Tall oil, maleated

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tall oil, maleated

EC Number:

268-859-6

EC Name:

Tall oil, maleated

Cas Number:

68152-93-2

Molecular formula:

UVCB

IUPAC Name:

3,7-dimethyl-14,16-dioxo-19-(propan-2-yl)-15-oxapentacyclo[10.5.2.0²,¹¹.0³,⁸.0¹³,¹⁷]nonadeca-13(17),18-diene-7-carboxylic acid; 8-(7-hexyl-1,3-dioxo-1,3,3a,4,7,7a-hexahydro-2-benzofuran-4-yl)octanoic acid

Test material form:

liquid

Details on test material:

Lot No.: HD0258QH13

Specific details on test material used for the study:

Identification: Maleated TOFA
Batch (Lot) Number: XF09SS
Expiry Date: 12 July 2022
Physical Description: Dark brown viscous liquid
Purity/Composition: 100%

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Wistar Han rats (Crl: WI(Han). )

Details on test animals or test system and environmental conditions:

Age at Initiation of Dosing: 11 - 15 weeks old
Body Weight Range at Initiation of Dosing: 181 – 254 g
Source (Main + DRF study): Charles River Deutschland, Sulzfeld, Germany
Number of Acclimation days: 5 – 6 days
Environmental Conditions: The actual daily mean temperature during the study period was 19 to 21 °C with an actual daily mean relative humidity of 50 to 76%. The values that were outside the targeted mean humidity range occurred for 5 days with a maximum of 76% and were without a noticeable effect on the clinical condition of the animals or on the outcome of the study.
Caging: Polycarbonate cages (Makrolon type MIII, height 18 cm) containing sterilized wooden fibers as bedding material (Lignocel S 8-15, JRS-J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. Animals will be individually housed. These housing conditions will be maintained unless deemed inappropriate by the Study Director and/or Clinical Veterinarian. The room(s) in which the animals will be kept will be documented in the study records. Cages will be arranged on the racks according to a Latin-square model.
Cage Identification: Color-coded cage card indicating at least Test Facility Study No., group, animal identification number.
Animal Enrichment: For psychological/environmental enrichment and nesting material, animals will be provided with paper and with aspen wooden sticks, except when interrupted by study procedures/activities. Results of analysis for contaminants are provided by the supplier and are on file at the Test Facility. It is considered that there are no known contaminants that would interfere with the objectives of the study.
Environmental Conditions: The target conditions for animal room environment will be as follows:
Temperature: 18 to 24 °C
Humidity: 40 to 70%
Light Cycle: 12-hours light and 12-hours dark (may be interrupted for designated procedures).
Ventilation: At least 10 air changes per hour.
Any variations to these conditions will be evaluated and maintained in the raw data.
Food: Diet: SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany Type: Pellets (alternate diet may be provided on individual animal basis as warranted as approved by the Study Director).
Frequency: Ad libitum, except during designated procedures.
Analysis: Results of analysis for nutritional components and environmental contaminants are provided by the supplier and are on file at the Test Facility. It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study.
Water:
Type: Municipal tap water.
Frequency/Ration: Freely available to each animal via water bottles.
Analysis: Periodic analysis of the water is performed, and results of these analyses are on file at the Test Facility. It is considered that there are no known contaminants in the water that could interfere with the outcome of the study.
Veterinary Care: Veterinary care will be available throughout the course of the study and animals will be examined by the veterinary staff as warranted by clinical signs or other changes. All veterinary examinations and recommended therapeutic treatments, if any, will be documented in the study records.
In the event that animals show signs of illness or distress, the responsible veterinarian may make initial recommendations about treatment of the animal(s) and/or alteration of study procedures, which must be approved by the Study Director. All such actions will be properly documented in the study records and, when appropriate, by Study Plan amendment. Treatment of the animal(s) for minor injuries or ailments may be approved without prior consultation with the Sponsor Representative when such treatment does not impact fulfillment of the study objectives. If the condition of the animal(s) warrants significant therapeutic intervention or alterations in study procedures, the Sponsor Representative will be contacted, when possible, to discuss appropriate action. If the condition of the animal(s) is such that emergency measures must be taken, the Study Director and/or attending veterinarian will attempt to consult with the Sponsor Representative prior to responding to the medical crisis, but the Study Director and/or veterinarian has authority to act immediately at his/her discretion to alleviate suffering. The Sponsor Representative will be fully informed of any such events.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Remarks:

supplied by (Merck, Darmstadt, Germany), Specific Gravity: 1.036

Details on exposure:

The objectives of this study are to determine the potential of Maleated TOFA to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female Wistar Han rats from Days 6 to 20 post-coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity will be evaluated.
A Dose Range Finder (Test Facility Reference No. 20252893) was conducted to select dose levels for the Main study. Four groups of 6 females were exposed to 0, 300, 600 and 1000 mg/kg/day Days 6 to 20 post-coitum inclusive by oral gavage.
Homogeneity and stability of the test item under test conditions was demonstrated in the analytical method development and validation study (Test Facility Study No. 20252890).
The dose levels were selected based on the results of an Acute Oral Toxicity Study, on the DRF phase of an OECD422 combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (ECHA dossier) and on a 14 day repeated dose study (Test Facility Reference No. 20252891) with Maleated TOFA in Wistar Han rats by oral gavage. In the Acute Oral Toxicity Study, the LD50 value was found to be above 2000 mg/kg in female rats. In the DRF phase of the OECD422 combination study, where 2 groups of 3 female Han Wistar rats were treated at 300 and 500 mg/kg/day for 10 days, animals were noted with a slight reduced body weight gain (2/3 animals) at 500 mg/kg/day and hypersalivation at both
doses of 300 and 500 mg/kg/day. In the OECD422 combination study, a parental, reproduction and developmental NOAEL was derived of at least 400 mg/kg/day.
In the 14 days repeated dose study, 2 groups of 3 female Wistar Han rats were treated by oral gavage for 14 days. Dose levels were 500 and 1000 mg/kg/day. Animals were noted with salivation and a slight increase in liver weight on both dose levels. No further adverse effect were noted. Based on these results, and in consultation with the Sponsor, dose levels for the current Dose Range Finder study were selected at 300, 600 and 1000 mg/kg/day.
Dosing of the DRF was initiated on 14 Apr 2021. The in-life phase of the DRF was completed on 29 Apr 2021. The test item and vehicle were administered to the appropriate animals by once daily oral gavage from Days 6 to 20 post-coitum, inclusive at 0, 300, 600, and 1000 mg/kg bw/d with 6 females per dose group. No mortality occurred during the study period. Based on the results of the DRF, selected dose levels for the Main study were 100, 300 and 1000 mg/kg/day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

An ultra performance liquid chromatographic method with mass spectrometric detection (UPLC-MS) for the quantitative analysis of he test item in vehicle was developed and applied (project 20252890).
Organic phase was acetonitrile and aqueous phase consisted of 10 mM ammonium acetate in water. The instrument used was Acquity UPLC system with an Acquity TQD mass spectrometer. The column used was an Acquity UPLC BEH C18 50 mm * 2.1 mm i.d., dp 1.7 μm at 40 °C. Injection volume was 2 μL and flow was 0.6 mL/min; MS detection by ESI-.
Calibration solutios were determined in duplicates for the required analytical range. Accuracy of calibrations and test solution in the range of 85 - 115% were given and repeatability of ≤10% was given (validity criteria).
coefficient r = >0.99 with a deviation ≤15%
Further details on analytical method can be found in the analytical report, appended to the final study report.

Details on mating procedure:

The test system, justification of test system, animal identification and environmental acclimation were identical as for the RF study.
Number of Females: 22 (time-mated) per dose group.
Number of Fetuses Expected: ~ 12 fetuses/female.
Untreated females will be mated at the Supplier and will be at Day 0 post-coitum on arrival at the Test Facility (Day 0 post-coitum is the day of successful mating).
At arrival, animals will be assigned to groups at random and animals will be identified using a chip. Animals in poor health will not be assigned to groups.

Duration of treatment / exposure:

The test item and vehicle was administered to the appropriate animals by one daily oral gavage from Day 6 to Day 20 post-coitum, inclusive.
Dose pot identification via Provantis was used as additional check to verify the dosing procedure according to Standard Operating Procedures.

Frequency of treatment:

Once daily

Duration of test:

daily exposure by oral gavage from Day 6 to Day 20 post-coitum, inclusive

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

control, vehicle only

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

low dose group

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

mid dose group

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

high dose group, maximum dose recommended

No. of animals per sex per dose:

22

Control animals:

yes, concurrent vehicle

Details on study design:

The study design followed OECD TG 414 protocol.
The preparation of test item was identical as for the DRFstudy. Preparations were visually inspected for homogeneity prior to use and all preparations were used within 24 hours after preparation of the formulation. For analytical verifications see above.
Homogeneity and stability of the test item under test conditions was demonstrated in the analytical method development and validation study (Test Facility Study No. 20252890).
Test System
The test system, justification of test system, animal identification and environmental acclimation was identical as for the DRF study. Number of Females in DRF study: 24 (time-mated) and in main study 88 (22 per dose group).
Untreated females were mated at the animal supplier and were at Day 0 post-coitum on arrival at the Test Facility (Day 0 post-coitum is the day of successful mating). At arrival, animals were assigned to groups (22 animals per dose group) at random and animals were identified using a chip. Animals in poor health will not be assigned to groups.
At Day 6 daily exposure by gavage started until Day 20, inclusive.
In-life Procedures, Observations, and Measurements - General In-life Assessments – F0-Animals
Mortality: All DRF animals at least twice daily (morning and afternoon) beginning upon arrival through termination/release / Animals will be observed within their cage unless necessary for identification or confirmation of possible findings.
Cageside Observations: All DRF animals; at least once daily, starting on Day 6 post-coitum onwards up to the day prior to necropsy. Directly post-dose. / Animals will be observed within their cage unless necessary for identification or confirmation of possible findings. Cage debris will be examined to detect premature birth, if applicable.
Detailed Clinical Observations: All DRF animals; on Days 2, 6, 13 and 21 post-coitum / Animals are removed from the cage.
Individual Body Weights: All DRF animals; on Days 2, 6, 9, 12, 15, 18 and 21 post-coitum. / In order to monitor the health status animals may be weighed more often.
Food Consumption: All DRF animals; over Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post coitum. / Quantitatively measured.
Terminal Procedures – F1-Generation / F1-animals
Viable fetus of dam surviving until scheduled necropsy on Day 21 post-coitum: External Examination, Body Weight and External Sex Determination
Non-viable fetus of dam surviving until scheduled necropsy on Day 21 post-coitum: External Examination, Body Weight and External Sex Determination
Late resorption: External Examination
Fetus of dam not surviving until scheduled necropsy on Day 21 post-coitum (Dams that are found dead, sacrificed before planned necropsy or that started to deliver): External Examination
Live fetuses will be euthanized by decapitation. In case decapitation might affect a malformation, the fetus will be euthanized by interscapular injection of sodium pentobarbital.
• Malformed fetuses will be collected and fixed in the most appropriate fixative (based on type of malformation).
• Malformed late resorptions will be collected and fixed in 10% buffered formalin.
Fetuses and late resorptions without malformations will be discarded.

Examinations

Maternal examinations:

Comprising
- mortality
- clinical observations
- body weight & body weight gain
- food consumption
- thyroid hormone analysis
- thyroid weight
- macroscopic evaluations
- microscopic evaluations of thyroid gland
- maternal pregnancy data

Ovaries and uterine content:

see above

Blood sampling:

see above

Fetal examinations:

Comprising
- litter size
- sex ratio
- fetal body weights
- fetal anogenital distance
- fetal morphological examinations
- external malformations & variations
- visceral malformations & variations
- skeletal malformations & variations

Statistics:

The procedures of constructed variables, statistical analysis, computerized systems, amendments and deviations, and retention of records were identical in the Main study with DRF study. All statistical analyses were performed within the respective study phase, unless otherwise noted. Numerical data collected on scheduled occasions were summarized and statistically analyzed as indicated below according to sex and occasion.
Parametric/Non-parametric: Levene’s test will be used to assess the homogeneity of group variances. The groups will be compared using an overall one-way ANOVA F-test if Levene’s test is not significant or the Kruskal-Wallis test if it is significant. If the overall F-test or Kruskal-Wallis test is found to be significant, then pairwise comparisons will be conducted using Dunnett’s or Dunn’s test, respectively.
Non-Parametric: The groups will be compared using an overall Kruskal-Wallis test. If the overall Kruskal-Wallis test is found to be significant, then the above pairwise comparisons will be conducted using Dunn’s test.
ANCOVA: The data corresponding to a response variable of interest and to a related covariate will be submitted to an analysis of covariance (ANCOVA), including only groups with at least three non-missing paired values and if found to be significant, then pairwise comparisons will be conducted using Dunnett’s test.
Incidence:A Fisher’s exact test will be used to conduct pairwise group comparisons of interest.

Indices:

Incidence:A Fisher’s exact test will be used to conduct pairwise group comparisons of interest.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs of toxicity were noted during the observation period.
At 1000 mg/kg/day, two females were noted with abnormal breathing sounds on either Days 7-8 post-coitum or on Day 21 post-coitum. Given the low incidence, this was considered not toxicologically relevant.
Erected fur was noted for one female at 100 and 300 mg/kg/day each and for three females at 1000 mg/kg/day, but also for two control females, on 3-7 days between Days 10-20 post-coitum. In absence of a clear dose response relation, this finding was regarded unrelated to treatment with the test item.
Salivation was observed after dosing among most animals at 1000 mg/kg/day and a few animals at 300 and 100 mg/kg/day on 1-3 days between Days 8-12 post-coitum. A higher incidence was noted with increasing dose. Considering the nature and minor severity of this effect and its time of occurrence (i.e. after dosing) it was regarded to be a physiological response rather than a sign of systemic toxicity. Skin scab findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment with the test item.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

not applicable in gavage study

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, body weight gain was slightly lower compared to control between Days 9-12 and 18-21 post-coitum (not achieving statistical significance at the later time point). This resulted in an overall mean body weight gain over the treatment period (Days 6-21 post-coitum) that was 14% lower compared to the control mean. At the end of the treatment period on Day 21 post-coitum, mean body weight was 4% lower than control (no statistical significance was achieved).
Noteworthy, Female No. 79 showed slight body weight loss (5 gram) between Days 18-21 post-coitum. This female had a normal litter (i.e. 11 live fetuses, no resorptions).
Mean body weight gain corrected for gravid uterus was decreased at 1000 mg/kg/day (23% lower than control; not statistically significant). This could mainly be attributed to Female Nos. 73, 78 and 79 (adjusted BWG of 0.8, -1.9 and -1.0 gram, respectively). After exclusion of these females, the mean body weight gain corrected for gravid uterus at 1000 mg/kg/day was improved, but still lower than control (11% lower than control).
Noteworthy, a single female of the control group (No. 16) was also noted with a low body weight gain corrected for gravid uterus (adjusted BWG of 2.4 gram).
At 300 and 100 mg/kg/day, mean body weights, body weight gain and weight gain corrected for gravid uterus were considered to be in the same range as control over the treatment period. Mean gravid uterus weights was slightly decreased at 1000 mg/kg/day (11% lower than control; not statistically significant). This decrease in gravid uterus weight was mainly attributable to females with relative few live fetuses (Nos. 75 and 80 with 3 and 4 live fetuses, respectively). Mean gravid uterus weight at 300 and 100 mg/kg/day was considered to be in the same range as control.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, mean food consumption was decreased between Days 9-12 post-coitum (9% lower than control) and Days 18-21 post-coitum (7% lower than control; not statistically significant).
At 300 and 100 mg/kg/day, mean food consumption was considered to be in the same range as control.

Food efficiency:

not examined

Description (incidence and severity):

provided ad libitum

Water consumption and compound intake (if drinking water study):

not examined

Description (incidence and severity):

provided ad libitum

Ophthalmological findings:

not examined

Immunological findings:

effects observed, treatment-related

Description (incidence and severity):

Mean serum levels of total triiodothyronine (T3) showed an apparent dose-related trend towards a decrease across the dose groups, but reached statistical significance at 1000 mg/kg/day only (0.8x of control). Mean T3 remained within historical control range across the dose groups. Mean serum levels of total thyroxine (T4) and thyroid stimulating hormone (TSH) were considered to be unaffected by treatment with the test item up to 1000 mg/kg/day.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test item-related alterations in thyroid gland weights. There were no test item-related gross observations in the thyroid glands.
All recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross observations in the thyroid glands.
All recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related microscopic observations in the thyroid glands.

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The mean numbers of corpora lutea, implantation sites, early and late resorptions, and pre- and post-implantation loss in the control and test groups were considered similar and in the range of normal biological variation.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

The mean numbers of corpora lutea, implantation sites, early and late resorptions, and pre- and post-implantation loss in the control and test groups were considered similar and in the range of normal biological variation.

Early or late resorptions:

no effects observed

Description (incidence and severity):

The mean numbers of corpora lutea, implantation sites, early and late resorptions, and pre- and post-implantation loss in the control and test groups were considered similar and in the range of normal biological variation.

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

All females, except for Female No. 46 (300 mg/kg/day), were pregnant and had live fetuses at scheduled necropsy. Therefore, the number of females with viable litters for evaluation was 22, 22, 21 and 22 in the control, 100, 300 and 1000 mg/kg/day groups, respectively.

Other effects:

no effects observed

Description (incidence and severity):

No other maternal developmental effects were observed.

Details on maternal toxic effects:

The following figures and table are attached to this robust study summary that were discussed and summarized above:
Figure 1: Summary of Group Mean Body Weights: Gestation
Figure 2: Summary of Group Mean Food Consumption: Gestation
Table 1: Summary of Clinical Observations: Gestation
Table 2: Summary of Body Weights: Gestation
Table 3: Summary of Body Weight Gains (g): Gestation
Table 4: Summary of Gravid Uterine Weights and Corrected Body Weights: Gestation
Table 5: Summary of Food Consumption: Gestation
Table 6: Summary of Thyroid Hormone Values
Table 7: Summary of Thyroid Weights
Table 8: Summary of Macroscopic Pathology
Table 9: Summary of Microscopic Pathology
Table 10: Summary of Maternal Performance and Mortality
SEE ATTACHMENT - CONFIDENTIAL!

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Mean fetal body weights (male, female and combined) were considered unaffected by treatment with the test item up to 1000 mg/kg/day.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was considered unaffected by treatment with the test item up to 1000 mg/kg/day.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Litter size was considered unaffected by treatment with the test item up to 1000 mg/kg/day.
Mean litter sizes were 11.4, 12.0, 10.9 and 10.1 live fetuses/litter in the control, 100, 300 and 1000 mg/kg/day groups, respectively. There were no dead fetuses in this study. The slightly lower mean litter size for individual females in the mid and high dose groups was related to a slightly lower number of corpora lutea and implantation sites. As the treatment with test item is started after implantation (Day 6 post-coitum), this was considered not related to treatment with the test item. The mean litter size was considered to reflect normal biological variation.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

Anogenital distance (absolute and normalized for body weight) in male and female fetuses was considered not to be affected by treatment with the test item up to 1000 mg/kg/day.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

In this study, one external malformation was observed in one fetus of the 1000 mg/kg/day group, presenting with polydactyly. Due to its isolated occurrence, this was considered a chance finding.
No external variations were observed in any fetuses in this study.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations occurred in 0 (0), 1 (1), 2 (2) and 1 (1) fetuses (litters) in 0, 100, 300 and 1000 mg/kg/day groups, respectively. Two fetuses presented with sternoschisis, one in the low- and one in the mid-dose group. The single occurrences were considered chance findings and not indicative of a test item-related effect. The bent humerus identified in a mid-dose fetus was discovered in isolation and also considered a chance finding. Lastly, the supernumerary phalange was consistent with the external observation of polydactyly in the same fetus, already discussed above (see section 7.4.1). Due to its isolated occurrence, this
was considered a chance finding.
All skeletal variations occurred in the absence of a dose-related incidence trend and/or infrequently. Therefore, they were considered not to be test item-related.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Two visceral malformations were discovered: one fetus in the 300 and 1000 mg/kg/day group each were found to have a small eye, during cephalic examination. Single occurrence at each dose level is not indicative of a test item-related effect and therefore, the small eyes were considered chance findings.
Visceral variations observed in this study affected the liver (supernumerary lobes) and ureter (convoluted and dilated). In the case of liver findings, left lateral supernumerary lobe was only noted in the control group, and left medial and right medial supernumerary lobes in the test item groups were not found to have significantly deviated from the control group and as such did not indicate any test item-relationship. Findings regarding the ureter were incidental and therefore also ruled out as a test item-related effect.

Details on embryotoxic / teratogenic effects:

The following figures and table are attached to this robust study summary that were discussed and summarized above:
Table 11: Summary of Ovarian and Uterine Examinations and Litter Observations
Table 12. Summary of Fetal Abnormalities by Finding
Table 13: Summary of Fetal Abnormalities by Classification
SEE ATTACHMENT - CONFIDENTIAL!

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

Table: Summary of Clinical Observations: Gestation

Sex: Females from Day 2 to 21 Observation Type: All Types	0 mg/kg/day Group 1	100 mg/kg/day Group 2	300 mg/kg/day Group 3	1000 mg/kg/day Group 4
Salivation   Number of Animals Affected   Number of Times Recorded   % of Affected Animals   First to Last seen
	0	1	7	21
	0	2	11	53
	0	5	32	95
	-	10 - 11	8 - 12	7 - 12
Breathing, Abnormal Sounds   Number of Animals Affected   Number of Times Recorded   % of Affected Animals   First to Last seen
	0	0	0	2
	0	0	0	3
	0	0	0	9
	-	-	-	7 - 21
Fur, Erected   Number of Animals Affected   Number of Times Recorded   % of Affected Animals   First to Last seen
	2	1	1	3
	11	3	7	20
	9	5	5	14
	14 - 20	18 - 20	10 - 16	10 - 20
Skin, Scab, Generalized   Number of Animals Affected   Number of Times Recorded   % of Affected Animals   First to Last seen
	0	0	1	0
	0	0	1	0
	0	0	5	0
	-	-	21 - 21	-
Skin, Scab, Ventral Cervical   Number of Animals Affected   Number of Times Recorded   % of Affected Animals   First to Last seen
	0	0	0	2
	0	0	0	10
	0	0	0	9
	-	-	-	11 - 17

 

 

Table: Summary of Body Weights: Gestation / Bodyweight (g)

Sex: Female		Day(s) Relative to Mating (Litter: A)
		2	6	9	12	15	18	21
0 mg/kg/day Group 1	Mean	207.5	219.0	228.3	245.0	256.9	286.6	321.4
	SD	12.4	12.5	13.2	14.4	15.8	18.5	20.0
	N	22	22	22	22	22	22	22
100 mg/kg/day Group 2	Mean	211.5	224.9	235.1	251.3	264.5	294.3	331.5
	SD	13.5	13.8	13.8	14.9	16.5	18.2	22.9
	N	22	22	22	22	22	22	22
	%Diff	1.9	2.7	3.0	2.6	3.0	2.7	3.1
300 mg/kg/day Group 3	Mean	203.6	216.3	226.4	242.8	256.4	284.0	317.2
	SD	12.9	13.6	14.1	14.7	16.8	20.8	24.6
	N	21	21	21	21	21	21	21
	%Diff	-1.9	-1.2	-0.8	-0.9	-0.2	-0.9	-1.3
1000 mg/kg/day Group 4	Mean	206.9	219.5	227.5	241.3	255.0	282.7	307.5
	SD	14.9	16.2	16.7	19.2	20.4	24.6	30.5
	N	22	22	22	22	22	22	22
	%Diff	-0.3	0.2	-0.3	-1.5	-0.7	-1.4	-4.3

 

Table: Summary of Body Weight Gains (g): Gestation / Bodyweight Gain (Interval)

 

Sex: Female		Day(s) Relative to Mating (Litter: A)
		6 → 9 [G]	9 → 12 [G]	12 → 15 [G]	15 → 18 [G]	18 → 21 [G1]	6 → 21 [G]
0 mg/kg/day Group 1	Mean	9.3	16.8	11.9	29.7	34.8	102.4
	SD	4.0	3.6	5.3	5.9	5.7	11.6
	N	22	22	22	22	22	22
100 mg/kg/day Group 2	Mean	10.3	16.2	13.2	29.7	37.2	106.6
	SD	4.4	3.6	5.9	3.8	7.5	13.1
	N	22	22	22	22	22	22
300 mg/kg/day Group 3	Mean	10.1	16.3	13.7	27.6	33.1	100.9
	SD	3.8	2.9	4.4	5.3	8.4	15.3
	N	21	21	21	21	21	21
1000 mg/kg/day Group 4	Mean	8.0	13.8*	13.8	27.6	24.8	88.0**
	SD	3.0	3.8	4.0	7.4	13.5	17.5
	N	22	22	22	22	22	22

[G] - Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01

[G1] - Kruskal-Wallis & Dunn

 

 

Table: Summary of Gravid Uterine Weights and Corrected Body Weights: Gestation

Sex: Female Day(s) Relative to Mating (Litter: A)		0 mg/kg/day Group 1	100 mg/kg/day Group 2	300 mg/kg/day Group 3	1000 mg/kg/day Group 4
Bodyweight on Day 6 (g) [G]	Mean	219.0	224.9	216.3	219.5
	SD	12.5	13.8	13.6	16.2
	N	22	22	21	22
	%Diff	-	2.7	-1.2	0.2
Terminal Body Weight (g) [G]	Mean	321.4	331.5	317.2	307.5
	SD	20.0	22.9	24.6	30.5
	N	22	22	21	22
	%Diff	-	3.1	-1.3	-4.3
Gravid Uterus Weight (g) [G]	Mean	77.27	82.19	74.78	68.69
	SD	12.21	11.95	16.12	18.40
	N	22	22	21	22
	%Diff	-	6.36	-3.22	-11.11
Adjusted BWG (6-abw) (g) [G]	Mean	25.14	24.45	26.12	19.26
	SD	8.39	7.08	7.30	10.82
	N	22	22	21	22
	%Diff	-	-2.73	3.93	-23.36

 

 

Table: Summary of Food Consumption: Gestation / Food Mean Daily Consumption (g/animal/day)

 

Sex: Female		Day(s) Relative to Mating (Litter: A)
		6 → 9	9 → 12	12 → 15	15 → 18	18 → 21	6 → 21
0 mg/kg/day Group 1	Mean	18.58	19.50	20.11	20.82	19.21	19.64
	SD	2.55	2.23	1.94	2.28	3.68	1.96
	N	22	22	22	22	22	22
100 mg/kg/day Group 2	Mean	19.76	20.42	20.95	20.80	19.94	20.38
	SD	2.25	2.51	2.36	2.56	2.89	1.94
	N	22	22	22	22	22	22
	%Diff	6.36	4.74	4.22	-0.07	3.79	3.73
300 mg/kg/day Group 3	Mean	18.92	19.14	20.49	20.30	18.59	19.49
	SD	2.01	1.67	1.50	2.09	2.42	1.31
	N	21	21	21	21	21	21
	%Diff	1.86	-1.83	1.92	-2.48	-3.25	-0.78
1000 mg/kg/day Group 4	Mean	17.88	17.74*	20.45	20.67	17.85	18.92
	SD	2.07	2.60	1.71	2.29	3.25	1.76
	N	22	22	22	22	22	22
	%Diff	-3.75	-9.01	1.73	-0.73	-7.10	-3.69

Anova & Dunnett: * = p ≤ 0.05

 

Table: Summary of Thyroid Hormone Values / Day: 21 Relative to Mating (Litter: A)

Sex: Female		Reporting Special Chemistry
		T3 (ng/mL) [G]	T4 (ng/mL) [G]	TSH (mU/L) [G]
0 mg/kg/day Group 1	Mean	0.406	21.55	0.3086
	SD	0.088	4.24	0.1713
	N	22	22	22
100 mg/kg/day Group 2	Mean	0.380	19.75	0.2790
	SD	0.079	4.94	0.1362
	N	22	22	22
	tCtrl	0.94	0.92	0.90
300 mg/kg/day Group 3	Mean	0.361	21.42	0.2735
	SD	0.092	5.76	0.1376
	N	21	21	21
	tCtrl	0.89	0.99	0.89
1000 mg/kg/day Group 4	Mean	0.324**	21.92	0.2978
	SD	0.075	4.64	0.1734
	N	22	22	22
	tCtrl	0.80	1.02	0.96

[G] - Anova & Dunnett: ** = p ≤ 0.01

 

Table: Summary of Thyroid Weights

 

Sex: Female Day(s) Relative to Mating (Litter: A)		0 mg/kg/day Group 1	100 mg/kg/day Group 2	300 mg/kg/day Group 3	1000 mg/kg/day Group 4
Terminal Body Weight (g) [G]	Mean	321.4	331.5	317.2	307.5
	SD	20.0	22.9	24.6	30.5
	N	22	22	21	22
	%Diff	-	3.1	-1.3	-4.3
Gland, Thyroid Weight (g) [G1]	Mean	0.01329	0.01423	0.01398	0.01367
	SD	0.00277	0.00216	0.00291	0.00240
	N	22	22	20	22
	%Diff	-	7.11598	5.18303	2.87376
Gland, Thyroid (%bw) [G1]	Mean	0.00412	0.00430	0.00443	0.00453
	SD	0.00076	0.00061	0.00099	0.00116
	N	22	22	20	22
	%Diff	-	4.26134	7.35242	9.84167

[G] - Anova & Dunnett

[G1] - Anova & Dunnett

 

Table: Summary of Macroscopic Pathology

Removal Reason(s): TERMINAL EUTHANASIA Summary: Incidence	Female
	0 mg/kg/day Group 1	100 mg/kg/day Group 2	300 mg/kg/day Group 3	1000 mg/kg/day Group 4
Number of Animals:	22	22	22	22
GLAND, THYROID   Submitted   No Visible Lesions   Small
	22	22	22	22
	21	22	22	22
	1	0	0	0
LIVER   Submitted   Abnormal appearance; lateral lobe
	1	0	0	0
	1	.	.	.
PLACENTA   Submitted   Abnormal appearance
	1	0	0	0
	1	.	.	.
SKIN   Submitted   Scab; flank
	0	0	1	0
	.	.	1	.

 

Table: Summary of Microscopic Pathology

Removal Reason(s): TERMINAL EUTHANASIA Summary: Incidence	Female
	0 mg/kg/day Group 1	100 mg/kg/day Group 2	300 mg/kg/day Group 3	1000 mg/kg/day Group 4
Number of Animals:	22	22	22	22
GLAND, THYROID   Examined   No Visible Lesions   Ectopia; thymic   .... minimal   Hypertrophy; follicular cell   .... minimal
	22	22	22	22
	19	21	20	20
	2	0	0	0
	2	0	0	0
	1	1	2	2
	1	1	2	2

 

Table: Summary of Maternal Performance and Mortality

Sex: Female Day(s) Relative to Mating (Litter: A)		0 mg/kg/day Group 1	100 mg/kg/day Group 2	300 mg/kg/day Group 3	1000 mg/kg/day Group 4
Group Size - Females		22	22	22	22
Number of Females Pregnant [f]	N+ve	22	22	21	22
	%	100.0	100.0	95.5	100.0
Female with Live Fetuses [f]	N+ve	22	22	21	22
	%	100.0	100.0	100.0	100.0
Total Resorptions [f] N+ve	0	0	0	0
	%	0.0	0.0	0.0	0.0
Female with all Nonviable [f]	N+ve	0	0	0	0
	%	0.0	0.0	0.0	0.0
Terminal Euthanasia [f]	N+ve	22	22	22	22
	%	100.0	100.0	100.0	100.0
Unscheduled Death/Euthanasia [f]	N+ve	0	0	0	0
	%	0.0	0.0	0.0	0.0

[f] - Fisher's Exact

 

Table: Summary of Ovarian and Uterine Examinations and Litter Observations

Sex: Female Day(s) Relative to Mating (Litter: A)		0 mg/kg/day Group 1	100 mg/kg/day Group 2	300 mg/kg/day Group 3	1000 mg/kg/day Group 4
Female with Live Fetuses	N+ve	22	22	21	22
	%	100.0	100.0	100.0	100.0
Number of Corpora Lutea [k]	Mean	13.1	13.1	12.4	12.3
	SD	1.3	1.9	1.6	2.1
	N	22	22	21	21
	%Diff	-	0.0	-5.4	-6.2
Number of Implantations [k]	Mean	11.9	12.4	11.5	10.9
	SD	1.6	1.8	2.1	2.5
	N	22	22	21	22
	%Diff	-	4.6	-2.9	-8.4
Pre-implantation Loss (%) [k]	Mean	8.93	4.99	7.17	10.99
	SD	12.08	5.56	11.99	15.30
	N	22	22	21	21
	%Diff	-	-44.08	-19.65	23.05
Total Number of Fetuses [k]	Mean	11.4	12.0	10.9	10.1
	SD	1.8	1.9	2.4	3.0
	N	22	22	21	22
	%Diff	-	5.6	-4.8	-11.2
Number of Live Fetuses [k]	Mean	11.4	12.0	10.9	10.1
	SD	1.8	1.9	2.4	3.0
	N	22	22	21	22
	%Diff	-	5.6	-4.8	-11.2
Number of Dead Fetuses [k]	Mean	0.0	0.0	0.0	0.0
	SD	0.0	0.0	0.0	0.0
	N	22	22	21	22
	%Diff	-	-	-	-
Number of Early Resorptions [k]	Mean	0.5	0.3	0.7	0.7
	SD	0.7	0.6	0.9	1.0
	N	22	22	21	22
	%Diff	-	-30.0	46.7	60.0
Number of Late Resorptions [k]	Mean	0.0	0.0	0.0	0.0
	SD	0.0	0.2	0.0	0.0
	N	22	22	21	22
	%Diff	-	-	-	-
Total Number of Resorptions [k]	Mean	0.5	0.4	0.7	0.7
	SD	0.7	0.6	0.9	1.0
	N	22	22	21	22
	%Diff	-	-20.0	46.7	60.0
Post-implantation Loss (%) [k]	Mean	3.85	2.98	6.33	8.23
	SD	5.63	4.80	8.92	14.13
	N	22	22	21	22
	%Diff	-	-22.60	64.35	113.68
Number of Live Male Fetuses [k]	Mean	5.8	6.3	5.3	4.8
	SD	1.8	2.1	1.9	2.0
	N	22	22	21	22
	%Diff	-	8.6	-8.3	-17.2
Number of Live Female Fetuses [k]	Mean	5.6	5.7	5.5	5.3
	SD	1.7	2.2	1.7	2.4
	N	22	22	21	22
	%Diff	-	2.4	-1.2	-4.9
Live Male Fetus/Litter (%) [k]	Mean	50.72	52.77	48.96	47.78
	SD	12.69	15.36	11.64	16.06
	N	22	22	21	22
	%Diff	-	4.04	-3.47	-5.80
Live Female Fetuses/Litter (%) [k]	Mean	49.28	47.23	51.04	52.22
	SD	12.69	15.36	11.64	16.06
	N	22	22	21	22
	%Diff	-	-4.15	3.57	5.97
Mean Fetal Weight males (g) [G]	Mean	5.144	5.140	5.206	5.074
	SD	0.281	0.210	0.281	0.378
	N	22	22	21	22
	%Diff	-	-0.071	1.223	-1.361
Mean Fetal Weight females (g) [G]	Mean	4.848	4.861	4.968	4.830
	SD	0.275	0.233	0.242	0.359
	N	22	22	21	22
	%Diff	-	0.279	2.480	-0.377
Mean Fetal Weight all (g) [G]	Mean	4.991	5.012	5.078	4.949
	SD	0.235	0.202	0.232	0.347
	N	22	22	21	22
	%Diff	-	0.423	1.748	-0.832
Mean Fetal AGD males (mm) [G1]	Mean	2.84	2.81	2.85	2.76
	SD	0.17	0.20	0.18	0.18
	N	22	22	21	22
	%Diff	-	-1.13	0.39	-2.58
Mean Fetal AGD females (mm) [G1]	Mean	1.40	1.44	1.42	1.37
	SD	0.22	0.23	0.20	0.19
	N	22	22	21	22
	%Diff	-	2.69	1.94	-1.95
Mean Normalized Fetal AGD m [G]	Mean	1.646	1.627	1.645	1.611
	SD	0.115	0.106	0.100	0.101
	N	22	22	21	22
	%Diff	-	-1.175	-0.075	-2.154
Mean Normalized Fetal AGD f [G]	Mean	0.827	0.848	0.836	0.811
	SD	0.132	0.132	0.120	0.103
	N	22	22	21	22
	%Diff	-	2.474	0.998	-2.007

[k] - Kruskal-Wallis & Dunn

[G] - Anova & Dunnett

[G1] - Anova & Dunnett

 

Tables: Summary of Fetal Abnormalities by Finding

Exam Type: External		0 mg/kg/day Group 1	100 mg/kg/day Group 2	300 mg/kg/day Group 3	1000 mg/kg/day Group 4
Number of Fetuses Examined:		251	265	228	223
Number of Fetuses Evaluated:		251	266	228	223
Number of Litters Examined:		22	22	21	22
Number of Litters Evaluated:		22	22	21	22
Paw/digit
Hindpaw, Left, Polydactyly – Malformation	Fetuses N(%)	0(0.00)	0(0.00)	0(0.00)	1(0.41)
	Litters N(%)	0(0.0)	0(0.0)	0(0.0)	1(4.5)

[Fetuses %] - Kruskal-Wallis & Dunn

FetusesN(%) N=Group Fetal Incidence;(%)=Mean Litter % of Fetuses with the Abnormality

 

		0 mg/kg/day Group 1	100 mg/kg/day Group 2	300 mg/kg/day Group 3	1000 mg/kg/day Group 4
Number of Fetuses Examined:		128	132	114	109
Number of Fetuses Evaluated:		251	266	228	223
Number of Litters Examined:		22	22	21	22
Number of Litters Evaluated:		22	22	21	22
Exam Type: Fixed Head
Eye
Eye, Left, Small – Malformation	Fetuses N(%)	0(0.00)	0(0.00)	1(0.68)	1(0.91)
	Litters N(%)	0(0.0)	0(0.0)	1(4.8)	1(4.5)
Exam Type: FreshVisBody
Liver
Lobe, Left lateral, Supernumerary - Variation	Fetuses N(%)	1(0.76)	0(0.00)	0(0.00)	0(0.00)
	Litters N(%)	1(4.5)	0(0.0)	0(0.0)	0(0.0)
Lobe, Left medial, Supernumerary - Variation	Fetuses N(%)	4(3.33)	13(10.84)	5(3.74)	4(3.33)
	Litters N(%)	2(9.1)	7(31.8)	3(14.3)	3(13.6)
Lobe, Right medial, Supernumerary - Variation	Fetuses N(%)	2(1.67)	7(5.05)	4(6.51)	9(9.18)
	Litters N(%)	2(9.1)	7(31.8)	3(14.3)	6(27.3)
Ureter
Ureter, Left, Convoluted - Variation	Fetuses N(%)	0(0.00)	1(0.76)	1(0.68)	0(0.00)
	Litters N(%)	0(0.0)	1(4.5)	1(4.8)	0(0.0)
Ureter, Left, Dilatation, Minimal - Variation	Fetuses N(%)	0(0.00)	0(0.00)	1(0.68)	1(0.65)
	Litters N(%)	0(0.0)	0(0.0)	1(4.8)	1(4.5)
Ureter, Right, Convoluted, Minimal - Variation	Fetuses N(%)	0(0.00)	0(0.00)	1(0.95)	1(2.27)
	Litters N(%)	0(0.0)	0(0.0)	1(4.8)	1(4.5)

 

 

		0 mg/kg/day Group 1	100 mg/kg/day Group 2	300 mg/kg/day Group 3	1000 mg/kg/day Group 4
Number of Fetuses Examined:		123	133	114	114
Number of Fetuses Evaluated:		251	266	228	223
Number of Litters Examined:		22	22	21	22
Number of Litters Evaluated:		22	22	21	22
Exam Type: Skeletal
Forelimb
Humerus, Right, Bent - Malformation	Fetuses N(%)	0(0.00)	0(0.00)	1(2.38)	0(0.00)
	Litters N(%)	0(0.0)	0(0.0)	1(4.8)	0(0.0)
Hindlimb
Hindpaw phalanges, 1 or more, Supernumerary - Malformation	Fetuses N(%)	0(0.00)	0(0.00)	0(0.00)	1(0.76)
	Litters N(%)	0(0.0)	0(0.0)	0(0.0)	1(4.5)
Pelvic girdle
Ilium, Both, Misaligned – Variation	Fetuses N(%)	5(4.85)	3(2.27)	5(5.71)	7(5.42)
	Litters N(%)	4(18.2)	3(13.6)	5(23.8)	6(27.3)
Rib
Costal cartilage, 1 or more, Fused - Variation	Fetuses N(%)	0(0.00)	0(0.00)	0(0.00)	1(0.91)
	Litters N(%)	0(0.0)	0(0.0)	0(0.0)	1(4.5)
Rib, 1 or more, Wavy rib – Variation	Fetuses N(%)	32(26.89)	43(33.61)	25(24.17)	34(28.74)
	Litters N(%)	16(72.7)	16(72.7)	12(57.1)	14(63.6)
Scapula
Scapula, Both, Bent – Variation	Fetuses N(%)	1(0.76)	0(0.00)	1(0.95)	0(0.00)
	Litters N(%)	1(4.5)	0(0.0)	1(4.8)	0(0.0)
Scapula, Right, Bent – Variation	Fetuses N(%)	0(0.00)	2(1.48)	2(3.17)	0(0.00)
	Litters N(%)	0(0.0)	2(9.1)	2(9.5)	0(0.0)
Skull
Frontal, Both, Incomplete ossification – Variation	Fetuses N(%)	0(0.00)	0(0.00)	2(1.59)	3(2.58)
	Litters N(%)	0(0.0)	0(0.0)	1(4.8)	3(13.6)
Exam Type: Skeletal
Interparietal, Incomplete ossification - Variation	Fetuses N(%)	24(18.27)	23(17.65)	13(12.78)	18(15.91)
	Litters N(%)	11(50.0)	12(54.5)	7(33.3)	9(40.9)
Parietal, Both, Incomplete ossification - Variation	Fetuses N(%)	10(7.60)	10(8.15)	12(10.12)	13(11.17)
	Litters N(%)	7(31.8)	9(40.9)	7(33.3)	6(27.3)
Parietal, Left, Incomplete ossification - Variation	Fetuses N(%)	0(0.00)	1(0.76)	0(0.00)	1(0.91)
	Litters N(%)	0(0.0)	1(4.5)	0(0.0)	1(4.5)
Parietal, Right, Incomplete ossification - Variation	Fetuses N(%)	8(6.19)	4(3.41)	1(0.95)	2(1.67)
	Litters N(%)	6(27.3)	4(18.2)	1(4.8)	2(9.1)
Squamosal, Both, Incomplete ossification - Variation	Fetuses N(%)	4(2.81)	4(3.41)	6(5.16)	4(3.30)
	Litters N(%)	4(18.2)	4(18.2)	2(9.5)	4(18.2)
Squamosal, Left, Incomplete ossification	- Variation	Fetuses N(%)	1(0.91)	1(1.14)	0(0.00)
	Litters N(%)	1(4.5)	1(4.5)	0(0.0)	3(13.6)
Squamosal, Right, Incomplete ossification - Variation	Fetuses N(%)	3(2.58)	2(1.52)	1(0.79)	1(0.76)
	Litters N(%)	3(13.6)	2(9.1)	1(4.8)	1(4.5)
Supraoccipital, Incomplete ossification – Variation	Fetuses N(%)	8(6.60)	6(4.89)	5(4.37)	7(6.06)
	Litters N(%)	6(27.3)	6(27.3)	3(14.3)	5(22.7)
Zygomatic arch, Both, Incomplete ossification - Variation	Fetuses N(%)	1(0.91)	3(2.80)	6(4.65)	5(4.55)
	Litters N(%)	1(4.5)	3(13.6)	2(9.5)	3(13.6)
Zygomatic arch, Left, Incomplete ossification - Variation	Fetuses N(%)	2(1.56)	2(1.67)	2(1.59)	1(0.91)
	Litters N(%)	2(9.1)	2(9.1)	2(9.5)	1(4.5)
Zygomatic arch, Right, Incomplete ossification - Variation	Fetuses N(%)	7(5.54)	2(1.33)	2(1.75)	2(1.82)
	Litters N(%)	7(31.8)	2(9.1)	2(9.5)	2(9.1)
Sternebra
Sternebra, 1 or more, Branched - Variation	Fetuses N(%)	1(0.76)	2(1.41)	0(0.00)	0(0.00)
	Litters N(%)	1(4.5)	2(9.1)	0(0.0)	0(0.0)
Sternebra, 1 or more, Misaligned - Variation	Fetuses N(%)	3(2.16)	1(0.76)	6(5.09)	3(3.79)
	Litters N(%)	3(13.6)	1(4.5)	5(23.8)	3(13.6)
Sternebra, 1 or more, Sternoschisis - Malformation	Fetuses N(%)	0(0.00)	1(0.76)	1(0.79)	0(0.00)
	Litters N(%)	0(0.0)	1(4.5)	1(4.8)	0(0.0)
Sternebra, 1 or more, Unossified - Variation	Fetuses N(%)	0(0.00)	0(0.00)	0(0.00)	1(2.27)
	Litters N(%)	0(0.0)	0(0.0)	0(0.0)	1(4.5)
Sternebra, 1 or more, Wide - Variation	Fetuses N(%)	1(0.76)	1(0.76)	0(0.00)	0(0.00)
	Litters N(%)	1(4.5)	1(4.5)	0(0.0)	0(0.0)
Sternebra, 1 or more, Incomplete ossification – Variation	Fetuses N(%)	1(0.65)	0(0.00)	1(1.19)	2(2.84)
	Litters N(%)	1(4.5)	0(0.0)	1(4.8)	2(9.1)
Supernumerary rib
Cervical, 1 or more, Full - Variation	Fetuses N(%)	4(3.38)	0(0.00)	1(1.19)	1(0.91)
	Litters N(%)	3(13.6)	0(0.0)	1(4.8)	1(4.5)
Cervical, 1 or more, Short - Variation	Fetuses N(%)	5(4.13)	3(2.42)	6(5.16)	6(6.36)
	Litters N(%)	3(13.6)	3(13.6)	4(19.0)	5(22.7)
Thoracolumbar, 1 or more, Full - Variation	Fetuses N(%)	6(4.70)	9(6.63)	9(8.02)	12(9.19)
	Litters N(%)	5(22.7)	5(22.7)	8(38.1)	7(31.8)
Thoracolumbar, 1 or more, Short - Variation	Fetuses N(%)	74(60.24)	78(59.12)	61(52.53)	69(59.68)
	Litters N(%)	21(95.5)	21(95.5)	19(90.5)	20(90.9)
Vertebra
Thoracic centrum, 1 or more, Incomplete ossification - Variation	Fetuses N(%)	2(1.67)	5(4.03)	2(1.98)	1(0.76)
	Litters N(%)	2(9.1)	2(9.1)	2(9.5)	1(4.5)

[Fetuses %] - Kruskal-Wallis & Dunn

FetusesN(%) N=Group Fetal Incidence;(%)=Mean Litter % of Fetuses with the Abnormality

 

Tables: Summary of Fetal Abnormalities by Classification

	0 mg/kg/day Group 1	100 mg/kg/day Group 2	300 mg/kg/day Group 3	1000 mg/kg/day Group 4
Number of Fetuses Examined:	251	265	228	223
Number of Fetuses Evaluated:	251	266	228	223
Number of Litters Examined:	22	22	21	22
Number of Litters Evaluated:	22	22	21	22
Exam Type: External
Malformation
Number of Fetuses	0	0	0	1
Litter % of Fetuses [k]	0.00	0.00	0.00	0.41
Number of Litters	0	0	0	1
All classifications
Number of Fetuses	0	0	0	1
Litter % of Fetuses [k]	0.00	0.00	0.00	0.41
Number of Litters	0	0	0	1

 

	0 mg/kg/day Group 1	100 mg/kg/day Group 2	300 mg/kg/day Group 3	1000 mg/kg/day Group 4
Number of Fetuses Examined:	128	132	114	109
Number of Fetuses Evaluated:	251	266	228	223
Number of Litters Examined:	22	22	21	22
Number of Litters Evaluated:	22	22	21	22
Exam Type: Fixed Head
Malformation
Number of Fetuses	0	0	1	1
Litter % of Fetuses [k]	0.00	0.00	0.68	0.91
Number of Litters	0	0	1	1
All classifications
Number of Fetuses	0	0	1	1
Litter % of Fetuses [k]	0.00	0.00	0.68	0.91
Number of Litters	0	0	1	1

[k] - Kruskal-Wallis & Dunn

 

	0 mg/kg/day Group 1	100 mg/kg/day Group 2	300 mg/kg/day Group 3	1000 mg/kg/day Group 4
Number of Fetuses Examined:	128	132	114	109
Number of Fetuses Evaluated:	251	266	228	223
Number of Litters Examined:	22	22	21	22
Number of Litters Evaluated:	22	22	21	22
Exam Type: FreshVisBody
Variation
Number of Fetuses	7	20	10	15
Litter % of Fetuses [k]	5.76	15.74	11.20	15.43
Number of Litters	3	12	7	10
All classifications
Number of Fetuses	7	20	10	15
Litter % of Fetuses [k]	5.76	15.74	11.20	15.43
Number of Litters	3	12	7	10

[k] - Kruskal-Wallis & Dunn

 

	0 mg/kg/day Group 1	100 mg/kg/day Group 2	300 mg/kg/day Group 3	1000 mg/kg/day Group 4
Number of Fetuses Examined:	123	133	114	114
Number of Fetuses Evaluated:	251	266	228	223
Number of Litters Examined:	22	22	21	22
Number of Litters Evaluated:	22	22	21	22
Exam Type: Skeletal
Variation
Number of Fetuses	102	109	83	91
Litter % of Fetuses [k]	82.53	82.72	73.03	78.83
Number of Litters	22	22	20	21
Malformation
Number of Fetuses	0	1	2	1
Litter % of Fetuses [k]	0.00	0.76	3.17	0.76
Number of Litters	0	1	2	1
All classifications
Number of Fetuses	102	109	83	91
Litter % of Fetuses [k]	82.53	82.72	73.03	78.83
Number of Litters	22	22	20	21

[k] - Kruskal-Wallis & Dunn

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the results of this prenatal developmental toxicity study in time-mated female Wistar Han rats the maternal and developmental No Observed Adverse Effect Levels (NOAELs) for Maleated TOFA were established as being at least 1000 mg/kg/day.

Executive summary:

The objectives of this study were to determine the potential of Maleated TOFA to induce developmental toxicity after maternal exposure during the critical period of organogenesis and to characterize maternal toxicity at the exposure levels tested when given orally by gavage to time-mated female Wistar Han rats from Days 6 to 20 post-coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated.

Time-mated female Wistar Han rats were treated with Maleated TOFA from Days 6 to 20 post-coitum, inclusive by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg/day. The rats of the control group received the vehicle, propylene glycol. Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.

No maternal toxicity was observed up to the highest dose level tested (1000 mg/kg/day) and all animals survived until scheduled necropsy.

At 1000 mg/kg/day, a minor decrease in mean food consumption between Days 9-12 and 18-21 post-coitum, and consequently a lower mean body weight and body weight gain was noted. Based on the small effect, this decrease was considered to be not adverse.

A decrease in corrected body weight gain and gravid uterus was noted at 1000 mg/kg/day. Based on the contribution of a few individual females and occurrence in the control group, this effect was considered not adverse at the magnitude of change observed.

Mean serum levels of total triiodothyronine (T3) showed an apparent dose-related trend towards a decrease across the dose groups, with statistical significance at 1000 mg/kg/day. As mean total T3 remained within the historical control data range at these dose levels, this decrease was considered not to represent an adverse effect.

No test item-related or toxicologically significant changes were noted in any of the remaining maternal parameters investigated in this study (i.e. clinical appearance, thyroid hormone levels (thyroxine (T4), thyroid stimulating hormone (TSH)), thyroid gland weights, macroscopic evaluation, microscopic evaluation of the thyroid gland, uterine contents, corpora lutea, implantation sites and pre- and post-implantation loss).

No developmental toxicity was observed up to the highest dose level tested (1000 mg/kg/day).

No test item-related changes were noted in any of the developmental parameters investigated in this study (i.e. litter size, sex ratio, fetal body weights, anogenital distance, external, visceral and skeletal malformations and developmental variations).

In conclusion, based on the results of this prenatal developmental toxicity study in time-mated female Wistar Han rats the maternal and developmental No Observed Adverse Effect Levels (NOAELs) for Maleated TOFA were established as being at least 1000 mg/kg/day.