Tris(2-ethylhexyl) phosphate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22-May-2019 - 01-Jul-2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted in accordance with international guidlines and in accordance with GLP. All relevant validity criteria were met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

1998

Deviations:

yes

Remarks:

Female#55 at 300 mg/kg/day was dosed slightly longer i.e. on day of necropsy. Fetuses of litter A035 at 100 mg/kg/day had an early delivery on post- coitum Day 27 were examined externally.The uterus weight was not determined for females#70 at 1000 mg/kg.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

2008

Deviations:

yes

Remarks:

Female#55 at 300 mg/kg/day was dosed slightly longer i.e. on day of necropsy. Fetuses of litter A035 at 100 mg/kg/day had an early delivery on post- coitum Day 27 were examined externally.The uterus weight was not determined for females#70 at 1000 mg/kg.

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2018

Deviations:

yes

Remarks:

Female#55 at 300 mg/kg/day was dosed slightly longer i.e. on day of necropsy. Fetuses of litter A035 at 100 mg/kg/day had an early delivery on post- coitum Day 27 were examined externally.The uterus weight was not determined for females#70 at 1000 mg/kg.

Principles of method if other than guideline:

Yes

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Tris(2-ethylhexyl) phosphate

EC Number:

201-116-6

EC Name:

Tris(2-ethylhexyl) phosphate

Cas Number:

78-42-2

Molecular formula:

C24H51O4P

IUPAC Name:

tris(2-ethylhexyl) phosphate

Test material form:

liquid

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: On 19 Jul 2019, time-mated female New Zealand White rabbits were received from Charles River (Chatillon sur Chalaronne, France).
- Females (if applicable) nulliparous and non-pregnant: Time mated female were used, Day 1-4 post-coitum (Day 0 post-coitum is defined as the day of successful mating).
- Age at study initiation: 17-20 weeks old
- Weight at study initiation: Females: 3.052 - 4.062 kg.
- Fasting period before study: No
- Housing: shelters: Individually in cages with perforated floors (Ebeco, Germany, dimensions 67 x 62 x 55 cm) equipped with water bottles.
- Water (e.g. ad libitum): Municipal tap water was freely available to each animal via water bottles/containers.
- Acclimation period: 2 days prior to the commencement of treatment.

DETAILS OF FOOD AND WATER QUALITY: Pelleted diet for rabbits (Global Diet 2030 from Harlan Teklad®, Mucedola, Milanese, Italy) was provided ad libitum throughout the study, except during designated procedures. In addition, pressed hay (Tecnilab-BMI bv, Someren, The Netherlands) was provided during the study period. The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. It was considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility. It was considered that there were no known contaminants in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 20ºC
- Humidity (%): 61 to 97 %
- Air changes (per hr): =>10 air changes/hour with 100% fresh air (no air recirculation).
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle.
IN-LIFE DATES: From: 19th Jul 2019 To Day 29 post-coitum

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1% Aqueous carboxymethyl cellulose (CMC) with 0.1% Tween 80

Details on exposure:

 PREPARATION OF DOSING SOLUTIONS:
Dosing formulations were prepared at least every 2 weeks and formulated in daily portions used within 6 hours after after removal from the refrigerator. 

VEHICLE
- Justification for use and choice of vehicle (if other than water): Trial preparations were performed to select the suitable vehicle and to establish a suitable formulation procedure. 1% Aqueous carboxymethyl cellulose (CMC) with 0.1% Tween 80 was considered suitable.
- Concentration in vehicle: concentration range of 0 to 200 mg/mL.
- Amount of vehicle (if gavage): 5 mL/kg (c.f. 5 ml/kg).
- Lot/batch no. (if required): No specified.
- Purity: Not specified.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and stability of the test item under test conditions was demonstrated in the analytical method development and validation study (Test Facility Study No. 20173310).

Details on mating procedure:

Mating procedure was not disclosed but the females arrived on Day 1 post-coitum (Day 0 post-coitum is defined as the day of successful mating).

Duration of treatment / exposure:

Day 6 to Day 28 post-coitum

Frequency of treatment:

Once daily

Duration of test:

23 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Group 4

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day

Remarks:

Group 3

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day

Remarks:

Group 2

Doses / concentrations 4

Dose / conc.:

0 mg/kg bw/day

Remarks:

Group 1

No. of animals per sex per dose:

22

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
A preliminary dose range finding study (DRF) in pregnant female New Zealand White rabbits was carried out using 6 rabbits per group with the test item by oral gavage at a dose volume of 5 mL/kg body weight at 0, 125, 250 and 500 mg/kg/day were administered. At 500 mg/kg/day, 1 out of 6 animals showed notable weight loss of 5% over Days 6-9 post-coitum, followed by (on most occasions) weight gain on subsequent days. This same animal was found to be non-gravid. All other animals of this dose group showed a similar level of weight gain as recorded among control animals. Food intake at 250 and 500 mg/kg/day was temporarily lower over Days 6-9 post-coitum, but recovered to levels similar to control means as treatment progressed. No necropsy findings were recorded that were considered to be related to treatment. No effects on examined developmental parameters were recorded in this study. For comparison, in the Tolerability Study (non-pregnant rabbits; Test Facility Study No. 20173147), 3 out of 3 animals showed weight loss up to 5% during the first 3-4 days of treatment at 500 mg/kg/day. These data suggest that –in contrast to what would normally be expected – pregnant animals show a lower degree of toxicity than non- pregnant animals. At subsequent dose levels tested in this Tolerability Study (750 and 1000 mg/kg/day), a similar degree of weight loss was recorded for most or all animals, I.e. no clear dose related-response was observed over the dose levels tested. Overall, these data indicate that the dose level of 500 mg/kg/day used as high dose in the Dose Range Finding Study may not result in a sufficient degree of toxicity in the Main Study. Therefore, based on the results of the Tolerability Study in combination with the observed lower degree of toxicity in pregnant animals vs non-pregnant animals, the limit dose of 1000 mg/kg/day was selected as high dose for the Main Study.
- Rationale for animal assignment (if not random): NA
 - Other: The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week from Day 6 to Day 28 post-coitum, inclusive. The dose volume for each animal was based on the most recent body weight measurement. The doses were given using a plastic catheter attached to a plastic disposable syringe.
The dosing formulations were stirred continuously during dose administration.
A dose control system was used as additional check to verify the dosing procedure according to Standard Operating Procedures.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
 - Time schedule: during clinical observations
 - Cage side observations checked in table [No.?]: Yes

DETAILED CLINICAL OBSERVATIONS: Yes 
 - Time schedule: From Day 6 post-coitum onwards up to the day prior to necropsy, animals will be observed at least once daily.
Animals were observed for specific clinical signs. The time of onset, grade and duration of any observed and graded for severity with the maximum grade predefined at 1, 3 or 4. Grades coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (i.e. maximum grade 1)were scored.

 BODY WEIGHT: Yes 
 - Time schedule for examinations: Days 6, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum.

 FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes 
 - Compound intake calculated as time-weighted averages from the consumption and body weight gain data: On days 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27 and 27-29 post-coitum. .

 WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: monitored on regular basis throughout the study by visual inspection of the water bottles/containers.
 
 POST-MORTEM EXAMINATIONS: Yes
 - Sacrifice on gestation day # GD 29
- Organs examined: Yes, external and visceral organs

 OTHER: The following data were recorded i.e.
* The number of corpora lutea.
* The weight of the (gravid) uterus (not for animals sacrificed before planned necropsy).
* The number of implantation sites.
* The number and distribution of live and dead fetuses.
* The number and distribution of embryo-fetal deaths.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:

- Gravid uterus weight: Yes 

- Number of corpora lutea: Yes 

- Number of implantations: Yes 

- Number of early resorptions: Yes 

- Number of late resorptions: Yes

 - Other: Yes, gross evaluation of placenta, pregnancy status

Fetal examinations:

- External examinations: Yes:
- Soft tissue examinations: Yes:
- Skeletal examinations: Yes:
- Head examinations: Yes:
- Other: The following litter data were recorded:
* Total number of fetuses
* Number of live fetuses
* Number of dead fetuses
* Individual fetal body weight
* Fetus sex

Statistics:

STATISTICAL ANALYSIS
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels. This included Dunnett-test (many-to-one-t-test), Steel-test (many-to-one rank test), Kruskal-Wallis nonparametric ANOVA test and Mann Whitney test.
An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Fisher’s exact test at the 5% significance level if the overall test was significant.
No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.

Indices:

Indices including; pre and post-implantation loss (%) and fetuses viability index (%).

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Incidental occurrence of clinical findings without any dose-related response were observed but considered not to be related to treatment with the test item.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period that was considered to be related to treatment with the test item.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Variation in body weight were observed but all within historical control range and as such not considered toxicologically relevant.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, lower mean absolute and relative food consumption was recorded over Days 6 to 9 (0.33x of control for relative food consumption; outside historical control data range) and 27 to 29 post-coitum (0.72x of control for relative food consumption; not statistically significant for relative food consumption and within historical control data range).
At 300 mg/kg/day, lower mean absolute and relative food consumption was recorded over Days 6 to 9 post-coitum (0.53x of control for relative food consumption; outside historical control data range).
At 100 mg/kg/day, absolute and relative food consumption was similar to control levels.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

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:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Details on results:

Morality:
- One control group animal (No. 4) was sacrificed for humane reasons on post-coitum Day 15. This animal showed weight loss (up to 5%) between post-coitum Days 6 and 15 and did (almost) not consume food during this period. Other than notably reduced faeces production, this animal showed no relevant clinical signs. Necropsy showed a black-brown focus (1x3 mm) on the glandular mucosa of the stomach. This female was pregnant.
- One animal at 100 mg/kg/day (No. 35) had an early delivery on post-coitum Day 27. This animal showed piloerection and lean appearance prior to early delivery, showed weight loss (up to 3%) between post-coitum Days 21 and 24 and did (almost) not consume food between post-coitum Days 21 and 27. Except for reduced faeces production, none of the clinical signs recorded for this animal were observed among other animals of this dose group. Necropsy showed no lesions.
- One animal at 300 mg/kg/day (No. 52) was found dead on post-coitum Day 29. This animal showed piloerection, reduced faeces production, and diarrhoea prior to being found dead, and did (almost) not consume food between post-coitum Days 21 and 29. Necropsy showed a beginning stage of autolysis. This female was pregnant.
- One animal at 1000 mg/kg/day (No. 82) was euthanized for humane reasons on Day 12 post-coitum. She was noted with faeces containing mucus (starting on Day 10 post-coitum) and red fluid on the manure tray mixed with faeces (starting on Day 11 post-coitum). In addition, she had severely reduced /no food intake from Days 6 to 12 post-coitum, along with a 12% body weight loss over days 6 to 12 and severely reduced faeces production from Day 9 post-coitum onwards. This female was examined by the veterinarian and found to be cold by touch. At necropsy, reddish and gelatinous content of the rectum was noted. This female was not pregnant.

Clinical Observations:
Reduced faeces production was recorded at increased incidence over the dose groups, when compared to the control group, and occurred from post-coitum Day 9 onwards.
One female at 300 mg/kg/day (No. 66) showed diarrhoea on post-coitum Days 22 and 23 and one other female at this dose (No. 65) showed pale faeces and lean appearance on post-coitum Day 28. Given the incidental occurrence of these findings, and since these did not show a dose-related response, these were considered not to be related to treatment with the test item.
Other findings occurred within the range of background findings to be expected for rabbits of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.

Body Weights and Body Weight Gains:

At 1000 mg/kg/day, slight mean weight loss was recorded over post-coitum Days 6 to 9 (-2%). Also, statistically significantly lower mean body weights were recorded on post- coitum Days 27 and 29 (5% lower than control means on both occasions). Although mean body weight gain at this dose was statistically significantly lower throughout most of the treatment period, means did not show a clear dose-related response over the dose groups. Overall mean weight gain over the treatment period was 6% at 1000 mg/kg/day vs. 11% in the control group. Mean body weights remained well within the historical control data range.
Corrected mean weight gain for gravid uterus at 1000 mg/kg/day was slightly outside the historical range for a total of 6 out of 20 animals. The mean corrected weight remained within the historical control range.
At 300 mg/kg/day, slight mean weight loss was recorded over post-coitum Days 6 to 9 (-1%). On subsequent days, body weight gain remained essentially similar to control means and means recorded at 100 and 1000 mg/kg/day (i.e. a clear dose-related trend was absent). Mean body weights remained well within the historical control data range. Therefore, the statistically significantly lower mean body weight gain on post-coitum Days 21 and 24 was considered not to represent a test item-related effect.
At 100 mg/kg/day, body weights and body weight gain remained similar to the control group. Corrected mean weight gain for gravid uterus at 100 and 300 mg/kg/day remained within the historical control range (in the control group, 100 and at 300 mg/kg/day, a total of 1/19, 2/20 and 2/20 values were slightly outside the historical control range, respectively).

Food Consumption:
At 1000 mg/kg/day, lower mean absolute and relative food consumption was recorded over Days 6 to 9 (0.33x of control for relative food consumption; outside historical control data range) and 27 to 29 post-coitum (0.72x of control for relative food consumption; not statistically significant for relative food consumption and within historical control data range).
At 300 mg/kg/day, lower mean absolute and relative food consumption was recorded over Days 6 to 9 post-coitum (0.53x of control for relative food consumption; outside historical control data range).
At 100 mg/kg/day, absolute and relative food consumption was similar to control levels.

Gross Pathology:
Macroscopic observations at necropsy of animals that survived until the scheduled treatment duration did not reveal any alterations that were considered to have arisen as a result of treatment with the test item.

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

not specified

Details on maternal toxic effects:

A total of 2 control animals (Nos. 7 and 20), one animal at 100 mg/kg/day (No. 26), one animal at 300 mg/kg/day (No. 64) and two animals at 1000 mg/kg/day (Nos. 82 (euthanized on Day 12) and 88) were not pregnant. The incidence of non-pregnancy did not show a relationship to treatment with the test item and was within the historical control range. These incidences of non-pregnancy still allowed a meaningful assessment of the data.
The numbers of pregnant females, corpora lutea and implantation sites, pre- and postimplantation loss and early and late resorptions in the control and test groups were similar and in the range of normal biological variation.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: No effects was observed up to the highest tested group

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Fetal body weights were considered not affected by treatment with the test item.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was 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):

There were no test item-related effects on litter size of any group.

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

External malformations were recorded in one control fetus and two fetuses each at 100, 300 and 1000 mg/kg/day. Some of these malformations were not previously encountered in historical control fetuses, or the incidence slightly exceeded this historical data range. However, the single occurrence, group distribution and/or occurrence in a control fetus of these malformations did not indicate a relationship to treatment with the test item. Therefore, these malformations were considered to be spontaneous in origin.
At 1000 mg/kg/day, these malformations consisted of a hind limb with syndactyly, brachydactyly and tarsal flexure in one fetus (A070-07). The latter malformation also occurred in this group in fetus A080-02 that had gastroschisis as well, and a flexure of both carpals occurred in one fetus at 300 mg/kg/day (A053-06). There was no underlying skeletal anomaly for both affected fetuses at 1000 mg/kg/day. The other externally affected fetus at 300 mg/kg/day (A059-01) had omphalocele that also occurred in one fetus at 100 mg/kg/day (A039-01) and in a late resorption at 1000 mg/kg/day (A080-04). Remaining malformations in this study were facial cleft and cleft palate (one fetus at 100 mg/kg/day (A027-02)) and short tail (control fetus A003-03; confirmed at skeletal examination).

Skeletal malformations:

no effects observed

Description (incidence and severity):

There were no test item-related effects on skeletal morphology following treatment with 1000
mg/kg/day.

Visceral malformations:

no effects observed

Other effects:

not specified

Details on embryotoxic / teratogenic effects:

Litter Size:
Mean litter sizes were 10.2, 8.9, 8.7 and 8.9 fetuses/litter for the control, 100, 300 and 1000 mg/kg/day groups, respectively.
Sex ratio:
Mean sex ratios (males:females) were 60:40, 50:50, 49:51 and 56:44 for the control, 100, 300 and 1000 mg/kg/day groups, respectively.

Fetal Body Weights:
Mean combined (male and female) fetal body weights were 37.7, 38.7, 40.5 and 38.4 gram for the control, 100, 300 and 1000 mg/kg/day groups, respectively.
Fetal Morphological Examinations:
The numbers of fetuses (litters) available for fetal morphological examination were 193 (19), 178 (20), 174 (20) and 178 (20) in the control, 100, 300 and 1000 mg/kg/day Groups, respectively. External and visceral examinations were done for all fetuses, soft tissue cephalic examination was done for approximately half of the fetuses of the same litter for all groups, and skeletal examination was done for all fetuses of Groups 1 and 4.
Dams A004, A052 and A082 (control, 300 and 1000 mg/kg/day group, respectively) did not survive until the planned day of necropsy. In addition, one dam at 100 mg/kg/day (A035) had an early delivery
External Malformations and Variations:
External malformations were recorded in one control fetus and two fetuses each at 100, 300 and 1000 mg/kg/day. Some of these malformations were not previously encountered in historical control fetuses, or the incidence slightly exceeded this historical data range. However, the single occurrence, group distribution and/or occurrence in a control fetus of these malformations did not indicate a relationship to treatment with the test item. Therefore, these malformations were considered to be spontaneous in origin.

At 1000 mg/kg/day, these malformations consisted of a hind limb with syndactyly, brachydactyly and tarsal flexure in one fetus (A070-07). The latter malformation also occurred in this group in fetus A080-02 that had gastroschisis as well, and a flexure of both carpals occurred in one fetus at 300 mg/kg/day (A053-06). There was no underlying skeletal anomaly for both affected fetuses at 1000 mg/kg/day. The other externally affected fetus at 300 mg/kg/day (A059-01) had omphalocele that also occurred in one fetus at 100 mg/kg/day (A039-01) and in a late resorption at 1000 mg/kg/day (A080-04). Remaining malformations in this study were facial cleft and cleft palate (one fetus at 100 mg/kg/day (A027-02)) and short tail (control fetus A003-03; confirmed at skeletal examination).

Visceral Malformations and Variations:
Visceral malformations were recorded in one control fetus and two fetuses (two litters) at 100 mg/kg/day, 2 fetuses (one litter) at 300 mg/kg/day and five fetuses (four litters) at 1000 mg/kg/day. Although some of these malformations were not previously encountered in historical control fetuses, or the incidence slightly exceeded this historical data range, the single occurrence and/or group distribution of these malformations did not indicate a relationship to treatment with the test item. Therefore, these malformations were considered to be spontaneous in origin. At 1000 mg/kg/day, the fetus with gastroschisis as external malformation (A080-02) also had abnormal liver lobation and a malpositioned kidney. The other affected fetuses in this group had tetralogy of Fallot (A067-06), absent kidney and ureter (A080-1), a large heart and fluid-filled body cavities (A081-07) and internal hydrocephaly (A086-09). At 300 mg/kg/day, one of the affected fetuses (A060-03) also had a malpositioned kidney, while a littermate (A060-05) had a cyst associated with the diaphragm in the thoracic cavity. At 100 mg/kg/day, visceral malformations observed were abnormal lung lobation (A038-05) and a multiple cardiovascular abnormality (A040-05). One control fetus (A005-02) had a malpositioned kidney and testis.
All visceral variations noted were considered unrelated to treatment with the test item as they occurred in the absence of a dose-related trend, occurred infrequently, in control fetuses only and/or at frequencies that were within the range of available historical control data.
Skeletal Malformations and Variations:

Skeletal malformations occurred in one control and five fetuses (four litters) at 1000 mg/kg/day. Although the incidence of some of these malformations slightly exceeded the historical data range, the single occurrence and/or group distribution of these malformations did not indicate a relationship to treatment with the test item. At 1000 mg/kg/day, these malformations consisted of a vertebral anomaly with or without associated rib anomaly in three fetuses (A075-05, A080-02 and -06), and the other two fetuses had either a costal cartilage anomaly (A067-06) or rib anomaly (A070-04). The affected control fetus (A008- 09) had a sternal anomaly. Although vertebral anomalies occurred in three fetuses at 1000 mg/kg/day only, this was considered not test-item related as a vertebral anomaly is the most common skeletal malformation among historical control fetuses.
Moreover, two cases were observed in litter A080 (-02 and -06) and because in that litter other malformations occurred in fetuses A080-01 and -02 and late resorption A080-04 (see above), it could be considered that the malformations had a maternal or hereditary origin. The other malformations at 1000 mg/kg/day (costal cartilage and rib anomaly) also occurred incidentally and incidences remained within the historical control data range. Therefore, these malformations were considered chance findings.
Noteworthy were the two related variations 13th full ribs and caudal shift of pelvic girdle that occurred at significantly higher incidences at 1000 mg/kg/day. Mean litter incidences were 71.7% and 38.2% per litter, respectively, compared to respective control values 50.5% and 13.4% per litter. Nevertheless, because the high-dose values were near the historical control maximum values of 71.4% and 38.5% for 13th full ribs and caudal shift of pelvic girdle, respectively, they were considered not test item-related.
All other variations noted were also considered not to be related to treatment with the test item as they occurred infrequently, occurred in control fetuses only and/or at frequencies that were within the range of available historical control data.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed up to limit dose of 1000 mg/kg bw/day

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Table 4. Summary of Clinical Signs in Females

Sign (Max.Grade Location)	Treatment
	Week	1	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	4	.	.
	Day	1	2	3	4	5	6	7	1	2	3	4	5	6	7	1	2	3	4	5	6	7	1	2	3
Group 1(Control)
Skin/fur Alopecia (3)	G	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	1	1	1	1	1
	%	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	1	1	1	1	0
Skin/fur Scabs (3) (Mouth)	G	1	1	1	1	1	1	1	1	1	1	1	.	.	.	.	.	.	.	.	.	.	.	.	.
	%	0	0	0	0	0	0	0	0	0	0	0	.	.	.	.	.	.	.	.	.	.	.	.	.
Skin/fur Scabs (3) (Nose)	G	.	.	1	1	1	1	1	1	1	1	1	1	1	.	.	.	.	.	.	.	.	.	.	.
	%	.	.	0	0	0	0	0	0	0	0	0	0	0	.	.	.	.	.	.	.	.	.	.	.
Skin/furScabs (3) (Flews)	G	1	1	1	1	1	1	1	1	1	1	.	.	.	.	.	.	.	.	.	.	.	1	1	1
	%	0	1	1	1	1	1	1	0	0	0	.	.	.	.	.	.	.	.	.	.	.	0	0	0
Skin/fur Wound (3) (Nose)	G	.	1	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
	%	.	0	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
Skin/fur Wound (3) (Flews)	G	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	1	.	.	.
	%	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	0	.	.	.
Red Staining (1) (Vagina)	G	.	.	.	.	.	.	.	.	.	1	.	.	.	.	.	.	.	.	.	.	.	.	.	.
	%	.	.	.	.	.	.	.	.	.	0	.	.	.	.	.	.	.	.	.	.	.	.	.	.
Secretion / excretion Watery discharge from eye (3) (Eye left)	G	.	.	.	1	1	1	1	1	3	3	2	1	2	2	2	2	2	2	1	1	1	1	1	1
	%	.	.	.	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
Secretion / excretion Faeces production reduced (3)	G	.	.	.	2	2	2	2	2	2	1	1	1	1	1	1	1	1	1	1	2	2	2	2	2
	%	.	.	.	1	1	1	1	1	1	5	5	5	4	4	4	2	2	2	2	2	2	2	5	5
Group 2 (100 mg/kg/day)
Skin / fur Piloerection (1)	G	.	.	.	.	.	.	.	.	.	.	.	.	.	.		.	.	.	.	.	.	1	1	1
	%	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	0	0	0
Skin/fur Scabs (3) (Nose)	G	1	1	1	1	1	1	1	1	1	1	1	1	1	1	.	.	.	.	.	.	.	.	.	.
	%	0	0		1	1	1	1	1	1	1	1	1	1	0	.	.	.	.	.	.	.	.	.	.
Skin/furScabs (3) (Flews)	G	1	1	1	1	1	1	1	1	1	1	1	1	1	1	.	.	.	.	.	.	.	.	.	.
	%	0	0	0	1	1	1	1	1	1	1	0	0	0	0	.	.	.	.	.	.	.	.	.	.
Skin/furScabs (3) (Snout)	G	.	.	.	.	.	.	.	.	.	.1	1	1	1	1	1	1	1	1	1	1	1	1	.	.
	%	.	.	.	.	.	.	.	.	.	0	0	0	0	0	0	0	0	0	0	0	0	0	.	.
Secretion / excretion Faeces production reduced (3)	G	.	.	.	1	1	1	1	1	1	2	2	2	2	2	2	2	2	2	2	1	1	1	2	1
	%	.	.	.	5	5	5	2	2	2	6	6	5	5	5	5	3	3	3	5	5	5	5	66
Various Lean (1)	G	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	1	1	.
	%	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	0	0	.
Broken (1) (Upper incisors)	G	.	.	.	.	.	.	.	.	.	.	.	.	.	.	1	1	1	1	.	.	.	.	.	.
	%	.	.	.	.	.	.	.	.	.	.	.	.	.	.	0	0	0	0	.	.	.	.	.	.
Group 3 (300 mg/kg/day)
Skin / fur Piloerection (1)	G	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	1	1	.
	%	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	0	0	.
Skin / fur Alopecia (3)	G	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	1	.
	%	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	0	.
Skin / fur Scars (3) (Flews)	G	.	.	.	.	.	.	.	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	1	.
	%	.	.	.	.	.	.	.	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	.
Skin / fur Scabs (3) (Mouth)	G	.	.	.	.	.	.	.	.	1	1	1	1	1	1	1	1	.	.	.	.	.	.	.	.
	%	.	.	.	.	.	.	.	.	0	0	0	0	0	0	0	0	.	.	.	.	.	.	.	.
Skin / fur Scabs (3) (Nose)	G	.	1	1	1	1	1	1	1	1	1	1	1	.	.	.	.	.	.	.	.	.	.	.	.
	%	.	0	0	0	0	0	0	0	0	0	0	0	.	.	.	.	.	.	.	.	.	.	.	.
Skin / fur Scabs (3) (Flews)	G	1	1	1	1	1	1	1	1	1	1	1	1	1	.	.	.	.	.	.	.	.	.	.	.
	%	0	1	1	0	1	1	1	0	0	0	0	0	0	.	.	.	.	.	.	.	.	.	.	.
Secretion / excretion Diarrhoea (1)	G	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	1	1	.	.	.	.	.	1
	%	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	0	0	.	.	.	.	.	A
Secretion / excretion Faeces pale (1)	G	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	1	.
	%	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.		.	.	0	.
Secretion / excretion Faeces production reduced(1)	G	.	.	.	2	2	2	1	1	1	1	1	1	1	1	1	2	2	2	2	2	2	1	1	.
	%	.	.	.	7	7	7	1	1	1	6	6	5	4	4	4	2	2	2	3	3	3	6	6	.
Various Lean (1)	G	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	1	.
	%	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	0	.
Group 4 (1000 mg/kg/day)
Skin / fur Scabs (3) (Mouth)	G	1	1	1	1	1	1	1	1	1	1	1	1	.	.	.	.	.	.	.	.	.	.	.	.
	%	0	0	0	0	0	0	1	1	0	0	0	0	.	.	.	.		.	.	.	.	.	.	.
Skin / fur Scabs (3) (Nose)	G	.	.	.	.	.	.	1	1	1	1	1	1	1	1	.		.	.	.	.	.	.	.	.
	%	.	.	.	.	.	.	0	0	0	0	0	0	0	0	.	.	.	.	.	.	.	.	.	.
Skin / fur Scabs (3) (Flews)	G	1	1	1	1	1	1	1	.	.	.	.	.	.	1	1	1	1	1	1	1	1	1	1	1
	%	0	0	0	1	1	1	1	.	.	.	.	.	.	0	0	0	0	0	0	0	0	0	0	0
Secretion / excretion Faeces containing mucus (1)	G	.	.	.	.	1	1	1	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
	%	.	.	.	.	0	0	0	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
Secretion / excretion Faeces production reduced (3)	G	.	.	.	2	2	2	2	1	1	2	2	2	2	2	2	2	1	1	1	2	2	2	1	1
	%	.	.	.	A	A	A	4	3	3	5	5	5	5	5	5	5	3	3	3	4	4	4	6	6
Secretion / excretionRed fluid on manure tray (3)	G	.	.	.	.	.	.	2	1	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.
	%	.	.	.	.	.	.	0	0	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.	.

G: median value of the highest individual dailyy grades; %: percentage of afftected animals (0=less than 5%, 1= bewteen 5% and 15%,..., A=more than 95%); .: Observation performed, sign not presnt.

 

Table 5. Summary of Mean Body Weights  and body weight gains of F0 generations.

Post Coitum day	Body Weights (g)				Body weight gains (%)
	Group 1 (Control)	Group 2 (100 mg/kg/day)	Group 3 (300 mg/kg/day)	Group 4 (1000 mg/kg/day)	Group 1 (Control)	Group 2 (100 mg/kg/day)	Group 3 (300 mg/kg/day)	Group 4 (1000 mg/kg/day)
0	3606	3637	3579	3574	-	-	-	-
6	3575	3554	3562	3550	0	0	0	0
9	3640	3587	3530	3474	2	1	-1**	-2**
12	3687	3656	3629	3573	3	3	2	1**
15	3742	3721	3677	3648	5	5	3	3
18	3809	3743	3715	3672	7	5	4	3*
21	3855	3766	3742	3720	8	6	5*	5*
24	3890	3809	3778	3753	9	7	6*	6*
27	3927	3807	3807	3740*	10	7	7	5**
29	3957	3845	3820	3754*	11	9	7	6**

*/** Dunnett-test based on pooled variance sigificant at 5%(*) or 1% (**) level; - Not applicable.

 

Table 6. Summary of Mean food consumption  and relative food consuption of F0 generations.

Post Coitum Day	Food consumption (g/animal/day)				Relative food consumption (g/kgbw/day)
	Group 1 (control)	Group 2 (100 mg/kg/day)	Group 3 (300 mg/kg/day)	Group 4 (1000 mg/kg/day)	Group 1(Control)	Group 2 (100 mg/kg/day)	Group 3 (300 mg/kg/day)	Group 4 (1000 mg/kg/day)
6-9	132	111	66**	40**	36	31	19**	12**
9-12	120	128	125	101	33	35	34	28
12-15	87	79	85	81	23	21	23	22
15-18	106	91	101	93	28	24	27	25
18-21	125	117	117	121	32	31	31	33
21-24	111	96	98	97	28	25	26	26
24-27	90	77	87	72	23	20	23	19
27-29	99	84	88	69*	25	22	23	18
Mean of means	109	98	96	84	29	26	24	23

*/** Dunnett-test based on pooled variance sigificant at 5%(*) or 1% (**) level

 

Table 7. Summary Macroscopic Findings in F0 generations.

Post Coitum	Group 1 (control)	Group 2 (100 mg/kg/day)	Group 3 (300 mg/kg/day)	Group 4 (1000 mg/kg/day)
Animals examined	22	22	22	22
Animals without findings	18	21	20	18
Animalw affected	4	1	2	4
General observations: Beginning autolysis	4	1	2	4
General Observations: Early delivery	0	0	1	0
Lung: focus/Foci	0	1	0	0
Stomach: focus/foci	0	0	0	1
Rectum	1	0	0	0
Contents: Discolouration	0	0	0	1
Oviduts Cyst(s)	0	0	0	1
Skin	2	0	0	2
Scab formation	1	0	0	0
scar (s)	0	0	1	0

# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level

 

Table 8. Summary of Maternal Survival & Pregnacy Status (%).

Dose Group	1 (0 mg/kg)		2 (100 mg/kg)		3 (300 mg/kg)		4 (1000 mg/kg)
Females on study	No.	%	No.	%	No.	%	No.	%
Female that aborted or delivered	0	0.0	1	4.5	0	0.0	0	0.0
Female that died	0	0.0	0	0.0	1	4.5	0	0.0
Females that aborted	0	0.0	0	0.0	0	0.0	0	0.0
Nongravid	0	0.0	0	0.0	0	0.0	0	0.0
Gravid	0	0.0	0	0.0	0	0.0	0	0.0
Female that were euthanized	1	4.5	0	0.0	0	0.0	0	0.0
Nongravid	0	0.0	0	0.0	0	0.0	0	0.0
Gravid	0	100.0	0	0.0	0	0.0	0	0.0
Female examined at sechduled necropsy	21	95.5	21	95.5	21	95.5	21	95.5
Nongravid	2	9.5	1	4.8	1	4.8	1	4.8
Gravid	19	90.5	20	95.2	20	95.2	20	95.2
With resorption only	1	100.0	0	0.0	0	0.0	0	0.0
with viable fetuses	19	100.0	20	100.0	20	100.0	20	100.0
Total female gravid	20	90.9	21	95.5	21	95.5	20	95.5

 

Table 9. Mean Summary of Fetal Data at Scheduled Necropsy.

Group	Sex		Viable fetuses	Dead Fetuses	Resorption		Post Implantation Loss	Implantation	Corpora Lutea	Pre Implatation Loss	Fetal Weight (g)	No. of Gravid Female
	Male	Female			Early	Late
Group 1 (0 mg/kg)	6.0	4.2	10.2	0.0	0.3	0.2	0.5	10.6	10.9	0.3	37.7	19
2 (100 mg/kg)	4.6	4.6	8.9	0.0	0.4	0.4	0.8	9.7	10.7	1.0	38.7	20
3 (300 mg/kg)	4.4	4.3	8.7	0.0	0.2	0.1	0.3	9.0	9.4	0.4	40.5	20
4 (1000 mg/kg)	4.9	4.1	8.9	0.0	0.5	0.2	0.7	9.6	10.5	0.9	38.4	20

 

Table 10. Mean Summary of Fetal Data at Scheduled Necropsy (% per litter).

Group	Copora lutea	Implantation site	viable fetuses	Dead fetuses	Early resorption	Late resorption	Total resorption	Pre-implantation	Post-implantation	Male	Female	Male fetal weight	Female fetal weight	Combined fetal weight
1 (0 mg/kg)	10.9	10.6	95.7	0.0	2.4	1.9	4.3	2.6	4.3	59.9	40.1	38.4	36.4	37.7
2 (100 mg/kg)	10.7	9.7	93.0	0.0	3.7	3.4	7.0	9.7	7.0	49.6	50.4	39.1	37.8	38.7
3 (300 mg/kg	9.4	9.0	97.6	0.0	1.5	0.9	2.4	5.2	2.4	49.3	50.7	40.6	40.4	40.5
4 (1000 mg/kg)	10.5	9.6	93.0	0.0	5.4	1.6	7.0	8.6	7.0	56.2	43.8	38.6	37.9	38.4

Proportional (%) data compared using the mann-whitney test fetal weights compared using dunnett's test
none significantly different from control group

Table 10. Mean Summary Of Fetuses And Litters With Malformations [Absolute No.]

Dose group	Fetuses				Litters
	1 (0 mg/kg)	2 (100 mg/kg)	3 (300 mg/kg)	4 (1000 mg/kg)	1 (0 mg/kg)	2 (100 mg/kg)	3 (300 mg/kg)	4 (1000 mg/kg)
Number examined externally	193	178	174	178	19	20	20	20
Tail- absent, short or filamentous	1	0	0	0	1	0	0	0
Carpal and/or tarsal flexure	0	0	1	2	0	0	1	2
Facial cleft	0	1	0	0	0	1	0	0
Cleft palate	0	1	0	0	0	1	0	0
Trunk- omphalocele	0	1	1	0	0	1	1	0
Trunk- gastroschisis	0	0	0	1	0	0	0	1
Syndactyly	0	0	0	1	0	0	0	1
Brachydactyly	0	0	0	1	0	0	0	1
Number examined viscerally	193	178	174	178	19	20	20	20
Tetralogy of fallot	0	0	0	1	0	0	0	1
Kidney(s)- malpositioned	1	0	1	1	1	0	1	1
Testis- malpositioned	1	0	0	0	1	0	0	0
Kidney(s) and/or ureter(s)- absent	0	0	0	1	0	0	0	1
Liver- abnormal lobation	0	0	0	1	0	0	0	1
Heart- large	0	0	0	1	0	0	0	1
Body cavity fluid-filled0	0	0	0	1	0	0	0	1
Lung- abnormal lobation	0	0	1	0	0	0	0	1
Diaphram - cyst	0	0	1	0	0	0	1	0
Aortic arch- dilated	0	1	0	0	0	1	0	0
Pulmonary trunk- narrow	0	1	0	0	0	1	0	0
Ventricular septum defect	0	1	0	0	0	1	0	0
Hydrocephaly- internal	0	0	0	1	0	0	0	1
Number examined skeletally	193	-	-	178	19	-	-	20
Costal cartilage anomaly	0	-	-	1	0	-	-	1
Rib anomaly	0	-	-	1	0	-	-	1
Vertebral anomaly with or without associated rib anomaly	0	-	-	3	0	-	-	2
Sternal anomaly	1	-	-	0	1	-	-	0
Total number with malformations external	1	2	2	2	1	2	2	2
Soft tissue	1	2	2	5	1	2	1	2
Skeletal	1	-	-	5	1	-	-	4
Combined	3	4	4	9	3	4	3	6

 

Table 11. Mean Summary Of Litter Proportions of  Malformations [% per Litter]

Dose group	1 (0 mg/kg)	2 (100 mg/kg)	3 (300 mg/kg)	4 (1000 mg/kg)
Number examined externally	19	20	20	20
Tail- absent, short or filamentous	0.4	0.0	0.0	0.0
Carpal and/or tarsal flexure	0.0	0.0	0.5	1.5
Facial cleft	0.0	0.6	0.0	0.0
Cleft palate	0.0	0.6	0.0	0.0
Trunk- omphalocele	0.0	0.5	1.7	0.0
Trunk- gastroschisis	0.0	0.0	0.0	0.8
Syndactyly	0.0	0.0	0.0	0.6
Brachydactyly	0.0	0.0	0.0	0.6
Tetralogy of fallot	0.0	0.0	0.0	0.6
Kidney(s)- malpositioned	0.5	0.0	0.5	0.8
Testis- malpositioned	0.5	0.0	0.0	0.0
Kidney(s) and/or ureter(s)- absent	0.0	0.0	0.0	0.8
Liver- abnormal lobation	0.0	0.0	0.0	0.8
Heart- large	0.0	0.0	0.0	0.4
Body cavity fluid-filled	0.0	0.0	0.0	0.4
Lung- abnormal lobation	0.0	0.6	0.0	0.0
Diaphram - cyst	0.0	0.0	0.5	0.0
Aortic arch- dilated	0.0	0.6	0.0	0.0
Pulmonary trunk- narrow	0.0	0.6	0.0	0.0
Ventricular septum defect	0.0	0.6	0.0	0.0
Hydrocephaly- internal	0.0	0.0	0.0	0.5
Costal cartilage anomaly	0.0	-	-	0.6
Rib anomaly	0.0	-	-	0.6
Vertebral anomaly with or without associated rib anomaly	0.0	-	-	2.1
Sternal anomaly	0.0	-	-	0.0
Percent per litter with external malformation	0.4	1.1	2.2	1.5
Percent per litter with soft tissue malformation	0.5	1.2	0.9	3.2
Percent per litter with skeletal malformation	0.6	-	-	3.3
Total percent per litter with malformation	1.5	2.2	3.1	5.7

 

Table 12. Mean Summary Of Fetuses Litter with variations [Absolute No.]

Dose group	Fetuses				Litters
	1 (0 mg/kg)	2 (100 mg/kg)	3 (300 mg/kg)	4 (1000 mg/kg)	1 (0 mg/kg)	2 (100 mg/kg)	3 (300 mg/kg)	4 (1000 mg/kg)
									Number examined viscerally	193	178	174	178	19	20	20	20
Number with findings	0	0	0	0	0	0	0	0
Number examined viscerally	193	178	174	178	19	20	20	20
Left carotid- originating from brachiocephalic trunk	5	2	3	9	3	1	2	4
Gallbladder- absent or small	2	4	0	1	2	4	0	1
Retrocaval ureter	7	5	1	2	6	4	1	2
Lung- absent accessory lobe	3	6	4	4	2	2	2	2
Aortic arch- supernumerary artery	0	1	0	5	0	1	0	3
Gallbladder- large	0	0	1	0	0	0	1	0
Adrenal gland- displaced	1	0	0	0	1	0	0	0
Renal papilla(e)- absent and/or small	1	1	0	0	1	1	0	0
Right subclavian- retroesophageal	0	1	0	0	0	1	0	0
Number examined skeletally	193	-	-	178	19	-	-	20
13th full rib(s)	96	-	-	125	19	-	-	20
Sternebra(e) malaligned	35	-	-	20	15	-	-	12
Sternebrae fused	2	-	-	0	2	-	-	0
Sternebra(e) #5 and/or #6 unossified	64	-	-	35	13	-	-	13
Pelvic girdle- caudal shift	24	-	-	65	7	-	-	14
13th rudimentary rib(s)	14	-	-	6	9	-	-	5
7th cervical ossification site(s)	2	-	-	2	1	-	-	2
Vertebral centra- reduced ossification	1	-	-	4	1	-	-	4
Metacarpal (s) and /or metatarsal (s) unossified	8	-	-	10	4	-	-	4
Tarsal (s) unossified	1	-	-	1	1	-	-	1
Caudal vertebral anomaly	1	-	-	1	1	-	-	1
Rib(s)- nodulated	0	-	-	1	0	-	-	1
Skull- supernumerary site	1	-	-	1	1	-	-	1
Number examined skeletally	193	-	-	178	19	-	-	20
Sternum- supernumerary ossification site	2	-	-	0	1	-	-	0
Hyoid body and/or arches unossified	0	-	-	1	0	-	-	1
Reduced ossification of the skull	0	-	-	1	0	-	-	1
hyoid arch(es) bent	1	-	-	0	1	-	-	0

 

Table 13. Mean Summary Of Fetuses Litter with variations [% per litter]

Dose Group	1 (0 mg/kg)	2 (100 mg/kg)	3 (300 mg/kg)	4 (1000 mg/kg)
Number examined	19	20	20	20
Numbers of litters with findings	0	0	0	0
Left carotid- originating from brachiocephalic trunk	2.7	1.0	1.3	5.2
Gallbladder- absent or small	0.9	2.2	0.0	0.6
Retrocaval ureter	3.8	2.6	0.4	1.0
Lung- absent accessory lobe	1.5	3.3	1.7	2.8
Aortic arch- supernumerary artery	0.0	0.5	0.0	2.7
Gallbladder- large	0.0	0.0	0.5	0.0
Adrenal gland- displaced	0.5	0.0	0.0	0.0
Renal papilla(e)- absent and/or small	0.0	0.6	0.0	0.0
Right subclavian- retroesophageal	0.0	0.6	0.0	0.0
Number examined skeletally	19	-	-	20
13th full rib(s)	50.5	-	-	71.1*
Sternebra(e) malaligned	18.9	-	-	11.5
Sternebrae fused	1.1	-	-	0.0
Sternebra(e) #5 and/or #6 unossified	34.6	-	-	19.2
Pelvic girdle- caudal shift	13.4	-	-	38.2*
13th rudimentary rib(s)	6.7	-	-	3.0
7th cervical ossification site(s)	1.0	-	-	1.0
Vertebral centra- reduced ossification	0.4	-	-	2.6
Metacarpal (s) and /or metatarsal (s) unossified	4.0	-	-	4.4
Tarsal (s) unossified	0.5	-	-	0.6
Caudal vertebral anomaly	0.5	-	-	0.7
Rib(s)- nodulated	0.5	-	-	0.7
Skull- supernumerary site	0.5	-	-	0.5
Number examined skeletally	19	-	-	20
Sternum- supernumerary ossification site	1.0	-	-	0.0
Hyoid body and/or arches unossified	0.0	-	-	0.8
Reduced ossification of the skull	0.0	-	-	0.8
hyoid arch(es) bent	0.4	-	-	0.0
Number of litter examined	19	20	20	20
Total variations Percetage per litter with external variation	0.0	0.0	0.0	0.0
Total variations percentage per litter with soft tissue varations	8.3	9.7	3.5	12.3
Total variations - percentage per litter with sekeletal variations	78.0	-	-	86.2

Applicant's summary and conclusion

Conclusions:

Based on the results in this prenatal developmental toxicity study, the maternal and developmental No Observed Adverse Effect Level (NOAEL) for Tris (2- ethylhexyl)phosphate was established as being at least 1000 mg/kg/day.

Executive summary:

The potential of Tris (2-ethylhexyl)phosphate 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 New Zealand White rabbits from Day 6 to 28 post- coitum, inclusive. In addition, the No Observed Adverse Effect Levels (NOAELs) for maternal toxicity and developmental toxicity were evaluated. The dose levels the study were selected to be 0, 100, 300 and 1000 mg/kg/day, based on the results of the dose range finder (Test Facility Study No. 20173163).

The following parameters and endpoints were evaluated in this study for the F0-generation: mortality/moribundity, clinical signs, body weights, food consumption, gross necropsy findings, number of corpora lutea, (gravid) uterine weight and uterine contents. In addition, the following parameters were determined for the F1-generation: the number of live and dead fetuses, early and late resorptions, total implantations, fetal body weights, sex ratio, and external, visceral and skeletal malformations and developmental variations.

Maternal findings
No adverse maternal findings were recorded up to a dose of 1000 mg/kg/day.

At 300 and 1000 mg/kg/day, a non-adverse slight mean weight loss (-1% or -2%) and lower mean absolute and relative food consumption (0.53x and 0.33x of control, respectively, for relative food intake) was recorded over post-coitum Days 6 to 9. From post-coitum Day 9 onwards, reduced faeces production was recorded at increased incidence over the dose groups. Food intake however remained similar to control levels during this period, except for post-coitum Days 27 to 29, when a lower mean absolute and relative food consumption (0.72x of control for relative food consumption) and lower mean body weights were recorded (0.95x of control) at 1000 mg/kg/day. In the period between PND 9 and 27, there were no clear differences in body weight and food intake between treated groups and the control group that could be ascribed to the test-item (statistically significant changes in that period occurred in the absence of a clear dose-related trend). Overall, mean weight gain at 1000 mg/kg/day over the treatment period was 6% vs. 11% in the control group, but mean body weights remained well within the historical control data range.

Also, corrected weight gain for gravid uterus at 1000 mg/kg/day was slightly outside the historical range for a total of 6 out of 20 animals (the mean corrected weight remained within the historical control range). Overall, it was considered that these changes in body weight and food intake did not represent adverse maternal effects, since these variations were not consistently seen with continuing treatment and generally remained within the historical control data range. Also, there were no indications among fetuses that pointed towards a secondary effect of these maternal variations in body weight and food intake.

At 100 mg/kg/day, no test item related maternal effects were recorded. No test item-related macroscopic lesions were recorded at any dose level. Fetal Examinations

No test item-related changes were noted up to 1000 mg/kg/day in any of the developmental parameters investigated in this study (i.e. litter size, post-implantation loss, sex ratio, fetal body weights, external, visceral and skeletal malformations and developmental variations).