O,O,O-triphenyl phosphorothioate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jan 2019 - 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

Jun 2018

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

Aug 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

O,O,O-triphenyl phosphorothioate

EC Number:

209-909-9

EC Name:

O,O,O-triphenyl phosphorothioate

Cas Number:

597-82-0

Molecular formula:

C18H15O3PS

IUPAC Name:

O,O,O-triphenyl thiophosphate

Test material form:

solid

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
For the test substance preparations, the specific amount of test substance was weighed, topped up with 0.5% CMC suspension in drinking water in a calibrated beaker and intensely mixed with a homogenizer.
- Final preparation of a solid:
Preparations prepared at the beginning and thereafter at intervals. Preparations kept at room temperature

FORM AS APPLIED IN THE TEST (if different from that of starting material)
- suspension

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH,
- Age at study initiation: 10-12 weeks
- Weight at study initiation: 162.6 - 233.6 g (pregnant animals)
- Fasting period before study: no
- Housing: individually
- Diet: mouse and rat maintenance diet “GLP”, meal, supplied by Granovit AG, Kaiseraugst, Switzerland, ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12 (illumination from 6 am to 6 pm)

IN-LIFE DATES: From: 29/30/31 Jan 2019 To: 18/19/20 Feb 2019

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% CMC in drinking water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Volume administered: 10 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the test substance preparations were sent four times (once at the beginning of administration and thrice at the end of the study) to the analytical laboratory for verification of the concentrations. Reserve samples were described by the suffix “R” in the report. The concentrations of the test item in the samples were calculated by means of their phosphor content. The method chosen was: determination of phosphor by inductively coupled plasma optical emission spectrometry (ICP-OES) after digestion with concentrated mineral acids.
Overall, the results of the analysis of the test substance preparations confirmed the correctness of the prepared concentrations. Almost all values of the measured concentrations corresponded to the expected values within the limits of the analytical method, i.e. were above 90% and below 110% of the nominal concentrations. The stability of the test substance in 0.5% CMC suspension in drinking water over a period of 7 days at room temperature was demonstrated. The homogeneous distribution of the test substance in the vehicle (0.5% CMC suspension in drinking water) was demonstrated.

Details on mating procedure:

The animals were paired by the breeder (“time-mated”); the day of evidence of mating (= detection of vaginal plug/sperm) was referred to as GD 0.

Duration of treatment / exposure:

From GD 6 to GD 19

Frequency of treatment:

once daily

Duration of test:

From implantation to one day prior to expected day of parturition

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: At the request of the sponsor
- Rationale for animal assignment (if not random): random (manually)
- Route selection rationale: The oral route was selected since this has proven to be suitable for the detection of a toxicological hazard.

Examinations

Maternal examinations:

OBSERVATIONS OF THE DAMS: Yes
- Time schedule: Twice daily
- Checked for mortality

CLINICAL SYMPTOMS: Yes
- Time schedule: once daily
- Prameters checked: signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20.

FOOD CONSUMPTION: Yes
- Time schedule: The consumption of food was recorded for the intervals GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20.

WATER CONSUMPTION: No:

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: reproductive organs (uterus), liver, thyroid glands
- Histopathology: Yes, thyroid glands

ORGAN WEIGHTS
- The following weights were determined in all dams sacrificed on schedule: liver, thyroid glands

HEMATOLOGY: Yes
- Time schedule for collection of blood: On GD 20
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Not specified
- How many animals: All females
- Parameters checked: Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, reticulocytes (RETA)

CLINICAL PTATHOLOGY: Yes
- Time schedule for collection of blood: On GD 20
- Animals fasted: Not specified
- How many animals: all females
- Parameters checked: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), Inorganic phosphate, Calcium, Urea, Creatinine, Glucose, Total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol
- In addition: Thyroid hormones were checked (T3, T4, and TSH)

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- gravid uterine weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Dead fetuses: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half litter

At necropsy each fetus was weighed, sexed, and external tissues and all orifices were examined macroscopically. The sex was determined by observing the distance between the anus and the base of the genitalia. Furthermore, the viability of the fetuses and the condition of placentas, umbilical cords, fetal membranes, and fluids were examined. The placentas were weighed and their individual weights were recorded. Thereafter, the fetuses were sacrificed by a subcutaneous injection of pentobarbital. The anogenital distance (defined as the distance from the center of the anal opening to the base of the genital tubercle) measurements were conducted, using a measuring ocular, on all liveborn fetuses. After these examinations, approximately one half of the fetuses per dam were eviscerated, skinned and fixed in ethanol; the other half was placed in Harrison’s fluid for fixation.

Statistics:

- Dunnett's test, Fisher's exact test, Wilcoxon test, Kruskal-Wallis test

Indices:

Conception rate, preimplantation loss, postimplantation loss, anogenital index

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any female of the test groups 0-3 (0, 100, 300 or 1000 mg/kg bw/d).

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no test substance-related or spontaneous mortalities in any females of all test groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weights and the average body weight gain of the low-, mid- and high-dose dams (100, 300 and 1000 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study period. The corrected body weight gain of test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d) revealed no difference of any biological relevance to the corresponding control group. Moreover, mean carcass weights of all test groups remained unaffected by the treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The mean food consumption of the dams in test groups 1-3 (100, 300 and 1000 mg/kg bw/d) was generally comparable to the concurrent control group throughout the entire study period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among hematological parameters were observed. At gestation day 21 in dams of test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d) absolute eosinophil counts were significantly decreased. Additionally, in dams of test groups 2 and 3, relative eosinophil counts were significantly decreased. However, the values were within historical control ranges (dams, absolute eosinophils 0.05-0.09 Giga/L; relative eosinophils 0.8-1.7 %). In dams of test group 1 (100 mg/kg bw/d) absolute monocyte counts were significantly decreased, but the alteration was not dose-dependent. Therefore, the mentioned changes were regarded as incidental and not treatment-related.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

In dams of test group 3 (1000 mg/kg bw/d), significantly decreased T3 values were measured. However, the values were within the historical control range (dams, T3: 0.56-1.06 nmol/L), whereas the study control T3 values were at the upper border of this range. Therefore, this change was regarded as incidental and not treatment related.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Uterus: The mean gravid uterus weights of the animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) were not influenced by the test substance. The differences between these groups and the control group revealed no dose-dependency and were assessed to be without biological relevance.
Thyroid: When compared to control group 0 (=100%), the mean absolute weight of the thyroids was significantly decreased in females treated with 300 mg/kg. Since this change had no dose-dependent relationship, it was regarded as not treatment-related.
Liver: When compared to control group 0 (=100%), the mean relative weight of the liver was significantly increased in females treated with 300 (103%) and 1000 mg/kg (108%). This increase is regarded as treatment-related but not adverse as no clinical chemical alterations of the hepatic parameters were noted.

Gross pathological findings:

no effects observed

Description (incidence and severity):

One individual finding occurred and was considered incidental or spontaneous in origin and without any relation to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

A minimal follicular hypertrophy/hyperplasia occurred in all test groups with a minimal dose-dependent increased incidence in test groups 2 and 3 (300 and 1000 mg/kg bw/day). These changes were of low magnitude and were not consistent with hormonal changes. Therefore, histopathological findings in dams of test groups 2 (300 mg/kg bw/d) and 3 (1000 mg/kg bw/d) were assumed to be possibly treatment-related but not adverse.

Histopathological findings: neoplastic:

no effects observed

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:

not examined

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The conception rate reached 96% in the test groups 0 and 1000 mg/kg bw/d and 100 % in the test groups 100 and 300 mg/kg bw/d.

Other effects:

no effects observed

Description (incidence and severity):

There were no test substance related and/or biologically relevant differences between the test groups 0-3 in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and post-implantation losses, the number of resorptions and viable fetuses. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean fetal weights of test groups 1, 2 and 3 were not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses in test groups 1-3 (100, 300 and 1000 mg/kg bw/d) was comparable to the control fetuses.

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

External malformations were detected in 3 fetuses of three different litters exclusively in test group 2 (300 mg/kg bw/d), all were associated with skeletal malformations. These events added up to a statistically significantly increased total incidence of external malformations in test group 2. In absence of a dose-relationship, none of these malformations were assessed as treatment-related. One external variation, i.e. limb hyperextension, was recorded in two control fetuses. There were no external variations in any of the treated groups.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations were detected in five fetuses of 300 mg/kg bw/d and in one single fetus of 1000 mg/kg bw/d. However, findings can be found in historical control data at comparable incidences, they showed no dose-response relationship and thus, were assessed as a spontaneous, isolated event.
Observed skeletal variations were related to several parts of fetal skeletons and appeared without a relation to dose. The overall incidences of skeletal variations were comparable to the historical control data.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

- 1000 mg/kg bw/day: Only one fetus showed multiple visceral malformations. However, these findings were isolated events and were well within the range of historical control data, hence, were assessed as not treatment-related. Three soft tissue variations were detected, however, findings were within historical control data and were not considered to be treatment-related.

Other effects:

no effects observed

Description (incidence and severity):

The mean placental weights of test groups 1-3 were comparable to the concurrent control group. Neither on anogenital distance nor on anogenital index test substance-related effects were noted in all fetuses of test groups 1-3.

Details on embryotoxic / teratogenic effects:

Overall, there was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose. Under the conditions of this study the test item is not teratogenic.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

Organ weights

Absolute weights	Females
Test group	1	2	3
(mg/kg bw/d)	(100)	(300)	(1000)
Thyroid glands	95%	88%**	97%

* : p <= 0.05, **: p <= 0.01

Relative weights	Females
Test group	1	2	3
(mg/kg bw/d)	(100)	(300)	(1000)
Liver	100%	103%*	108%**

* : p <= 0.05, **: p <= 0.01

All other mean relative weight parameters did not show significant differences when compared to the control group 0. The significantly decreased absolute weight of the thyroid glands in females of test group 2 had no dose-dependent relationship and was therefore considered not treatment-related. The significantly increased relative weight of the liver in females of test group 3 (4.845%) was marginally above the historical control range (4.343 – 4.801%) and was regarded as treatment-related, whereas the relative weight increase in test group 2 (4.621%) was within the control range and was considered not treatment-related.

Summary of histopathology

	Female animals
Test group (mg/kg bw/d)	0 (0)	1 (100)	2 (300)	3 (1000)
No. of dams	24	25	25	24
Hypertrophy/hyperplasia, follicular	4	2	6	9
·       Grade1	4	2	6	9

A minimal follicular hypertrophy/hyperplasia occurred in all test groups with a minimal dose-dependent increased incidencein test groups 2 and 3. These changes were of low magnitude and were not consistent with hormonal changes. Therefore, histopathological findings in dams of test groups 2 (300 mg/kg bw/d) and 3 (1000 mg/kg bw/d) were assumed to be possibly treatment-related but not adverse. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

External Examnation of fetuses

Individual fetal external malformations:

Test group	Dam No.-Fetus No., Sex	Finding
0 (0 mg/kg bw/d)	none
1 (100 mg/kg bw/d)	none
2 (300 mg/kg bw/d)	54-08 F^a)	exencephaly, microphthalmia
	63-13 M^a)	multiple external malformations
	70-06 F^a)	exencephaly, macroglossia, microphthalmia
3 (1000 mg/kg bw/d)	none

No. = number; M = male; F = female

^a) fetus with additional skeletal malformation (see below)

Total external malformations:

		Test group 0 0 mg/kg	Test group 1 100 mg/kg	Test group 2 300 mg/kg	Test group 3 1000 mg/kg
Litter	N	24	25	25	24
Fetuses	N	268	287	266	263
Fetal incidence	N (%)	0	0	3 (1.1)	0
Litter incidence	N (%)	0	0	3 (12)	0
Affected fetuses/litter	Mean%	0	0	1.0*	0

N = number; % = per cent, * = p <= 0.05 (Wilcoxon-test [one-sided])

External malformations were detected in 3 fetuses of three different litters exclusively in test group 2 (300 mg/kg bw/d), all were associated with skeletal malformations. These events added up to a statistically significantly increased total incidence of external malformations in test group 2. In absence of a dose-relationship, none of these malformations were assessed as treatment-related. One external variation, i.e. limb hyperextension, was recorded in two control fetuses. There were no external variations in any of the treated groups. No external unclassified observations were recorded.

Soft Tissue Examination

Individual fetal soft tissue malformations:

Test group	Dam No.-Fetus No., Sex	Finding
0 (0 mg/kg bw/d)	none
1 (100 mg/kg bw/d)	none
2 (300 mg/kg bw/d)	none
3 (1000 mg/kg bw/d)	99-12 M	multiple visceral malformations

No. = number; M = male; F = female^a)

Total soft tissue malformations:

		Test group 0 0 mg/kg	Test group 1 100 mg/kg	Test group 2 300 mg/kg	Test group 3 1000 mg/k
Litter	N	24	25	25	24
Fetuses	N	130	136	122	126
Fetal incidence	N (%)	0	0	0	1 (0.8)
Litter incidence	N (%)	0	0	0	1 (4.3)
Affected fetuses/litter	Mean%	0	0	0	0.6

N = number; % = per cent

One fetus of test group 3 (1000 mg/kg bw/d) had multiple visceral malformations, such as torsion of stomach, aortic arch atresia and abnormal lung lobation. However, these findings were isolated events in one single fetus and no cluster of any of these individual malformations were seen in the other offspring of this or the other treated groups. Furthermore, the value of affected fetuses per litter of this finding in test group 3 (0.6 %) was well within the range of the historical control data (affected fetuses per litter: 0.0 – 0.7 %). Thus, it was not assessed as treatment-related. The total incidence of soft tissue malformations in treated animals did not differ significantly from the concurrent control group and was covered by the historical control data.

Total soft tissue variations:

		Test group 0 0 mg/kg	Test group 1 100 mg/kg	Test group 2 300 mg/kg	Test group 3 1000 mg/kg
Litter	N	24	25	25	24
Fetuses	N	130	136	122	126
Fetal incidence	N (%)	3 (2.3)	3 (2.2)	2 (1.6)	10 (7.9)
Litter incidence	N (%)	2 (8.3)	3 (12)	2 (8.3)	8 (35)^Fi*
Affected fetuses/litter	Mean%	2.9	2.3	1.5	7.9^Wi*

N = number; % = per cent

^Fi* = p <= 0.05 (Fisher’s exact test [one-sided])

^Wi* = p <= 0.05 (Wilcoxon-test [one-sided])

Three soft tissue variations were detected, i.e. short innominate in test group 3, dilated renal pelvis and dilated ureter in all test groups. The incidences of ‘dilated renal pelvis’ (2.9/1.6/1.5/7.2* mean% affected fetuses/litter) and ‘dilated ureter’ (2.1/1.5/1.5/6.5* mean% affected fetuses/litter) were statistically significantly higher in test group 3 (1000 mg/kg bw/d). For both findings, the mean values of affected fetuses per litter were well within the range of the historical control data (affected fetuses per litter, dilated renal pelvis: range: 0.7 - 11.5 %; dilated ureter: 0.0 - 6.9 %). Consequently, the findings summed up to a statistically significantly higher total incidence of fetal soft tissue variations in the high-dose group. However, the mean value was well within the range of the historical control data (total soft tissue variations: mean: 0.7 - 12.6 %). Overall, based on the discussion above, these findings in test group 3 were not assessed as treatment-related. No soft tissue unclassified observations were recorded.

Skeletal examination of the fetuses

Individual fetal skeletal malformations:

Test group	Dam No.-Fetus No., Sex	Finding
0 (0 mg/kg bw/d)	none
1 (100 mg/kg bw/d)	none
2 (300 mg/kg bw/d)	51-05 M	cleft sternum
	54-08 F^a)	severely malformed skull bones, severely malformed vertebral column
	63-13 M^a)	severely malformed skull bones, severely malformed vertebral column
	70-06 F^a)	severely malformed skull bones
	75-09 M	shortened humerus
3 (1000 mg/kg bw/d)	77-07 M	shortened scapula, shortened humerus

No. = number; M = male; F = female

^a)fetus with additional external malformation (see above)

Total skeletal malformations:

		Test group 0 0 mg/kg	Test group 1 100 mg/kg	Test group 2 300 mg/kg	Test group 3 1000 mg/kg
Litter	N	24	25	25	24
Fetuses	N	138	151	144	137
Fetal incidence	N (%)	0	0	5 (3.5)	1 (0.7)
Litter incidence	N (%)	0	0	5 (20)^Fi*	1 (4.2)
Affected fetuses/litter	Mean%	0	0	3.0^Wi*	1

N = number; % = per cent

^Fi* = p <=0.05 (Fisher’s exact test [one-sided])

^Wi* = p <=0.05 (Wilcoxon-test [one-sided])

Skeletal malformations were detected in five fetuses of test group 2 (300 mg/kg bw/d) and in one single fetus of test group 3 (1000 mg/kg bw/d). In test group 2, different findings without relation to dose were observed: three fetuses (No. 54-08, No. 63-13, No. 70-06) of test group 2 were multiple malformed and had additional external malformations. The finding ‘severely malformed skull bones’ which occurred only in these three multiple malformed fetuses, was statistically significantly increased in test group 2. However, the finding can be found in the historical control data at comparable incidences and it occurred without any relation to dose. Therefore, it was not assessed as treatment-related. The same was true for the finding ‘severely malformed vertebral column’ showing no dose-response-relationship.

The finding cleft sternum in one single fetus of the mid-dose group was assessed as incidental, isolated event since it occurred without relation to dose. The malformations in the remaining fetuses (‘shortened humerus’ and ‘shortened scapula’) of mid- and high-dose are not assessed as treatment-related since they were isolated events in single fetuses without any relation to dose. All of them were well within the range of the historical control data (e.g. shortened humerus: affected fetuses per litter: range of 0.0 – 1.3%). The different findings in the mid-dose summed up to a statistically significant increase of total incidence of skeletal malformations in test group 2. However, consequently to the above- mentioned discussion of the individual findings, this increase occurred without a relation to dose and was well within the historical control range (total skeletal malformations: 0.0 - 3.1%). Therefore, this was not assessed as treatment-related.

Total fetal skeletal variations:

		Test group 0 0 mg/kg	Test group 1 100 mg/kg	Test group 2 300 mg/kg	Test group 3 1000 mg/kg
Litter	N	24	25	25	24
Fetuses	N	138	151	144	137
Fetal incidence	N (%)	133 (96)	147 (97)	136 (94)	135 (99)
Litter incidence	N (%)	24 (100)	25 (100)	25 (100)	24 (100)
Affected fetuses/litter	Mean%	96.6	97.3	94.9	98.6

N = number; % = per cent

For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared without a relation to dose. The overall incidences of skeletal variations were comparable to the historical control data. All skeletal variations with statistically significant differences between the control and any treated group are compiled in the table below. All incidences were expressed on a fetus per litter basis.

Occurrence of statistically significantly increased fetal skeletal variations:

Finding	Test group 0 0 mg/kg	Test group 1 100 mg/kg	Test group 2 300 mg/kg	Test group 3 1000 mg/kg	HCD Mean % (range)
Incomplete ossification of parietal; unchanged cartilage	2.2	6.2	4.9	7.2*	8.1 (0.7 - 20.0)
Dumbbell ossification of thoracic centrum; dumbbell-shaped cartilage of centrum	0	0	3.2*	0.7	2.7 (0.0 - 10.1)

HCD = Historical control data; % = per cent

* = p <= 0.05 (Wilcoxon-test [one-sided])

Concerning the statistically significant findings, an association to the test substance and a toxicological relevance is not assumed. As described in the table above, the mean value of the finding in the high-dose group was well within the range of the historical control data. The finding in the mid-dose group was without relation to dose and well within the historical control range.

Additionally, some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations,occurred in all test groups. The observed unclassified cartilage findings were related to the skull, the vertebral column, the ribs and the sternum and did not show any relation to dosing.

Assessment of all fetal external, soft tissue and skeletal observations

There were noted external, soft tissue and skeletal malformations in five fetuses of test group 2 and two fetuses of test group 3. Three fetuses of test group 2 and two fetuses of test group 3 had more than one malformation or were multiple-malformed across the different examination areas. Other malformations, such as cleft sternum and shortened humerus were scattered observations in individual fetuses of test group 2. They were not dose-related and all of them can be found in the historical control data at comparable or higher frequency. In test group 3, the observed malformations were either single isolated events without any hints of a cluster seen in the other offspring (e.g. ‘multiple visceral malformations’) and/or without relation to dose (e.g. ‘shortened humerus’). All of them were well within the historical control data. Therefore, they are not assessed as treatment-related, adverse events. In test group 2, three fetuses were multiple malformed. The findings showed no relation to dose and were not assessed as treatment-related. The different findings in test group 2 summed up to statistically significant incidence of total malformations showing no relation to dose. The mean value of affected fetuses per litter was well within the historical control range (HCD total malformations: 0.00 - 2.03 %). Therefore, it is not assessed as treatment-related.

Total malformations:

		Test group 0 0 mg/kg	Test group 1 100 mg/kg	Test group 2 300 mg/kg	Test group 3 1000 mg/kg
Litter	N	24	25	25	24
Fetuses	N	268	287	266	263
Fetal incidence	N (%)	0	0	5 (1.9)	2 (0.8)
Litter incidence	N (%)	0	0	5 (20)^Fi*	2 (8.3)
Affected fetuses/litter	Mean%	0	0	1.7^Wi*	0.8

N = number; % = per cent

^Fi* = p <= 0.05 (Fisher’s exact test [one-sided])

^Wi* = p <= 0.05 (Wilcoxon-test [one-sided])

One external variation, three soft tissue variations and a range of skeletal variations were noted in all test groups including the controls. The majority of individual variations are equally distributed across the different test groups, if normal biological variation is taken into account, and can be found in the historical control data at a comparable frequency. In test group 3, the incidences of two soft tissue variations, ‘dilated renal pelvis’ and ‘dilated ureter’ were statistically significantly higher in test group 3 (1000 mg/kg bw/d). For both findings, the mean values of affected fetuses per litter were well within the range of the historical control data (see above). Consequently, the findings summed up to a statistically significantly higher incidence in total fetal soft tissue variations in the high-dose group. However, this mean value was also well within the range of the historical control data. Overall, as discussed above, these findings in test group 3 were not assessed as treatment-related.

Total fetal variations:

		Test group 0 0 mg/kg	Test group 1 100 mg/kg	Test group 2 300 mg/kg	Test group 3 1000 mg/kg
Litter	N	24	25	25	24
Fetuses	N	268	287	266	263
Fetal incidence	N (%)	136 (51)	150 (52)	138 (52)	145 (55)
Litter incidence	N (%)	24 (100)	25 (100)	25 (100)	24 (100)
Affected fetuses/litter	Mean%	51.1	52.3	53.6	56.7**

N= number; % = per cent

** = p <= 0.01 (Wilcoxon-test [one-sided])

Unclassified external and unclassified soft tissue observations did not occur in any of the fetuses in this study. A spontaneous origin is assumed for the unclassified skeletal cartilage observations which were observed in several fetuses of all test groups (0, 100, 300 and 1000 mg/kg bw/d). The distribution and type of these findings do not suggest any relation to treatment. Finally, fetal examinations revealed that there is no effect of the compound on the respective morphological structures up to 1000 mg/kg bw/d.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this prenatal developmental toxicity study, the oral administration of the test item to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) caused no treatment-related, adverse findings up to the highest dose level of 1000 mg/kg bw/d. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is the highest dose level of 1000 mg/kg bw/d. There was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose. Under the conditions of this study the test item is not teratogenic. In conclusion, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is the highest dose of 1000 mg/kg bw/d.

Executive summary:

The test substance was tested for its prenatal developmental toxicity in Wistar rats. The test substance was administered as an aqueous preparation to groups of 25 time-mated female Wistar rats by gavage at doses of 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 19. The control group, consisting of 25 females, was dosed with the vehicle (0.5% Sodium carboxymethyl cellulose [CMC] suspension in drinking water) in parallel. A standard dose volume of 10 mL/kg body weight was used for each test group. At terminal sacrifice on GD 20, 24-25 females per group had implantation sites. Food consumption and body weights of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day. On GD 20, blood samples were obtained from all females by retrobulbar venous puncture following isoflurane anesthesia. After blood sampling all females were sacrificed by decapitation (under isoflurane anesthesia) and assessed by gross pathology (including sampling of thyroid glands (with parathyroid glands) and weight determinations of the liver, thyroid glands (with parathyroid glands), unopened uterus and placentas). For each dam, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for external findings. Anogenital distance measurements were conducted on all liveborn fetuses. Thereafter, one half of the fetuses of each litter were examined for soft tissue findings and the remaining fetuses for skeletal (inclusive cartilage) findings. Under the conditions of this prenatal developmental toxicity study, the oral administration of the test item to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) caused no treatment-related, adverse findings up to the highest dose level of 1000 mg/kg bw/d. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is the highest dose level of 1000 mg/kg bw/d. There was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose. Under the conditions of this study the test item is not teratogenic. In conclusion, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is the highest dose of 1000 mg/kg bw/d.