levels (fetuses)
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 150 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- fetal/pup body weight changes
Overall developmental toxicity
- Key result
- Developmental effects observed:
- yes
- Lowest effective dose / conc.:
- 450 mg/kg bw/day
- 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:
- not specified
Any other information on results incl. tables
Table 1. Maternal food consumption (g/animal/day)
Post coitum |
|
Dose level (mg/kg bw/day) |
|||
|
|
0 (control) |
50 |
150 |
450 |
Days 2- 6 |
Mean |
20 |
23 |
21 |
21 |
|
SD |
2.4 |
9.8 |
1.7 |
2.5 |
|
N |
20 |
22 |
21 |
21 |
|
|
|
|
|
|
Days 6 -9 |
Mean |
19 |
18 |
15** |
12** |
|
SD |
2.6 |
2.1 |
2.6 |
2.1 |
|
N |
20 |
22 |
21 |
21 |
|
|
|
|
|
|
Days 9-12 |
Mean |
20 |
19 |
17** |
14** |
|
SD |
2.2 |
1.8 |
2.8 |
3.8 |
|
N |
20 |
22 |
21 |
21 |
|
|
|
|
|
|
Days 12-15 |
Mean |
21 |
21 |
20 |
19 |
|
SD |
2.4 |
2.2 |
2.4 |
2.1 |
|
N |
20 |
22 |
21 |
20 |
|
|
|
|
|
|
Days 15 -18 |
Mean |
23 |
23 |
22 |
21 |
|
SD |
1.9 |
2.5 |
2.5 |
2.9 |
|
N |
20 |
22 |
21 |
20 |
|
|
|
|
|
|
Days 18-21 |
Mean |
21 |
21 |
20 |
19 |
|
SD |
3.1 |
2.3 |
3.4 |
3.9 |
|
N |
20 |
22 |
21 |
20 |
Mean of means |
|
20 |
21 |
19 |
18 |
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**)
|
|||||
Table 2. Maternal thyroid hormone determination – Pregnant females
|
|
Dose level (mg/kg bw/day) |
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
0 (control) |
50 |
150 |
450 |
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TSH (µIU/mL) |
Mean |
0.384 |
0.479 |
0.930 |
2.369** |
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
SD |
0.186 |
0.231 |
0.715 |
1.696 |
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
N |
20 |
22 |
21 |
19 |
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
|
|
|
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Total T3 (ng/dL) |
Mean |
61.1 |
53.6 |
42.4 |
53.6 |
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
SD |
9.3 |
10.1 |
-- |
-- |
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
N# |
17 |
13 |
1 |
1 |
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
|
|
|
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Total T4 (µg/dL) |
Mean |
2.12 |
2.37 |
1.55* |
1.86 |
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
SD |
0.65 |
0.53 |
0.48 |
0.96 |
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
N## |
19 |
19 |
19 |
5 |
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) # Missing values were below the detection limit of 40 ng/dL ## Missing values were below detection limit of 1.00 µg/dL Historical control data for thyroid hormone analyses in pregnant Wister Han rats (period 2018-2019): TSH mean = 0.440, P5–P95 = 0.195–0.819 (n=30) Total T3 mean = 66.1, P5–P95 = 51.0–84.5 (n=28) Total T4 mean = 2.08, P5–P95 = 1.47–3.03 (n=29) Table 3. Maternal organ weights (g) – Pregnant females
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Applicant's summary and conclusion
- Conclusions:
- Timed-pregnant Wistar rats were treated with the test item, reaction mass of diisopropyl-1,1’-biphenyl and tris-(1-methylethyl)-1,1’biphenyl, by daily oral gavage from gestation Days 6 up to and including Day 20. Groups of 22 females each received the test item formulated in corn oil at a volume of 4 mL. No test item-related mortality was noted during the study. Piloerection was observed in 8/22 females at 450 mg/kg bw/day on two or more consecutive days, mainly at the end of the treatment period and in a single female at 150 mg/kg bw/day on the last day of treatment. Maternal toxicity was observed at 450 mg/kg bw/day, consisting of body weight loss observed in approximately half of the females between post-coitum Day 6-9, followed by statistically significant reduced body weight gain throughout the treatment period. The observed body weight effects coincided with statistically significant reduced food consumption at 450 mg/kg bw/day between post-coitum Day 6-12.
Macroscopic evaluation revealed enlarged livers in females treated at 450 mg/kg bw/day. Moreover, a test item-related increase in liver weights was observed at all dose levels (up to 1.18, 1.36 and 1.84x compared to concurrent controls at 50, 150 and 450 mg/kg bw/day, respectively for relative liver weights). Also, in line with these findings are the increased ALAT and ASAT values at 450 mg/kg bw/day and the increased ALAT value at 150 mg/kg bw/day. Taking into account the magnitude of the effect on the liver weights and the increased liver enzymes at 150 and 450 mg/kg bw/day, adversity of these finding could not be excluded. In the absence of increased liver enzymes at 50 mg/kg bw/day and as the increase in liver weights was less than 20 percent, the liver findings at 50 mg/kg bw/day were considered non-adverse.
A marked increase in serum levels of TSH was observed for high dose females and a similar, not statistically significant trend was observed in mid dose females. Additionally, a decrease in total T4 and total T3 was observed in mid and high dose females. The observed decrease in total T3/T4 together with the increase in TSH is likely related to the increased liver weights observed at 150 and 450 mg/kg bw/day and the non-adverse follicular cell hypertrophy in the thyroid glands that was noted at increased incidence and severity at 450 mg/kg bw/day. Possible adversity of the effects on thyroid hormones could not be assessed within this type of study and was therefore not taken into account when determining the parental NOAEL.
At 450 mg/kg bw/day, one female was suspected of delivering her litter early on Day 20 post-coitum although no pups were found. As an early delivery at Day 20 post-coitum is rare for rats a relation with the test item could not be excluded.
A test item-related increase in the mean % of early resorptions, and consequently % total resorptions and % post-implantation loss per litter was observed at 450 mg/kg bw/day.
Mean male and female fetal body weights were significantly reduced at 450 mg/kg bw/day.
Skeletal examination showed an increase in the number of high dose fetuses with bent ribs. Additionally, several ossification parameters indicated reduced ossification at 450 mg/kg bw/day compared to the control group. These variations were considered not a direct effect of the test item but secondory the the observed lower fetal weights.
No treatment-related toxicologically significant changes were noted in any of the remaining developmental parameters investigated in this study (i.e. litter size, sex ratio, (corrected) anogenital distance, external, visceral malformations and developmental variations).
Based on the above reported findings the no-observed-adverse-effect level (NOAEL) for maternal general toxicity was considered to be 50 mg/kg bw/day, the NOAEL for developmental and embryo-fetal toxicity was considered to be 150 mg/kg bw/day.
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