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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:
12 February 2021 to 15 April 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
OECD guideline no. 414 “Prenatal Developmental Toxicity Study” of 25 June 2018.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline
EC Number:
233-215-5
EC Name:
4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline
Cas Number:
10081-67-1
Molecular formula:
C30H31N
IUPAC Name:
4-(2-phenylpropan-2-yl)-N-[4-(2-phenylpropan-2-yl)phenyl]aniline
Test material form:
solid
Details on test material:
Test substance name: 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline
Scymaris reference number: 1108TS001
CAS number: 10081-67-1
Batch/Lot number: EL0D14H276
Purity: 97%
Physical state and appearance: White to off-white powder
Expiry/re-test date: 14 April 2023
Storage conditions: Room Temperature
Specific details on test material used for the study:
No further details specified in the study report.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
Animal supply and acclimatisation
A total of 116 Sprague Dawley SD virgin female rats, 9 weeks old (200-225 g) were ordered from Charles River Italia S.p.A., Calco (Lecco), Italy. The male rats used were from the same supplier, and were at least 11 weeks old (at least 350 g). After arrival the weight range was determined and the females were uniquely identified by tattoo on the hind feet. A health check was then performed by a veterinarian. An acclimatisation period of approximately 2 weeks was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations.

Animal husbandry
The rats were housed in a limited access rodent facility. Animal room controls were set to maintain temperature and relative humidity at 22°C ± 2°C and 55% ± 15% respectively; actual conditions were monitored, recorded and the records retained. No relevant deviations from these ranges were recorded during the study. There were approximately 15-20 air changes per hour and the rooms were lit by artificial light for 12 hours each day.
Before mating for all animals and after mating for males, the animals were housed no more than 5 of one sex to a cage in clear polysulfone cages measuring 59.5×38×20 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). During the mating period, one male rat was housed with one female rat in clear polysulfone cages measuring 42.5×26.6×18 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese) with a stainless steel mesh lid and floor. Each cage tray held absorbent material which was inspected and changed daily. After mating, the mated females were housed in clear polysulfone cages measuring 42.5×26.6×18 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese).
Nesting material was provided inside suitable bedding bags. In addition, suitable nesting material (Scobis 0Mucedola) was provided as necessary. Nesting material was changed at least 2 times a week.
Drinking water was supplied ad libitum to each cage via water bottles.
A commercially available laboratory rodent diet (4 RF 21,Mucedola S.r.l., Via G. Galilei 4, 20019 SettimoMilanese (MI), Italy) was offered ad libitum throughout the study.
There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet. Records of analyses of water and diet are kept on file at ERBC.
Dated and signed records of activities relating to the day to day running and maintenance of the study in the animal house were recorded.

Allocation to groups
On the day of allocation (Day 0 post coitum) all females were weighed and allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights. Each female was identified within the study by ear notch and housed no more than 5 per cage.
The cages were identified by a label recording the study number, animal numbers and details of treatment. The arrangement of cages in batteries was such that cages from each treatment group were evenly distributed across the battery to minimise possible environmental effects.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5%
Details on exposure:
The required amount of the test item was suspended in the vehicle to reach the required concentrations of 5, 25 and 100 mg/mL. The formulation was prepared up to weekly interval based on the stability data measurements. Concentrations were calculated and expressed in terms of test item as supplied.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis was performed in a separate study in order to validate the analytical method and the formulation procedure and to verify the stability of the formulations (ERBC Study no. A4101). A 28 hour stability at room temperature and a 8 day stability at +4°C were verified in the range from 5 to 100 mg/mL.
Samples of the formulations prepared during the current study (the first and the last week of treatment) were analysed to check the homogeneity and concentration. The results were within the acceptability limits stated in ERBC protocols for suspensions.
Details on mating procedure:
The females were paired with male rats. Females were paired one to one in the home cage of the male and left overnight. Vaginal smears were taken daily in themorning from the day after pairing until a positive identification of mating was made. The day of mating, as judged by the presence of spermin the vaginal smear or by the presence of a copulation plug, was considered as Day 0 of gestation (or Day 0 post coitum). Full mating records were maintained.
On the day of allocation (Day 0 post coitum) all females were weighed and allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights. Each female was identified within the study by ear notch and housed no more than 5 per cage.
The cages were identified by a label recording the study number, animal numbers and details of treatment. The arrangement of cages in batteries was such that cages from each treatment group were evenly distributed across the battery to minimise possible environmental effects.
Duration of treatment / exposure:
All animals were dosed from Day 3 through Day 19 post coitum.
Frequency of treatment:
All animals were dosed once a day.
Duration of test:
20 days
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control Level
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Low Level
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
Medium Level
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High Level
No. of animals per sex per dose:
Each group comprised 24 mated female rats.
Control animals:
yes, concurrent vehicle
Details on study design:
Choice of the species, administration route and dose levels
The Sprague Dawley SD rat was the species and strain of choice because it is accepted by many regulatory authorities and there are ample experience and background data on this species and strain.
The oral route was selected as it is a possible route of exposure of the test item in man.
Dose levels were selected by the Sponsor.

Administration of test item
The test item was administered orally by gavage at a dose volume of 10 mL/kg body weight.
Control animals received the vehicle alone at the same dose volume. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.

Examinations

Maternal examinations:
Mortality
Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.

Clinical signs
All clinical signs were recorded for individual animals. Each animal was observed daily and any clinical signs recorded starting from allocation until sacrifice.

Body weight
All animals were weighed on Days 0, 3, 6, 9, 12, 15, 18 and 20 post coitum.

Food consumption
Food consumption was measured on Days 3, 6, 9, 12, 15, 18 and 20 post coitum starting from Day 0 post coitum.

Terminal studies
Euthanasia
All animals at termination were killed by carbon dioxide inhalation on Day 20 post coitum and necropsied as detailed below. All foetuses were sacrificed by intraperitoneal injection of Sodium Thiopental followed by hypothermia.

Necropsy
The clinical history of the animal was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted and the abnormalities preserved in 10% neutral buffered formalin.

Organ weight, fixation and preservation
From all females completing the scheduled test period, the thyroid and the brain were weighed, fixed and preserved in 10% neutral buffered formalin. The thyroid weight was determined after fixation. The ratio of thyroid weight to brain weight was calculated for each animal.

Thyroid histopathological examination
After dehydration and embedding in paraffin wax, sections of the tissue were cut at 5 micrometre thickness and stained with haematoxylin and eosin. Sections were examined for evaluation of pathological changes.
Ovaries and uterine content:
The ovaries and uteri were examined to determine:
– Gravid uterine weight (not obtained from animals found dead or killed during the study);
– number of corpora lutea;
– number of implantation sites;
– number, sex and weight of all live foetuses;
– number and sex of dead foetuses (foetuses at termwithout spontaneous movements and breathing);
– number of intra-uterine deaths;
– gross evaluation of placentae.

Intra-uterine deaths were classified as:
– early resorptions: only placental remnants visible.
– late resorptions: placental and foetal remnants visible.
Uteri or individual uterine horns without visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of embryonic death at very early stages of implantation.
Blood sampling:
On Day 20 post coitum, blood samples for thyroid hormones determination (approximately 1 mL), were collected, randomizing (equalised) between treatment groups, from the sublingual vein of all females, under slight isoflurane anaesthesia. This procedure was performed within a short time frame (e.g. two hours, if possible) on the morning of the day of necropsy.
Samples were transferred into tubes containing no anticoagulant and centrifuged at room temperature. The serum obtained was divided in two aliquots (300 µL in the aliquot A, the remaining in the aliquot B, if possible) and stored at -20°C, pending analysis.

Immunoanalysis was carried out by the Test Site, BioVetim (Study Phase number: BIOVX1610),
according to immunoanalytical methods validated at the Test Site:
– RV-T3/R/S-BKM/RIA-002 for T3
– RV-T4/R/S-BKM/RIA-002 for T4
– RV-TSH/R/S-IZO/RIA-002 for TSH
Samples were assayed to determine the serum levels of Total triiodothyronine (total T3), Total thyroxine (total T4) and Thyroid stimulating hormone (TSH) by RadioImmunoAssay (RIA).
Total number of samples: 96 × 2 aliquots
The aliquots were maintained at -20°C until transfer to the Test Site indicated below, where the immunoanalysis was carried out:

BIOVETIM
VetAgro Sup, aile 1 niveau 3
1 avenue Bourgelat
69280Marcy l’Etoile
France

Prior to shipment, the Principal Investigator was notified by e-mail.
Samples were labelled as follows: study number, animal number, group and sex, sampling date. Samples were transported in dry ice, by a dedicated courier and were subject to the latter’s standard insurance policy. In the first instance, only the aliquots A were despatched to the Test Site. The backup samples (aliquots B) will disposed of after Study Director’s authorisation and following Sponsor’s approval.
A Final Delegated Phase Report with the results of these analyses was sent to ERBC for inclusion in the Final Study Report. The results were presented as individual data (mean of duplicates, where applicable), group mean and group standard deviation. Evaluation of the data and the statistical analysis was performed in ERBC.
Fetal examinations:
Examination of foetuses
All live foetuses were examined externally. Approximately one-half of the foetuses (i.e., routinely, every second live foetus) in each litter was preserved in Bouin’s solution for subsequent fixed-visceral examination. The remaining foetuses were eviscerated after which the carcasses were fixed in 95% (v/v) ethanol for subsequent skeletal examination.
Skeletal and fixed-visceral examinationswere performed in all groups. Structural deviations were classified as follows:

Malformations
Major abnormalities that are rare and/or affect the survival or health of the species under investigation.

Anomalies
Minor abnormalities that are detected relatively frequently.

Variants
A change that occurs within the normal population under investigation and is unlikely to adversely affect survival or health. This might include a delay in growth or morphogenesis that would have otherwise followed a normal pattern of development.

Anogenital distance (AGD):
The AGD of each live foetuses was measured on Day 20 post coitum. The AGD was normalized to the cube root of foetal body weight collected on Day 20 post coitum. Individual and mean data was reported.
Statistics:
For continuous variables the significance of the differences amongst group means was
assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
Statistical analysis of non-continuous variables was carried out by means of the Kruskal-
Wallis test and intergroup differences between the control and treated groups assessed by a
non-parametric version of theWilliams test. The criterion for statistical significance was
p<0.05. The mean values, standard deviations and statistical analysis were calculated from
the actual values in the computer without rounding off.
Indices:
Pre-implantation loss was calculated as a percentage from the formula:

Pre impl. Loss % = [(no. of corpora lutea – no. of implantations) / no. of corpora lutea] x 100

Post-implantation loss was calculated as a percentage from the formula:

Post impl. Loss% = [(no. of implantations – no. of foetuses) / no. of implantations] x 100

Total implantation loss was calculated as a percentage from the formula:

Total impl. Loss % = [(no. of corpora lutea – no. of live foetuses) / no. of corpora lutea] x 100

Sex ratios of the foetuses were calculated as the percentage of males.
All derived values (e.g., means, percentages, ratios) first were calculated within the litter and the group values derived as a mean of individual litter values. Foetal structural deviations were expressed as the percentage of affected foetuses relative to all foetuses examined per group, as well as in terms of the mean litter percentage of affected litters.
Historical control data:
No specified 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 treatment-related clinical signs were detected throughout the study. A palpable subcutaneous mass was recorded in one lowdose female (no. 73) during the last day of study. This formation did not show microscopically changes. Other signs like hairloss or scabs were occasionally recorded during the study in single animals, with no correlation with the dose.
Thus they were considered as minor clinical signs and not related to the administration with the test item.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
All females survived to the final sacrifice on gestation Day 20 and were found pregnant with the exception of two controls (no. 31, 33) and one low dose female (no. 81).
The final number of females with live foetuses on gestation day 20 was of:
– 22 in the control group (0 mg/kg/day)
– 23 in the low dose group (50 mg/kg/day)
– 24 in the mid-dose group (250 mg/kg/day)
– 24 in the high dose group (1000 mg/kg/day)
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No treatment-related effects were seen in maternal body weight/body weight gain throughout the study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No treatment-related effects were seen in food consumption throughout the study.
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:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
no effects observed
Description (incidence and severity):
Serum levels of T3, T4 and TSH did not differ between treated and control groups.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Body weight at termination, uterus weight and the absolute weight gain of females were unaffected by treatment.
No effects were seen in thyroid weight between treated and control groups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
At post mortem examination, no treatment-related macroscopic observations were noted in treated females receiving test item at ≥ 50 mg/kg/day, when compared to controls. Any macroscopic observations were within the range of expected spontaneous findings in SD rats of the same age considered incidental and unrelated to the test item.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment-related changes were noted in thyroid gland of females receiving 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline at ≥ 50 mg/kg/day, when compared to controls. The microscopic observations in control and treated females were consistent with those known to occur spontaneously in untreated SD rats of the same age and were considered incidental and unrelated to treatment.
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
not specified
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no differences in the number of implantations, corpora lutea and the intrauterine
deaths between control and treated groups.
Total litter losses by resorption:
not examined
Early or late resorptions:
not examined
Dead fetuses:
no effects observed
Description (incidence and severity):
The number of live foetuses, the mean litter and body weights and the percentage of males respect to females did not differ between control and treated groups.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The number of live foetuses, the mean litter and body weights and the percentage of males respect to females did not differ between control and treated groups.
Details on maternal toxic effects:
Treatment with the test item did not cause changes in body weight or food consumption at all dose levels when compared to controls throughout the study. In addition, the animals did not showtreatment-related clinical signs. The serumlevels of TSH, T3 and T4 hormones were comparable between control and treated groups. No treatment-related changes were noted at necropsy of females or at thyroid weight. The microscopic analysis of thyroid did not reveal findings at any dose level.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
changes in number of pregnant
clinical signs
dead fetuses
food consumption and compound intake
gross pathology
histopathology: neoplastic
maternal abnormalities
mortality
necropsy findings
organ weights and organ / body weight ratios
pre and post implantation loss

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The body weights did not differ between control and treated groups.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The number of live foetuses did not differ between control and treated groups.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The the percentage of males respect to females did not differ between control and treated groups.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The mean litter and body weights did not differ between control and treated groups.
Changes in postnatal survival:
not specified
External malformations:
no effects observed
Description (incidence and severity):
No treatment-related findings were recorded at external examination of foetuses. One single small foetus (body weight below 2.7g) was found in Group 3 as abnormality.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Minor abnormalities or variations occurred in all groups and included for example altered ossification (asymmetrical, incomplete or no ossification) of several bones of the skull, sternebrae, thoracic/sacral vertebrae and the presence supernumerary ribs (14th). The incidence of the affected litters in treated groups was similar or even lower than observed in the control group or without dose relation. Therefore, the findings were considered unrelated to treatment.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
A total of three major abnormalities were found in the study. Two were related to the heart (incomplete septum and/or persistent truncus arteriosus) and observed in one control foetus (foetus no. 10; Dam no. 9) and in one foetus of group 3 (foetus 10; Dam no. 125).
The other major abnormality was related to brain (extreme enlarged lateral ventricles) and it was found in one foetus of group 3 (Dam no. 139). These malformations occurred in single instances and in the absence of a dose dependency. Therefore, they were considered incidental and unrelated to treatment.
The incidences of foetuses or litters with minor abnormalities or variations did not suggest any test item effect.
The overall data confirm that there were no treatment-related findings at fixed visceral examination of foetuses.
Details on embryotoxic / teratogenic effects:
The number of corpora lutea, implantations, viable foetuses, intrauterine deaths and sex ratios were comparable between control and treated groups. No treatment-related effects were seen in the mean foetal weight. The external examination of foetuses including the measurement of the anogenital distance did not show differences in treated animals at all dose levels when compared to the control group.
No treatment-related findings were noted at skeletal and visceral examinations. Indeed, no evidence of teratogenicity was noted up to and including the dose level of 1000 mg/kg/day.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
changes in sex ratio
fetal/pup body weight changes
changes in litter size and weights
external malformations
skeletal malformations
visceral malformations

Overall developmental toxicity

Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
no

Any other information on results incl. tables

SUMMARY OF REPRODUCTION DATA – GROUP DATA
























 



Dosage Levels


mg/kg/day



Observations



0



50



250



1000



No. Dams Inseminated


No. Dams That Conceived


Percent Dams Conceived


No. Dams Died During Study


No. Dams with Resorptions only


No. of Litters


Mean Absolute Weight Gain


Total No. Corpora Lutea


Mean No. Corpora Lutea/Pregnancy


Total No. Implantation


Mean No. Implants/Pregnancy


Total No. Live Fetuses


Mean No. Live Fetuses/Pregnancy


Total No. Dead Fetuses


Mean No. Dead Fetuses/Pregnancy


No. Dams with Resorptions


Total No. Resorptions


Mean No. Resorptions/Pregnancy


Preimplantation Loss (%)


Post Implantation Loss (%)


Mean Live Foetal Weight (g)



24


22


91.7


0


0


22


66.9


326


14.8


320


14.5


317


14.4


0


0.0


3


3


0.1


2.1


1.0


3.97



24


23


95.8


0


0


23


63.6


345


15.0


339


14.7


333


14.5


0


0.0


5


6


0.3


1.7


1.8


3.96



24


24


100.0


0


0


24


80.5


349


14.5


347


14.5


336


14.0


0


0.0


4


11


0.5


0.8


3.2


4.03



24


24


100.0


0


0


24


63.2


368


15.3


362


15.1


357


14.9


0


0.0


4


5


0.2


1.4


1.3


3.98



 


CLINICAL SIGNS OF FEMALES – GROUP INCIDENCE



































Interval: 0 – 20 Days


Group


Observation



1


(24)



2


(24)



3


(24)



4


(24)



 



A



B



A



B



A



B



A



B



APPERANCE


Scab(s)


Presence if palpable mass



 


2


0



 


8.3


0.0



 


1


1



 


4.2


4.2



 


0


0



 


0.0


0.0



 


0


0



 


0.0


0.0



Key:


() = Number of animals alive at start of interval


A = Number of animals affected


B = Percent of animals with observations during interval


 


BODY WEIGHT (g) OF PREGNANT FEMALES – GROUP DATA





































































Group(s)



 



Day of Phase



0!



3”



6



9



12



15



18



20



1



(n)


Mean


SD



22


274.20


19.12



22


291.48


19.07



22


301.78


20.33



22


317.60


21.07



22


336.15


22.98



22


355.70


23.20



22


399.69


28.91



22


436.66


31.84



2



(n)


Mean


SD



23


272.39


12.58



23


285.38


12.44



23


300.41


13.42



23


312.48


13.97



23


331.93


14.84



23


351.01


17.37



23


392.40


20.65



23


430.58


24.53



3



(n)


Mean


SD



24


272.90


16.51



24.


294.75


18.34



24


308.83


17.96



24


323.23


18.55



24


342.84


19.29



24


363.27


22.68



24


409.54


26.36



24


448.51


30.88



4



(n)


Mean


SD



24


273.76


12.25



24


292.71


15.88



24


303.76


14.66



24


316.81


14.18



24


334.43


17.86



24


355.05


18.86



24


397.54


24.80



24


434.91


30.01



Note: ! = Gestation phase; “ = Dosing/Gestation phase


* = mean value of group is significantly different from control at p < 0.05


** = mean value of group is statistically different from control at p < 0.01


Statistical analysis: Dunnett’s test if group variances are homogenous; Modified t test if group variances are inhomogenous ($)


 


BODY WEIGHT GAIN PER DAY° (g) OF PREGNANT FEMALES – GROUP MEAN DATA
































































Group(s)



 



Day of Phase



3



6



9



12



15



18



20



1



(n)


Mean


SD



22


5.762


2.126



22


3.431


2.144



22


5.274


2.985



22


6.184


1.561



22


6.518


1.651



22


14.662


2.579



22


18.485


2.957



2



(n)


Mean


SD



23


4.333


2.875



23


5.008


2.997



23


4.024


1.611



23


6.484


1.554



23


6.359


1.548



23


13.798


2.233



23


19.091


3.190



3



(n)


Mean


SD



24


7.284


1.749



24


4.6914


2.049



24


4.801


1.795



24


6.539


1.735



24


6.810


2.109



24


15.422


2.629



24


19.485


3.184



4



(n)


Mean


SD



24


6.319


2.705



24


3.683


1.848



24


4.350


1.814



24


5.871


2.015



24


6.875


1.897



24


14.162


2.729



24


18.685


3.493



Note: Data for Dosing/Gestation phase


* = mean value of group is significantly different from control at p < 0.05


** = mean value of group is statistically different from control at p < 0.01


Statistical analysis: Dunnett’s test if group variances are homogenous; Modified t test if group variances are inhomogenous ($)


°= mean daily body weight gain over the previous period starting from gestation day 0


 


FOOD CONSUMPTION° (g/animal/day) OF FEMALES – GROUP MEAN CAGE DATA
































































Group(s)



 



Day of Phase



3



6



9



12



15



18



20



1



(n)


Mean


SD



9


22.47


2.50



9


25.08


1.70



9


26.59


1.99



9


26.85


1.89



9


27.61


2.53



9


29.97


3.20



9


30.58


2.99



2



(n)


Mean


SD



9


20.57


4.56



9


25.43


1.92



9


26.39


1.64



9


28.04


2.14



9


27.84


3.29



9


30.09


2.49



9


30.54


3.98



3



(n)


Mean


SD



9


23.96


1.37



9


25.99


1.70



9


27.30


1.28



9


27.94


1.38



9


29.05


1.79



9


31.70


2.06



9


32.29


2.58



4



(n)


Mean


SD



9


23.66


1.19



9


25.28


1.33



9


26.54


1.65



9


27.28


2.56



9


28.30


1.89



9


30.65


2.80



9


31.29


2.03



Note: Data for Dosing/Gestation phase


* = mean value of group is significantly different from control at p < 0.05


** = mean value of group is statistically different from control at p < 0.01


Statistical analysis: Dunnett’s test if group variances are homogenous; Modified t test if group variances are inhomogenous ($)


°= food consumed over the previous period starting from Day 0 post coitum


 


TERMINAL BODY WEIGHT, GRAVID UTERUS WEIGHT AND ABSOLUTE WEIGHT GAIN (g) OF PREGNANT FEMALES – GROUP MEAN DATA









































Group



 



Terminal body weight


(g)



Gravid uterus weight


(g)



Absolute weight gain#


(g)



1



Mean


SD


N



427.77


32.18


22



86.72


15.79


22



66.85


18.32


22



2



Mean


SD


N



423.44


25.60


23



87.50


13.16


23



63.56


24.12


23



3



Mean


SD


N



439.63


30.86


24



86.28


20.79


24



80.45*


16.53


24



4



Mean


SD


N



426.77


30.71


24



89.84


17.13


24



63.18


19.95


24



# = Body weight at necropsy minus gravid uterus weight, minus body weight at Day 0 of pregnancy


* = mean value of group is significantly different from control


Statistical analysis: Kruskall Wallis test; Williams test if group means are different from control at p < 0.05


 


THYROID HORMONE DETERMINATION ON DAY 20 POST COITUM – GROUP MEAN DATA






































Parameter/units



Group



1



2



3



4



Triiodothyronine


nmol/L



Mean


SD


n



1.310


0.185


24



1.298


0.190


24



1.276


0.174


24



1.287


0.205


24



Thyroxine


nmol/L



Mean


SD
n



20.4


4.2


24



19.8


4.1


24



19.0


1.9


24



18.5


1.9


24



THYROID STIMULATING HORMONE


ng/ml



Mean


SD


n



7.93


1.79


24



7.49


1.31


24



7.58


1.81


24



7.12


1.25


24



Controls from group(s): 1           Subgroup(s): 1


* = mean value of group is significantly different from control at p < 0.05


+ = mean value of group is significantly different from control at p < 0.01


 


LITTER DATA AND SEX RATIO OF FEMALES – GROUP MEAN DATA



















































































































Group



 



Corpora Lutea



Implan-tations



Uterine Deaths



Viable Young



% males



Implantation loss (%)



Litter Weight (g)



Mean Foetal Weight (g)



By sex



Combined



Early



Late



Total



Total



M



F



Pre



Post



Total



M



F



1



Mean


SD


(n)



14.82


2.40


22



14.55


2.67


22



0.14


0.35


22



0.00


0.00


22



0.14


0.35


22



14.41


2.70


22



7.18


2.28


22



7.23


1.93


22



49.43


12.29


22



2.06


5.38


22



0.99


2.59


22



3.04


5.60


22



57.27


11.74


22



4.09


0.21


22



3.85


0.23


22



3.97


0.21


22



2



Mean


SD


(n)



15.00


2.30


23



14.74


2.30


23



0.22


0.52


23



0.04


0.21


23



0.26


0.54


23



14.48


2.33


23



6.87


2.01


23



7.61


1.90


23



47.26


11.23


23



1.67


3.92


23



1.76


3.59


23



3.41


5.10


23



57.30


9.46


23



4.05


0.29


23



3.89


0.22


23



3.96


0.24


23



3



Mean


SD


(n)



14.54


2.92


24



14.46


3.05


24



0.46


1.64


24



0.00


0.00


24



0.46


1.64


24



14.00


3.44


24



7.67


2.87


24



6.33


2.41


24



54.50


13.51


24



0.81


2.77


24



3.16


10.98


24



3.97


11.10


24



56.25


14.09


24



4.11


0.23


24



3.93


0.26


24



4.03


0.24


24



4



Mean


SD


(n)



15.33


3.13


24



15.08


2.95


24



0.21


0.51


24



0.00


0.00


24



0.21


0.51


24



14.88


2.91


24



7.71


2.61


24



7.17


2.51


24



51.94


13.65


24



1.41


3.32


24



1.31


3.21


24



2.70


4.48


24



58.96


10.91


24



4.07


0.21


24



3.90


0.20


24



3.98


0.19


24



* = mean value of group is significantly different from control


Statistical analysis: Kruskall Wallis test; William’s test of group means are different from control at p < 0.05


 


ANOGENITAL DISTANCE ON DAY 20 POST COITUM – GROUP MEAN DATA


MALES






































Parameter/units



Group



1



2



3



4



ANOGENITAL DISTANCE


(mm)



Mean


SD


n



4.30


0.26


22



4.24


0.30


23



4.36


0.28


24



4.37


0.25


24



Foetus weight


(g)



Mean


SD


n



4.10


0.21


22



4.05


0.29


23



4.11


0.23


24



4.07


0.21


24



ANOGENITAL DISTANCE ^


(NORMALISED)


mm/g1/3



Mean


SD


n



2.69


0.16


22



2.66


0.18


23



2.72


0.16


24



2.74


0.16


24



^ = normalised for the cube root of the body weight collected on Day 20 post coitum (mm/g)


Statistical analysis: Kruskall Wallis test; T test if group mean differences are different from control at p < 0.05


* = mean value of group is significantly different from control


 


ANOGENITAL DISTANCE ON DAY 20 POST COITUM – GROUP MEAN DATA


FEMALES






































Parameter/units



Group



1



2



3



4



ANOGENITAL DISTANCE


(mm)



Mean


SD


n



2.54


0.53


22



2.48


0.60


23



2.53


0.55


24



2.69


0.50


24



Foetus weight


(g)



Mean


SD


n



3.85


0.23


22



3.89


0.22


23



3.93


0.26


24



3.90


0.20


24



ANOGENITAL DISTANCE ^


(NORMALISED)


mm/g1/3



Mean


SD


n



1.62


0.35


22



1.58


0.38


23



1.60


0.34


24



1.71


0.32


24



^ = normalised for the cube root of the body weight collected on Day 20 post coitum (mm/g)


Statistical analysis: Kruskall Wallis test; T test if group mean differences are different from control at p < 0.05


* = mean value of group is significantly different from control


 


ABSOLUTE ORGAN WEIGHTS (g) – GROUP MEAN DATA


FEMALES


















Organ: Brain



Controls from group: 1



Data homogenous by Bartlett’s test (Dunnett’s test)



Group


Number/group


Mean


Standard deviation


Group diff. at p > 0.05


Group diff. at p < 0.01



Control


24


1.969


0.105



2


24


1.944


0.083


0.065


0.081



3


24


1.976


0.104


0.065


0.081



4


24


1.967


0.078


0.065


0.081



Analysis of variance: F ratio = 0.52         Df = 3/92           F probability = 0.677


Note: a * indicates group mean is significantly different from control at level of significance shown.


















Organ: Thyroid



Controls from group: 1



Data homogenous by Bartlett’s test (Dunnett’s test)



Group


Number/group


Mean


Standard deviation


Group diff. at p > 0.05


Group diff. at p < 0.01



Control


24


0.0241


0.0048



2


24


0.0241


0.0056


0.0031


0.0039



3


24


0.0222


0.0042


0.0031


0.0039



4


24


0.0215


0.0032


0.0031


0.0039



Analysis of variance: F ratio = 2.13         Df = 3/92           F probability = 0.100


Note: a * indicates group mean is significantly different from control at level of significance shown.


 


ORGAN WEIGHTS° TO BRAIN WEIGHT – GROUP MEAN DATA


FEMALES


















Organ: Thyroid



Controls from group: 1



Data homogenous by Bartlett’s test (Modified t test)



Group


Number/group


Mean


Standard deviation


Group diff. at p > 0.05


Group diff. at p < 0.01



Control


24


1.228


0.248



2


24


1.241


0.299


0.164


0.223



3


24


1.122


0.193


0.133


0.180



4


24


1.092


0.165


0.126*


0.171



Analysis of variance: F ratio = 2.49         Df = 3/92           F probability = 0.064


Note: a * indicates group mean is significantly different from control at level of significance shown.


° = expressed as % organ to brain weight ratio


 


MACROSCOPIC OBSERVATIONS OF FEMALES – FINAL SACRIFICE – GROUP INCIDENCE






































 



-- Females --



Group:


Number in group:



1


24



2


24



3


24



4


24



Skin


            Subcutaneous mass(es)



 


0



 


1



 


0



 


0



Uterus


            Not pregnant



 


2



 


1



 


0



 


0



Whole animal


            No abnormalities detected



 


22



 


22



 


24



 


24



 


EXTERNAL EXAMINATION OF FOETUSES – GROUP INCIDENCE






































































Group



Organ



Cat



Observation(s)



No. Foetuses



No. Litters



Observed



Affected



%



Observed



Affected



%



1



Whole foetus



 



No abnormalities detected



317



317



100.00



22



22



100.00



2



Whole foetus



 



No abnormalities detected



333



333



100.00



23



23



100.00



3



Whole foetus


Whole foetus



 


AN



No abnormalities detected


Small



336


336



335


1



99.70


0.30



24


24



23


1



95.83


4.17



4



Whole foetus



 



No abnormalities detected



357



357



100.00



24



24



100.00



 


SKELETAL EXAMINATION OF FOETUSES – GROUP INCIDENCE






































































Group



Organ



Cat



Observation(s)



No. Foetuses



No. Litters



Obs



Aff



%



Obs



Aff



%



1



Forepaw(s)


Forepaw(s)


Pelvic girdle


Ribs


Ribs


Ribs


Sacral vertebrae


Skull


Skull


Skull


Skull


Skull


Skull


Skull


Skull


Sternebrae


Sternebrae


Sternebrae


Sternebrae


Sternebrae


Sternebrae


Sternebrae


Thoracic vertebrae


Thoracic vertebrae


Thoracic vertebrae



AN


AN


AN


AN


AN


VA


AN


AN


AN


AN


VA


VA


VA


VA


VA


AN


AN


AN


VA


VA


VA


VA


AN


VA


VA



Metacarpal(s) no ossification 4th


Metacarpal(s) incomplete ossification


Pubis incomplete ossification


Short


Rudimentary


Short 14th


Arch(es) incomplete ossification


General incomplete ossification


Hyoid no ossification


Temporal incomplete ossification


Nasal incomplete ossification


Parietal incomplete ossification


Supraoccipital incomplete ossification


Hyoid incomplete ossification


Interparietal incomplete ossification


Asymmetrical ossification


Fused


No ossification 6th


Incomplete ossification 6th


Incomplete ossification


No ossification 5th


Incomplete ossification 5th


Centrum asymmetrical ossification


Centrum incomplete ossification


Centrum dumb-bell shaped



165


165


165


165


165


165


165


165


165


165


165


165


165


165


165


165


165


165


165


165


165


165


165


165


165



68


1


1


4


1


6


8


2


17


6


1


2


18


16


49


6


1


28


51


1


38


32


1


19


1



41.21


0.61


0.61


2.42


0.61


3.64


4.85


1.21


1.30


3.64


0.61


1.21


10.91


9.70


29.70


3.64


0.61


16.97


30.91


0.61


23.03


19.39


0.61


11.52


0.61



22


22


22


22


22


22


22


22


22


22


22


22


22


22


22


22


22


22


22


22


22


22


22


22


22



19


1


1


3


1


4


4


1


8


4


1


2


10


11


16


5


1


13


19


1


16


14


1


10


1



86.36


4.55


4.55


13.64


4.55


18.18


18.18


4.55


36.36


18.18


4.55


9.09


45.45


50.00


72.73


22.73


4.55


59.09


86.36


4.55


72.73


63.64


4.55


45.45


4.55



2



Forepaw(s)


Ribs


Ribs


Sacral vertebrae


Skull


Skull


Skull


Skull


Skull


Skull


Skull


Sternebrae


Sternebrae


Sternebrae


Sternebrae


Sternebrae


Sternebrae


Sternebrae


Thoracic vertebrae


Thoracic vertebrae


Thoracic vertebrae


Thoracic vertebrae



AN


AN


VA


AN


AN


AN


AN


VA


VA


VA


VA


AN


AN


AN


VA


VA


VA


VA


AN


AN


VA


VA



Metacarpal(s) no ossification 4th


Rudimentary


Short 14th


Arch(es) incomplete ossification


Frontal incomplete ossification


Hyoid no ossification


Temporal incomplete ossification


Interparietal incomplete ossification


Parietal incomplete ossification


Supraoccipital incomplete ossification


Hyoid incomplete ossification


Asymmetrical ossification


No ossification 6th


Bipartite 5th


No ossification 5th


Incomplete ossification 6th


Incomplete ossification


Incomplete ossification 5th


Centrum no ossification


Centrum bipartite


Centrum incomplete ossification


Centrum dumb-bell shaped



171


171


171


171


171


171


171


171


171


171


171


171


171


171


171


171


171


171


171


171


171


171



58


3


3


5


1


15


1


33


1


13


9


11


17


2


43


41


3


53


1


2


15


1



33.92


1.75


1.75


2.92


0.58


8.77


0.58


19.30


0.58


7.60


5.26


6.43


9.94


1.17


25.15


23.98


1.75


30.99


0.58


1.17


8.77


0.58



23


23


23


23


23


23


23


23


23


23


23


23


23


23


23


23


23


23


23


23


23


23



20


3


3


5


1


9


1


16


1


6


6


10


8


1


16


18


2


19


1


2


9


1



86.96


13.04


13.04


21.74


4.35


39.13


4.35


69.57


4.35


26.09


26.09


43.48


34.78


4.35


69.57


78.26


8.70


82.61


4.35


8.70


39.13


4.35



3



Forepaw(s)


Ribs


Ribs


Ribs


Sacral vertebrae


Skull


Skull


Skull


Skull


Skull


Skull


Sternebrae


Sternebrae


Sternebrae


Sternebrae


Sternebrae


Sternebrae


Sternebrae


Thoracic vertebrae


Thoracic vertebrae



AN


AN


AN


VA


AN


AN


AN


VA


VA


VA


VA


AN


AN


AN


VA


VA


VA


VA


AN


VA



Metacarpal(s) no ossification 4th


Short


Rudimentary


Short 14th


Arch(es) incomplete ossification


Temporal incomplete ossification


Hyoid no ossification


Hyoid incomplete ossification


Interparietal incomplete ossification


Parietal incomplete ossification


Supraoccipital incomplete ossification


No ossification 6th


No ossification


Asymmetrical ossification


No ossification 6th


Incomplete ossification 6th


Incomplete ossification 5th


Incomplete ossification


Centrum bipartite


Centrum incomplete ossification



174


174


174


174


174


174


174


174


174


174


174


174


174


174


174


174


174


174


174


174



60


1


1


1


6


1


7


7


38


2


9


13


1


9


32


49


46


1


3


21



34.48


0.57


0.57


0.57


3.45


0.57


4.02


4.02


21.84


1.15


5.17


7.47


0.57


4.02


18.39


28.16


26.44


0.57


1.72


12.07



24


24


24


24


24


24


24


24


24


24


24


24


24


24


24


24


24


24


24


24



20


1


1


1


5


1


4


6


15


2


9


8


1


4


13


16


16


1


3


11



83.33


4.17


4.17


4.17


20.83


4.17


16.67


25.00


62.50


8.33


37.50


33.33


4.17


16.67


56.17


66.67


66.67


4.17


12.50


45.83



4



Forepaw(s)


Ribs


Ribs


Sacral vertebrae


Skull


Skull


Skull


Skull


Skull


Skull


Sternebrae


Sternebrae


Sternebrae


Sternebrae


Sternebrae


Sternebrae


Sternebrae


Thoracic vertebrae


Thoracic vertebrae


Thoracic vertebrae



AN


AN


VA


AN


AN


AN


VA


VA


VA


VA


AN


AN


AN


VA


VA


VA


VA


AN


VA


VA



Metacarpal(s) no ossification 4th


Rudimentary


Short 14th


Arch(es) incomplete ossification


Temporal incomplete ossification


Hyoid no ossification


Interparietal incomplete ossification


Hyoid incomplete ossification


Supraoccipital incomplete ossification


Parietal incomplete ossification


Asymmetrical ossification


Bipartite 5th


No ossification 6th


Incomplete ossification


Incomplete ossification 5th


No ossification 5th


Incomplete ossification 6th


Centrum bipartite


Centrum incomplete ossification


Centrum dumb-bell shaped



183


183


183


183


183


183


183


183


183


183


183


183


183


183


183


183


183


183


183


183



43


6


2


3


1


9


43


12


16


3


3


3


23


1


45


31


53


2


19


2



23.50


3.28


1.09


1.64


0.55


4.92


23.50


6.56


8.74


1.64


1.64


1.64


12.57


0.55


24.59


16.94


28.96


1.09


10.38


1.09



24


24


24


24


24


24


24


24


24


24


24


24


24


24


24


24


24


24


24


24



16


5


2


2


1


7


17


9


11


3


2


3


9


1


18


17


17


2


11


2



66.67


20.83


8.33


8.33


4.17


29.17


70.83


37.50


45.83


12.50


8.33


12.50


37.50


4.17


75.00


70.83


70.83


8.33


45.83


8.33



 


FIXED VISCERAL EXAMINATION OF FOETUSES – GROUP INCIDENCE






































































Group



Organ



Cat



Observation(s)



No. Foetuses



No. Litters



Obs



Aff



%



Obs



Aff



%



1



Abdomen


Brain


Heart


Heart


Heart


Heart


Kidneys


Kidneys


Testis


Ureter



VA


AN


AN


AN


MA


VA


AN


VA


AN


VA



Umbelical vein left


Ventricle enlarged moderate


Ventricle enlarged


Atrium enlarged


Septum incomplete


Ventricle enlarged


Ectopic


Pelvic dilation slight


Displaced


Enlarged slight



152


152


152


152


152


152


152


152


152


152



1


1


1


1


1


2


2


1


10


7



0.66


0.66


0.66


0.66


0.66


1.32


1.32


0.66


6.58


4.64



22


22


22


22


22


22


22


22


22


22



1


1


3


1


1


3


2


1


9


4



4.55


4.55


13.64


4.55


4.55


13.64


9.09


4.55


40.91


18.18



2



Abdomen


Abdomen


Great vessels


Heart


Kidneys


Kidneys


Testis


Thoracic cavity


Ureter



VA


VA


VA


AN


AN


VA


AN


VA


VA



Umbelical vein left


Haemorrhagic


Long innominate artery


Atrium enlarged


Ectopic


Pelvic dilation slight


Displaced


Haemorrhage


Enlarged slight



162


162


162


162


162


162


162


162


162



2


2


1


4


4


1


5


2


5



1.23


1.23


0.62


2.47


2.47


0.62


3.09


1.23


3.09



23


23


23


23


23


23


23


23


23



2


2


1


2


3


1


2


2


5



8.70


8.70


4.35


8.70


13.04


4.35


8.70


8.70


21.74



3



Abdomen


Abdomen


Brain


Great vessels


Heart


Heart


Heart


Heart


Kidneys


Kidneys


Testis


Testis


Thoracic cavity


Ureter


Ureter



VA


VA


MA


VA


AN


MA


MA


VA


AN


VA


AN


AN


VA


AN


VA



Haemorrhagic


Umbelical vein left


Ventricles enlarged extreme


Long innominate artery


Atrium enlarged


Septum incomplete


Persistant truncus areriosus


Pericardium Haemorragic


Ectopic


Pelvic dilation slight


Malpositioned


Displaced


Haemorrhage


Kinked moderate


Enlarged slight



162


162


162


162


162


162


162


162


162


162


162


162


162


162


162



3


1


1


1


2


1


1


1


4


2


1


5


5


1


4



1.85


0.62


0.62


0.62


1.23


0.62


0.62


0.62


2.47


1.23


0.62


3.09


3.09


0.62


2.47



24


24


24


24


24


24


24


24


24


24


24


24


24


24


24



3


1


1


1


2


1


1


1


3


2


1


5


3


1


4



12.50


4.17


4.17


4.17


8.33


4.17


4.17


4.17


12.50


8.33


4.17


20.83


12.50


4.17


16.67



4



Abdomen


Great vessels


Heart


Heart


Kidneys


Kidneys


Testis


Testis


Thoracic cavity


Thoracic cavity


Ureter


Ureter


Ureter



VA


VA


AN


VA


AN


VA


AN


AN


AN


VA


AN


AN


VA



Haemorrhagic


Long innominate artery


Atrium enlarged


Pericardium Haemorragic


Ectopic


Pelvic dilation slight


Hypoplasia


Displaced


Haemorrhage


Haemorrhage


Enlarged moderate


Kinked moderate


Enlarge slight



173


173


173


173


173


173


173


173


173


173


173


173


173



7


3


6


2


1


1


1


10


2


1


1


1


3



4.05


1.73


3.47


1.16


0.58


0.58


0.58


5.78


1.16


0.58


0.58


0.58


1.73



24


24


24


24


24


24


24


24


24


24


24


24


24



5


2


4


1


1


1


1


9


2


1


1


1


3



20.83


8.33


16.67


47.17


4.17


4.17


4.17


37.50


8.33


4.17


4.17


4.17


12.50



 

Applicant's summary and conclusion

Conclusions:
Based on the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for maternal and developmental toxicity can be set at 1000 mg/kg/day.
Executive summary:

Study design


The effects of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline were investigated in female Sprague Dawley SD rats during pregnancy and embryo-foetal development according to OECD guideline no. 414 “Prenatal Developmental Toxicity Study”. Females were in-house mated with sexually mature males of the same strain and then assigned to 4 groups of 24 females each as indicated below:


 


















Group Number



Treatment


(mg/kg/day)+



Level



Female Numbers


(odd only)



1


2


3


4



0


50


250


1000



Control


Low


Medium


High



1-47


49-95


97-143


145-191



+: in terms of test item supplied


 


The test item was suspended in 0.5 % methyl cellulose (0.5% MC) and administered by oral gavage at 50, 250 and 1000 mg/kg/day with the dose volume set at 10 mL/kg body weight.


The animals were treated throughout the gestation period from Day 3 up to Day 19 post coitum. Control females received the vehicle (0.5 % MC) at the same dose volume during the same treatment period.


 


Parameters assessed included clinical observations, body weight and food consumption during the in-life phase. Blood collection for hormones determination (T3, T4 and TSH) was performed on Day 20 post coitum before dispatch to necropsy.


 


All females were caesarean-sectioned on Day 20 post coitum and subjected to post mortem examination along with thyroid and brain weights. The ovaries were examined and the corpora lutea counted. Gravid uterus was weighed and the uterine content examined for the number of implantations, intrauterine deaths and live foetuses.


 


Live foetuses were weighed, sexed and observed for external abnormalities. In addition, the anogenital distance (AGD) was recorded in all live foetuses.


 


Approximately one half of the foetuses in each litter was examined for fixed-visceral and skeletal abnormalities.


 


Histopathology of thyroid was performed in all groups.


 


Fate of females


All females survived to the final sacrifice on gestation Day 20 and were found pregnant with the exception of two controls and one low dose female.


The final number of females with live foetuses on gestation Day 20 was as follows:


– 22 in the control group (0 mg/kg/day)


– 23 in the low dose group (50 mg/kg/day)


– 24 in the mid-dose group (250 mg/kg/day)


– 24 in the high dose group (1000 mg/kg/day)


 


Clinical signs


No treatment-related findings were recorded.


 


Maternal body weight and body weight gain


No treatment-related effects were seen in maternal body weight/body weight gain throughout the study.


 


Food consumption


No treatment-related effects were seen in food consumption throughout the study.


 


Thyroid hormone determination


Serum levels of T3, T4 and TSH did not differ between treated and control groups.


 


Terminal body weight, uterus weight and absolute weight gain


Body weight at termination, uterus weight and the absolute weight gain of females were unaffected by treatment.


 


Organ weight


No effects were seen in thyroid weight between treated and control groups.


 


Litter data and sex ratios


Litter data and sex ratios were unaffected by treatment.


 


Anogenital distance


No differences were seen in the anogenital distance between treated and control groups both in male and females foetuses.


 


Macroscopic and microscopic examinations


At post mortem examination, no treatment-related macroscopic observations were noted in treated females receiving test item at ≥ 50 mg/kg/day, when compared to controls. Any macroscopic observations were within the range of expected spontaneous findings in SD rats of the same age considered incidental and unrelated to the test item.


 


External examinations of foetuses


No treatment-related effects were seen at external examination of foetuses.


 


Skeletal examinations of foetuses


No treatment-related effects were seen at external examination of foetuses.


 


Visceral examination of foetuses


No treatment-related effects were seen at external examination of foetuses.


 


Conclusion


Based on the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for maternal and developmental toxicity can be set at 1000 mg/kg/day.