Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting Date: 24 March 2016 and Experimental Completion Date: 01 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
Please see below in "Test and materials method"
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: yellow-brown liquid
Details on test material:
Name: TOFA_TETA_PAA_BADGE_CGE_Adduct
Other synonyms: Euredur 450 BD and Aradur 450 BD
CAS number: 186321-96-0
Batch number: WA520
Test material form: yellow brown liquid
Molecular weight: 755
Date of receipt: 24 August 2012
Storage details: stored at 15 - 25°C, protected from light
Purity: 100%
Expiry date: February 2014

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
Details on test animals and environmental conditions:
TEST ANIMALS

Animal Information
A total of ninety-six time-mated female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were obtained from Charles River (UK) Limited, Margate, Kent. Animals were received in two deliveries two days apart and weighed on arrival (Day 0 or Day 1 of gestation). The first delivery consisted of two batches of animals at Day 1 and Day 0 of gestation respectively, the second delivery consisted of one batch of animals at Day 0 of gestation. The day that positive evidence of mating (sperm in the vaginal smear) was observed was designated Day 0 of gestation. At the start of treatment (Day 3 of presumed gestation) the females weighed 196 to 298g.

- Housing: The animals were housed individually in solid-floor polypropylene cages with stainless steel mesh lids furnished with softwood flakes
- Diet (e.g. ad libitum): The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global
Certified Diet, Envigo RMS (UK) Limited, Oxon, UK) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage.

- Water (e.g. ad libitum):
ENVIRONMENTAL CONDITIONS
The animals were housed in a single air-conditioned room
The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.
The Study Plan target ranges for temperature and relative humidity were 22 ± 3 ºC and 50 ± 20%. There were no deviations from these targets
The in-life phase of the study was conducted between 25 March 2016 (first day of treatment) and 13 April 2016 (final day of necropsy).

The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The in-life phase of the study was conducted between 25 March 2016 (first day of treatment) and 24 June 2016 (final day of necropsy).



Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
Polyethylene glycol 400
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

For the purpose of the study the test item was prepared at the appropriate concentrations as a solution in Polyethylene Glycol 400. The original intention was to use formulations prepared in corn oil to match the vehicle used in the read across study (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rat; Covance Laboratories Ltd Report No.: 8266708) but acceptable dosing formulation could not be produced using this vehicle. The homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK Analytical Services as part of this study. As the analysis method developed to analyze the concentration of Test Item within formulations was not stability indicating, it was not possible to measure the stability of the dosing formulations. In view of this, dose formulations were prepared on a daily basis and the animals were dosed as early as possible after preparation of the dosing formulations. Dosing was completed within 4 hours of dose preparation and it is assumed that Test Item formulations were stable for this period.

Representative samples of test item formulations were taken on two occasions and were analyzed for concentration of TOFA_TETA_PAA_BADGE_CGE_Adduct at Envigo Analytical Laboratory, Shardlow. As samples were only analyzed after the high dosage had been reduced to 450 mg/kg bw/day (112.5 mg/mL), samples from dosing formulations at 1000 mg/kg bw/day (250 mg/mL) and 600 mg/kg bw/day (150 mg/mL) were not analyzed. However formulations prepared at 250 mg/mL were analyzed as part of the overall formulation chemistry assessment. The results indicate that the prepared dosing formulations were within 89 to 115% of the nominal concentration. Formulation at 112.5 mg/ml were slightly higher than the normally accepted range but indicated that animals would have received at least their approximate expected dosage.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Representative samples of test item formulations were taken on two occasions and were analyzed for concentration of TOFA_TETA_PAA_BADGE_CGE_Adduct at Envigo Analytical Laboratory, Shardlow. As samples were only analyzed after the high dosage had been reduced to 450 mg/kg bw/day (112.5 mg/mL), samples from dosing formulations at 1000 mg/kg bw/day (250 mg/mL) and 600 mg/kg bw/day (150 mg/mL) were not analyzed.
However formulations prepared at 250 mg/mL were analyzed as part of the overall formulation chemistry assessment.
Analytical Services. The results indicate that the prepared dosing formulations were within 89 to 115% of the nominal concentration.

Apparatus: Analytical and top-pan balances.

Reagents:
Control vehicle: PEG 400
Tetrahydrofuran: Gradient HPLC grade
Dilution solvent: Tetrahydrofuran


Preparation of calibration standards:
Stock solutions of the test item dilution solvent were prepared for external standard calobration. An aliquot, approx. 0.025 g of test item was exactly weighed into a 200 mL volumetric flask and brought to vilume with dilution solvent to yield a solution with a concentration of 0.125 mg/mL. Aliquots of this stock standard solution were used to prepare working standard solutions in dilution solvent with a concentration of 0.01 mg/mL.
On each occasion stadard solutions derived from two stock standard solutions were used for calculation.
Calibration solutions were injetced onto the instrument, at the beginning and end of each sample analysis sequence as a minimum, using the conditions detailed in the instrument parameters.
to assess the calibration range of the method, a range of standard solutions were prepared in dilution solvent from a stock solution of 0.125 mg/mL by seriel dilution covering the concentration range 0.00506 mg/mL to 0.01265 mg/mL.

Preparation of test sample:
The formulation received were diluted with dilution solvent. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with dilution solvent this was then shaken to dissolve. Where neccessary, sample solutions were further diluted with dilution solvent to achieve the working concentration.

Preparation of Accuracy and precision Samples:
Sample of PEG 00 were accurately fortified with known amounts of test item equivalent to lh lowest and highest anticipated dose concentrations. These samples were then prepared for analysis as the sample section.

The concentration of test item in the final solution was quantified by spectrometer using UV detection.

Instrumentation Parameters:
Spectophotometer: Perkin-Elmer Lambda 20
Wavelenght: at 227 nm
Cell path lenght: 1 cm
Reference medium: Tetrahydrofuran

Data evaluation and calculations:
The paek area response for test item in each calibration standard chromatogram was measured. Calibration curves were constructed by linear regression of calibration standard response versus calibration standard concentration. The area response of the peak observed at the characteristic retention time for test item in sample and procedural recovery chromatograms was measured. The concentration of test item was determined using the following equations:

The equation "x" of the test item in an injected sample was calculated by the following equation:
x= y-a / m
Where:
x: concentration of the test item in the injected sample (mg/mL)
y: mean absorbance response of the test item in the injected sample (counts)
m: slope of the calibration plot (counts per mg/mL)
a: y-axis intercept (counts)

The concentration of the test item in the test sample was calculated using the following equation:
c= (x / mass) x F x SG
Where
c: concentration of the test item in the test sample (mg/mL)
x: concentration of the test item in the injected sample solution (mg/mL)
F. Factor dilution factor
SG: Specific Gravity (where applicable) (g/mL)
Mass: Mass taken (g)

The recovery rate (R) of the spiked recovery sample was calculated using the following equation:

R= (c / cfort) x 100
Where :
R= Recovery rate (%)
C= determined concentration of the test item in the spiked recovery sample (mg/mL)
C fort= determined concentration of the test item in the spiked recovery sample (mg/mL)

Concentration of Dose Formulations:
For each occasion, freshly prepared test formulations were analyzed. Duplicated samples were analyzed in accordance with the analytical procedure. Sample were disposed of once satisfactory results were achieved.

Major Computerized Systems:
The Computerized Systems that have been used in this project are:
PerkinElmer UV WinLab Enhanced Security Version 6.0.3.0730
Delta BMS

RESULTS:
The analytical procedure was succeflly validated for the test item in PEG 400 with respect to the specificty, the linearity of detector response, method accuracy and precision.
The specificity of teh analytical method was demonstrated by the absence of absorbance ate the characteristic wavelenght for test item in the control sample scan.

The data was found to have a linear correlation within the calibration range of 0.00506 mg/mL to 0.01265 mg/mL. The R2 fit of the calibration curve to the data was 0.09994, and was considered to be acceptable.

A mean recovery value of 101% (CV=1.54%, n=5) was obtained for 25 mg/mL and 107% (CV=7.67%, n=5) was obtained for 250 mg/mL.


Concentration of dose formulations and conclusion:
The mean concentrationsof the tets item in test formulations analysed for the study were within +/- 15 % of nominal concentrations, confirming accurate formulation. The formulations were deemed to be homogeneous by visual inspection.

Details on mating procedure:
A total of ninety-six time-mated female Sprague-Dawley Crl:CD (SD) IGS BR strain rats were obtained from a reliable breeder. Animals were received in two deliveries two days apart. The first delivery consisted of two batches of animals at Day 1
and Day 0 of gestation respectively, the second delivery consisted of one batch of animals at Day 1 of gestation. The day that positive evidence of mating (sperm in the vaginal smear) was observed was designated Day 0 of gestation.
Duration of treatment / exposure:
The test item was administered daily, from Day 3 to Day 19 of gestation, by gavage.
Control animals were treated in an identical manner with the vehicle alone.
Frequency of treatment:
Daily
Duration of test:
Between Days 3 and 19 of gestation by gavage.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
A further group of twenty-four time mated females was exposed to the vehicle only (Polyethylene Glycol 400) to serve as a control.
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Due to adverse maternal toxicity, the high dosage was subsequently lowered to 600 mg/kg bw/day (30 March 2016) and then to 450 mg/kg bw/day (1 April 2016).
No. of animals per sex per dose:
Only females used, 24 females per dose group.
Control animals:
yes, concurrent vehicle
Details on study design:
The animals were randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Examinations

Maternal examinations:
MATERNAL EXAMINATIONS

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

BODY WEIGHT: Yes
Individual scheduled body weights were recorded on Day 3 (before the start of treatment) and on Days 4, 5, 8, 11, 14 and 17 of gestation. Body weights were also recorded for surviving animals at terminal kill (Day 20).


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

Water Consumption: Yes
Water intake was observed daily by visual inspection of the water bottles for any overt changes.

CAGE SIDE OBSERVATIONS: No


DETAILED CLINICAL OBSERVATIONS: Yes
Following arrival, all animals were examined once daily during the gestation period. Additionally, during the dosing period, observations were recorded immediately before and soon after dosing and one hour post dosing.




TERMINAL INVESTIGATION

POST-MORTEM EXAMINATIONS: Yes
All surviving animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on Day 20 of gestation. All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded.



Ovaries and uterine content:
All surviving animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on Day 20 of gestation. All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded. The ovaries and uteri of pregnant females were removed, examined and the following data recorded:
i) Number of corpora lutea
ii) Number, position and type of intrauterine implantation
iii) Fetal sex
iv) External fetal appearance
v) Fetal weight
vi) Placental weight
vii) Gravid uterus weight
The uteri of any apparently non-pregnant females were immersed in 0.5% ammonium polysulphide solution to reveal evidence of implantation.

Implantation types were divided into:
- Early Death: No visible distinction between placental/decidual tissue and embryonic tissue
- Late Death: Separate embryonic/fetal and placental tissue visible
- Dead Fetus: A fetus that had died shortly before necropsy. There were no dead fetuses observed within this study

All implantations and viable fetuses were numbered according to their intrauterine position as follows (as an example):

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

V = viable fetus

The fetuses were killed by subcutaneous injection of sodium pentobarbitone. Fetuses from each litter were divided into two groups and examined for skeletal alterations and soft tissue alterations. Alternate fetuses were identified using an indelible marker and placed in Bouin’s fixative. Fetuses were subsequently transferred to distilled water and examined for visceral anomalies under a low power binocular microscope and then stored in 10% Buffered Formalin. The remaining fetuses were identified using cardboard tags marked with chinagraph pencil and placed into 70% IMS in distilled water. The fetuses were subsequently eviscerated, processed and the skeletons stained with alizarin red S before being transferred to 50% glycerol for examination of skeletal development and anomalies and storage.
Fetal examinations:
- External examinations: Yes: half per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data
Statistics:
The following parameters were analyzed statistically, where appropriate, using the test methods outlined below:

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

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

Fetal evaluation parameters, including skeletal or visceral findings: Kruskal-Wallis nonparametric analysis of variance and Mann-Whitney ‘U’ test. Probability values (p) are presented as follows:
p<0.001 ***
p<0.01 **
p<0.05 *
p≥0.05 (not significant)
Indices:
Pre and Post Implantation Loss

Percentage pre-implantation loss was calculated as:
((number of corpora lutea - number of implantations) / number of corpora lutea) x 100



Percentage post-implantation loss was calculated as:
((number of implantations - number of live fetuses) / number of implantations ) x100


Sex Ratio:
Sex ratio was calculated as:
% male fetuses (sex ratio) = (number of male fetuses / total number of fetuses) x 100
Historical control data:
No data

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
At 1000/600/450 mg/kg bw/day, 18 of the surviving 19 females showed incidences of increased post-dosing salivation and noisy respiration was also observed at some stage of the study for 11 females. Other clinical signs such as decreased respiration rate, hunched posture, staining around the mouth or ano-genital region and chromodacryorrhea were also observed but the incidence of these signs tended to be restricted to single animals on isolated
occasions.
At 300 mg/kg bw/day, 6 of the surviving 22 females showed incidences of increased postdosing salivation, however there were no other clinical signs apparent for surviving animals at this dosage.
At 100 mg/kg bw/day, no clinical signs were apparent for animals throughout gestation.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
There were two unscheduled deaths at 300 mg/kg bw/day and five at 1000/600/450 mg/kg bw/day.

Female 95 (1000/600mg/kg bw/day) was killed in extremis on Day 7 post coitum due to adverse body weight loss and adverse clinical signs including decreased respiratory rate, laboured and noisy respiration, pallor of the extremities and staining around the snout.
Clinical signs prior to Day 7 included decreased respiratory rate and noisy respiration on Days 5 and 6. Necropsy revealed oil-like fluid in the thoracic cavity and gaseous distension of the stomach.

Female 74 (1000/600 mg/kg bw/day) showed adverse body weight loss and noisy respiration and was killed in extremis on Day 8 post coitum. Necropsy revealed oil-like fluid in the thoracic cavity and gaseous distension of the stomach.

Female 90 (1000/600/450 mg/kg bw/day) showed adverse body weight loss and was killed in extremis on Day 6 post coitum. Necropsy revealed gaseous distension in the stomach and gastro-intestinal tract.

Female 75 (1000/600/450 mg/kg bw/day) was killed on Day 11 post coitum after showing adverse body weight loss. This animal had previously shown chromodacryorrhea on Day 9 and noisy respiration on Days 9 and 10 post coitum. Necropsy revealed gaseous distension in the stomach and gastro-intestinal tract.

Female 73 (1000/600/450 mg/kg bw/day) was killed in extremis on Day 12 post coitum due to continuing body weight loss and adverse clinical signs including decreased respiratory rate, noisy respiration, hunched posture, apparent dehydration, piloerection and staining around the mouth. Clinical signs prior to this included decreased respiration rate on Day 5, noisy respiration on Days 5 to 11 and increased post-dosing salivation on Day 10 post coitum. Necropsy revealed oil-like fluid in the thoracic cavity.

Female 69 (300 mg/kg bw/day) showed adverse body weight loss and noisy respiration and was killed in extremis on Day 5 post coitum. No findings were apparent at macroscopic necropsy examination.

Female 51 (300 mg/kg bw/day) was found dead on Day 13 post coitum; no clinical signs or body weight loss had been apparent for this animal prior to this occurrence. Necropsy revealed dark lungs and fluid in the thoracic cavity.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000/600/450 mg/kg bw/day, a mean body weight loss was apparent on the first day of
dosing and subsequently body weight gain was lower than control to Day 11 of gestation.
While differences in body weight gain from control only attained statistical significance
during the first two days of treatment (these differences would have been more marked if the
decedent animals had been included), by Day 11 the lower mean body weight of the females
was statistically significantly lower than control. Overall, the majority of animals showed
some degree of body weight loss during this period; although there was great variation in the
extent of the observed losses which were relatively transient in nature. Body weight gains of
surviving animals after Day 11 of gestation were similar to control and appeared unaffected
by treatment. However, the initial body weight loss and lower body weight gain resulted in
statistically significant lower cumulative mean body weight gain, compared to control,
throughout gestation and mean overall weight gain at the end of the study remained
statistically significantly lower than control, following adjustment for the contribution of the
gravid uterus.
At 300 mg/kg bw/day, mean body weight gain was lower than control during the first two
days of dosing, although statistical significance was only attained for the first day. While
body weight gain appeared similar to control on Day 8, lower body weight gain was again
apparent between Day 8 and 11 of gestation, although differences showed no statistical
significance. Occasional animals showed incidences of notable body weight loss similar to
that observed at the high dosage but, with the exception of the decedent animals, these losses were transient in nature. Subsequent mean body weight gains from Day 11 of gestation to
termination were considered to be similar to control; there was a suggestion of lower body
weight gain during Days 17 to 20 of gestation but this was considered to reflect a lower
contribution from the gravid uterus due to lower litter size compared to control at this dosage.
The lower body weight gain at the start of the study resulted in statistically significant lower
cumulative mean body weight gain, compared to control, throughout gestation and mean
overall weight gain at the end of the study remained statistically significantly lower than
control, following adjustment for the contribution of the gravid uterus.
At 100 mg/kg bw/day, there was no obvious effect of treatment on body weight or body
weight gain of pregnant females throughout gestation, including when overall body weight
gain was adjusted for the contribution of the gravid uterus.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
At 1000/600/450 mg/kg bw/day, food consumption was statistically significantly lower than
control during the first six days of treatment. Thereafter there were no statistically significant
differences in food consumption although food intake remained slightly lower than control to
termination.
At 300 mg/kg bw/day, food consumption was statistically significantly lower than control
during the first six days of treatment, although to a lesser degree than observed at the high
dosage. Thereafter food consumption remained slightly lower than control to Day 17 of
gestation although differences failed to attain statistical significance. Food consumption was
again statistically significantly lower than control during Days 17 to 20 of gestation, however
values may have been influenced by a lower demand on the pregnant females due to a lower
litter size at this dosage.
At 100 mg/kg bw/day, there was no obvious effect of treatment on food consumption during
gestation.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Visual assessment of water consumption did not reveal any obvious effects of treatment at 100, 300 or 1000/600/450 mg/kg bw/day.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings were apparent for surviving adult females at termination on Day 20 of gestation at 100, 300 or 1000/600/450 mg/kg bw/day.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
There was no effect of maternal treatment on litter data as assessed by numbers of implantations, in-utero offspring survival (as assessed by the mean numbers of early or late resorptions), live litter size, sex ratio and pre and post-implantation losses at 100, 300 or 1000/600/450 mg/kg bw/day.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There was no effect of treatment on post-impanation loss at any dosage but the lower number of corpora lutea and implantations resulted in statistically significant lower number of live young at Day 20 at 100 and 300 mg/kg bw/day. As this finding was related to the previous lower number of corpora lutea/implantations, it was clearly incidental and unrelated to treatment.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
not examined

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
Key result
Dose descriptor:
LOEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
mortality
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: Biological Relevance
Remarks:
Effects on body weight, food consumption and a simgle mortality was observed however, the effects receeded during the conduct of the study and thereby mayreflect natural biological variation. As a result of such uncertainty a conservative NOEL of 100 mg/kg bw d has been determined.

Results (fetuses)

Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There was no effect of maternal treatment on mean fetal, litter or placental weights at 100,
300 or 1000/600/450 mg/kg bw/day.
At 100 and 300 mg/kg bw/day, litter weights (and gravid uterus weights) and total placental
weights were lower than control, with differences for gravid uterus and total placental
weights attaining statistical significance. These differences reflected the lower litter size at
these dosages, which were unrelated to treatment. These differences
in litter, gravid uterus and total placental weights, in the absence of any effect on mean
offspring and placental weights were therefore clearly incidental and unrelated to maternal
treatment.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, non-treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): There was no effect of maternal treatment on mean fetal, litter or placental weights at 100, 300 or 1000/600/450 mg/kg bw/day.

At 100 and 300 mg/kg bw/day, litter weights (and gravid uterus weights) and total placental weights were lower than control, with differences for gravid uterus and total placental weights attaining statistical significance. These differences reflected the lower litter size at these dosages, which as previously indicated were unrelated to treatment. These differences in litter, gravid uterus and total placental weights, in the absence of any effect on mean offspring and placental weights were therefore clearly incidental and unrelated to maternal treatment.
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
There was no effect of maternal treatment on litter data as assessed by numbers of
implantations, in-utero offspring survival (as assessed by the mean numbers of early or late
resorptions), live litter size, sex ratio and pre and post-implantation losses at 100, 300 or
1000/600/450 mg/kg bw/day.
For all treated groups, the number of corpora lutea was lower than control, with differences at
300 and 1000/600/450 mg/kg bw/day attaining statistical significance. As the number of
corpora lutea would have been established prior to dosing on this study, this finding was
clearly incidental and unrelated to treatment. There was no effect of treatment on preimpanation
loss at any dosage but the lower corpora lutea counts in treated groups resulted in
a lower number of implantations in all treated groups, with differences at 300 and 1000/600/
450 mg/kg bw/day again attaining statistical significance. As this finding was related to the
previous lower number of corpora lutea, it was clearly incidental and unrelated to treatment.
There was no effect of treatment on post-impanation loss at any dosage but the lower number
of corpora lutea and implantations resulted in statistically significant lower number of live
young at Day 20 at 100 and 300 mg/kg bw/day. As this finding was related to the previous
lower number of corpora lutea/implantations, it was clearly incidental and unrelated to
treatment.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
Neither the type, incidence nor distribution of findings apparent during external examination of fetuses on Day 20 of gestation indicated any obvious effect of maternal treatment on fetal development at 100, 300 or 1000/600/450 mg/kg bw/day.
Skeletal malformations:
no effects observed
Description (incidence and severity):
Neither the type, incidence nor distribution of findings apparent during detailed skeletal examination of fetuses indicated any obvious effect of maternal treatment on fetal development at 100, 300 or 1000/600/450 mg/kg bw/day.
Visceral malformations:
no effects observed
Description (incidence and severity):
Neither the type, incidence nor distribution of findings apparent during detailed visceral examination of fetuses indicated any obvious effect of maternal treatment on fetal development at 100, 300 or 1000/600/450 mg/kg bw/day.
Other effects:
not examined
Details on embryotoxic / teratogenic effects:
No effect observed during external, visceral and skeletal examinations.
Neither the type, incidence nor distribution of findings apparent during examinationof fetuses indicated any obvious effect of maternal treatment on fetal development.

Effect levels (fetuses)

Dose descriptor:
NOEL
Effect level:
450 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
fetal/pup body weight changes
changes in litter size and weights

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

DISCUSSION:

Treatment at 1000/600/450 mg/kg bw/day was initially associated with lower body weight gain or body weight loss and a concomitant reduction in food consumption compared to control. The majority of animals showed some degree of body weight loss within the first week of treatment. While these body weight losses resulted in the early termination of five animals at this dosage, for some animals the body weight losses observed were quite transient in nature. In addition to body weight loss, four of the five decedent females showed noisy respiration on the day of, or day prior to, termination and similar noisy respiration was also observed during the study for eleven of the surviving females at this dosage. Necropsy of the five decedent animals revealed oil-like fluid in the thoracic cavity in one animal and gaseous

distension of the stomach and gastro-intestinal tract for the remaining four animals.

Collectively these findings suggest that the test item formulation at this concentration used for the high dosage group may be slightly irritant and it is considered that the accidental aspiration of test item during the dosing procedure may have been a contributory factor to the weight losses observed. Increased post-dosing salivation was observed for the majority of surviving animals at this dosage and this clinical sign is commonly observed when a slightly irritant test item is administered via the oral gavage route.

For surviving animals, body weight gains after Day 11 of gestation were similar to control, although overall body weight gain remained statistically significantly lower than control, including when adjusted for the contribution of the gravid uterus. Food consumption from Day 8 remained lower than control to termination but differences no longer attained statistical significance and may reflect normal biological variation.

Treatment at 300 mg/kg bw/day was associated with statistically significant lower body weight gain, compared to control, during the first day of dosing (Day 3 of gestation). Lower, but not statistically significant, body weight gain was also apparent compared to control the following day and during Days 8 to 11 of gestation. One female was killed due to bodyweight loss on Day 5 post coitum. Food consumption was statistically significantly lower than control to Day 8 of gestation.

Subsequent mean body weight gains from Day 11 of gestation were considered to be unaffected by treatment, although overall body weight gain remained statistically significantly lower than control, including when adjusted for the contribution of the gravid uterus. While food intake from Day 8 remained lower than control to termination, differences no longer attained statistical significance and may reflect normal biological variation, particularly as there was no dosage relationship for food consumption towards the end of gestation. However, one female at this dosage was found dead on Day 13 of gestation.

Body weight gain and food consumption was lower than control during Days 17 to 20 of gestation but this was considered to be due to lower litter size at this dosage, which was unrelated to treatment.

At 100 mg/kg bw/day clinical signs, body weight and body weight gains, food consumption and necropsy findings did not indicate any effect of treatment. This dosage was therefore considered to be the No Observed Effect Level (NOEL) for the pregnant female.

Despite the obvious effects of treatment at 1000/600/450 mg/kg bw/day observed for the pregnant dam, there was no indication of any effect on the litter or developing conceptus at this dosage. For all treated groups the number of implantations and live young was lower than control as a consequence of lower numbers of corpora lutea. As the number of corpora lutea would have been established prior to dosing on this study, this finding was clearly incidental and unrelated to treatment. The lower litter size also influenced litter weights, gravid uterus weights and total placental weights for all treated groups and, in the absence of any effect on mean offspring and placental weights, observed differences were considered to be clearly incidental and unrelated to maternal treatment. Neither the type, incidence nor

distribution of findings apparent during external examination, visceral examination and skeletal examination indicated any obvious effect of maternal treatment on fetal development at 100, 300 or 1000/600/450 mg/kg bw/day. A maternal dosage of at least 450 mg/kg bw/day was therefore considered to be the No Observed Effect Level (NOEL) for the growth survival and development of the offspring.

CONCLUSION

Based on this study the No Observed Effect Level (NOEL) for the pregnant female rat was considered to be 100 mg/kg bw/day.

A maternal dosage of at least 450 mg/kg bw/day was therefore considered to be the No Observed Effect Level (NOEL) for the growth survival and development of the offspring.

Applicant's summary and conclusion

Conclusions:
Based on this study the No Observed Effect Level (NOEL) for the pregnant female rat was considered to be 100 mg/kg bw/day.
A maternal dosage of at least 450 mg/kg bw/day was considered to be the No Observed Effect Level (NOEL) for the growth survival and development of the offspring.
Executive summary:

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

The study was designed to comply with the following guidelines:

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

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

- OECD Guidelines for Testing of Chemicals, No 414, ‘Prenatal Developmental Toxicity Study’ (adopted 22 January 2001) ommission Regulation (EC) No 440/2008 of 30 May 2008 test methods pursuant to

- Regulations (EC) No 1907/2006 of the European Parliament and of the Council on the

Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods

The test item was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, between Days 3 and 19 of gestation inclusive at dose levels 100, 300, and 1000 mg/kg bw/day. Due to adverse maternal toxicity, the high dosage was subsequently lowered to 600 mg/kg bw/day and then to 450 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle only (Polyethylene Glycol 400) to serve as a control.

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

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

Results

Mortality

At 1000/600/450 mg/kg bw/day, five females were terminated early for animal welfare considerations after showing adverse body weight loss, often accompanied by adverse clinical signs:

One female killed on Day 6 post coitum showed gaseous distension of the stomach and

gastro-intestinal tract at necropsy.

One female killed on Day 7 post coitum showed decreased respiratory rate, laboured and noisy respiration, pallor of the extremities, hunched posture, pilo-erection and staining around the snout. Decreased respiratory rate and noisy respiration were also observed during days 5 and 6. Necropsy revealed gaseous distension of the stomach and gastro-intestinal tract.

One female killed on Day 8 post coitum exhibited noisy respiration. Necropsy revealed gaseous distension of the stomach and gastro-intestinal tract.

One female killed on Day 11 post coitum showed chromodacryorrhea on Day 9 post-dosing salivation and noisy respiration on Days 9 and 10 post coitum. Necropsy revealed gaseous distension of the stomach and gastro-intestinal tract.

One female killed on Day 12 post coitum showed decreased respiratory rate, noisy respiration, hunched posture, apparent dehydration, piloerection and staining around the snout. Decreased respiratory rate had also been apparent on Day 5, noisy respiration on Days 5 to 11 and increased post-dosing salivation on Days 9 and 10 post coitum. Necropsy revealed oil-like fluid in the thoracic cavity.

At 300 mg/kg bw/day, there were two unscheduled deaths:

One female was killed after showing adverse body weight loss and noisy respiration on Day 5 post coitum. No findings were apparent at macroscopic necropsy examination.

One female was found dead on Day 13 post coitum; no clinical signs or body weight loss had been apparent for this animal prior to this occurrence. Necropsy revealed dark lungs and fluid in the thoracic cavity.

Clinical Observations

At 1000/600/450 mg/kg bw/day, 18/19 surviving females showed incidences of increased post-dosing salivation at some stage of the study. Noisy respiration was also observed at some stage of the study for 11/19 females. Decreased respiratory rate, hunched posture, staining around the mouth or ano-genital region, chromodacryorrhea and diarrhoea were also observed during the study at this dosage but these signs were generally restricted to single animals on isolated occasions.

At 300 mg/kg bw/day, 3/22 surviving females showed incidences of increased post-dosing salivation, and 7/22 showed incidences of noisy respiration.

At 100 mg/kg bw/day, no clinical signs were apparent for animals throughout gestation.

Body Weight

At 1000/600/450 mg/kg bw/day, mean body weight loss was apparent on the first day of dosing and lower body weight gain was observed to Day 11 of gestation, differences from control attaining statistical significance during the first two days of treatment. The initial body weight loss and lower body weight gain resulted in statistically significant lower cumulative mean body weight gain throughout gestation, compared to control. Mean overall weight gain at the end of the study remained statistically significantly lower than control, following adjustment for the contribution of the gravid uterus.

At 300 mg/kg bw/day, mean body weight gain was lower than control during the first two days of dosing and also between Day 8 and 11 of gestation, but statistical significance was only apparent for the first day of treatment. The lower body weight gain at the start of the study resulted in statistically significant lower cumulative mean body weight gain, compared to control, throughout gestation. Mean overall weight gain at the end of the study remained statistically significantly lower than control, following adjustment for the contribution of the gravid uterus.

At 100 mg/kg bw/day, there was no obvious effect of treatment on body weight or body weight gain of pregnant females throughout gestation, including when overall body weight gain was adjusted for the contribution of the gravid uterus.

Food Consumption

At 1000/600/450 mg/kg bw/day, food consumption was statistically significantly lower than control during the first six days of treatment; thereafter food intake remained slightly lower than control to termination.

At 300 mg/kg bw/day, food consumption was statistically significantly lower than control during the first six days of treatment. Food consumption remained slightly lower than control to termination attaining statistical significance during Days 17 to 20 of gestation, when values were probably influenced by lower litter size at this dosage.

At 100 mg/kg bw/day, there was no obvious effect of treatment on food consumption during gestation.

Water Consumption

Visual assessment of water consumption did not reveal any obvious effects of treatment at 100, 300 or 1000/600/450 mg/kg bw/day.

Post Mortem Studies

No macroscopic findings were apparent for surviving adult females at termination on Day 20 of gestation at 100, 300 or 1000/600/450 mg/kg bw/day.

Litter Data

There was no effect of maternal treatment on litter data as assessed by numbers of implantations, in-utero offspring survival (as assessed by the mean numbers of early or late resorptions), live litter size, sex ratio and pre and post-implantation losses at 100, 300 or 1000/600/450 mg/kg bw/day.

Litter Placental and Fetal Weights

There was no effect of maternal treatment on mean fetal, litter or placental weights at 100, 300 or 1000/600/450 mg/kg bw/day.

Fetal Examination

External Examinations

External examination of fetuses on Day 20 of gestation did not indicate any effect of maternal treatment on fetal development at 100, 300 or 1000/600/450 mg/kg bw/day.

Visceral Examinations

Visceral examination of fetuses did not indicate any effect of maternal treatment on fetal development at 100, 300 or 1000/600/450 mg/kg bw/day.

Skeletal Examinations

Skeletal examination of fetuses did not indicate any effect of maternal treatment on fetal development at 100, 300 or 1000/600/450 mg/kg bw/day.

Conclusion

Based on this study the No Observed Effect Level (NOEL) for the pregnant female rat was considered to be 100 mg/kg bw/day.

A maternal dosage of at least 450 mg/kg bw/day was therefore considered to be the No Observed Effect Level (NOEL) for the growth survival and development of the offspring.