Propyl acetate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Sep - Oct 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

Jan 2001

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

Aug 1998

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Propyl acetate

EC Number:

203-686-1

EC Name:

Propyl acetate

Cas Number:

109-60-4

Molecular formula:

C5H10O2

IUPAC Name:

propyl acetate

Test material form:

liquid

Details on test material:

TEST SUBSTANCE: propyl acetate

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: BASF SE, Ludwigshafen, Germany
- Batch No.of test material: 83294456P0
- Expiration date of the batch: 08 Nov 2018
- Purity: 99.9%
- Identitiy: Confirmed
- Physical state/appearance: liquid/colorless, clear

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions (in the vehicle): Guaranteed by analytical verifications in corn oil over a period of seven days at room temperature
- Homogeneity: given (visually)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance preparations were prepared at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability. The preparations were kept at room temperature. For the test substance preparations, the specific amount of test substance was weighed, topped up with corn oil in a graduated flask and intensely mixed with a magnetic stirrer or by shaking until it was completely dissolved. Before and during administration, the preparations were kept homogeneous with a magnetic stirrer.

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI[Han]

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: 144.5 - 195.6 g
- Fasting period before study: no
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12 (6 am to 6 pm illumination)

IN-LIFE DATES: From: 1st Cohort: 28 Sep 2017; 2nd Cohort: 29 Sep 2017 To: 1st Cohort: 18 Oct 2017; 2nd Cohort: 19 Oct 2017

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Volume applied: 4ml/kg

DIET PREPARATION
- Rate of preparation of diet (frequency): at the beginning and at intervals thereafter
- Mixing appropriate amounts: test substance was weighed, topped up with corn oil and intensely mixed.
- Storage temperature: room temperature

VEHICLE
- Justification for use and choice of vehicle (if other than water): non given

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the test substance preparations were sent thrice to the analytical laboratory for verification of the concentrations. Control procedures were Gas chromatography (GC) and H-NMR spectrum.

Details on mating procedure:

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. The animals arrived on the same day (GD 0) at the experimental laboratory. The following day was designated as “GD 1”.

Duration of treatment / exposure:

GD 6-19

Frequency of treatment:

daily

Duration of test:

14 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day

Remarks:

2.5 g/100 ml

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day

Remarks:

7.5 g/100 ml

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

25.00 g/100 ml

No. of animals per sex per dose:

25 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Reason for species selection:
The Crl:WI(Han) strain was selected since extensive historical control data is available from the test facility for Wistar rats. This specific strain has been proven to be sensitive to substances with a teratogenic potential.

Examinations

Maternal examinations:

MORTALITY: Yes
- Time schedule: twice daily (Mo-Fr), once a day (Sat, Sun)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily before and after treatment

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19, and 20

FOOD CONSUMPTION: Yes
- Time schedule: 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 data
- Time schedule for examinations: no data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Gross pathology

NECROPSY:
- uteri and ovaries were analyzed: weight of the unopened uterus, number of corpora lutea, number and distribution of implantation sites classified as live fetuses and dead implantations

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
- Dead fetuses: Yes

Fetal examinations:

- External examinations: Yes:
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: No data
- Weight, sex, external tissues, and all orifices were examined
- Viability
- Condition and weight of placentas, umbilical cords, fetal membranes, and fluids

Statistics:

Food consumptiona), body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight: Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two-sided) forthe hypothesis of equal means

Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings: Pairwise comparison of each dose group with the control group using FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions

Proportions of fetuses with malformations, variations and/or unclassified observations in each litter: Pairwise comparison of each dose group with the control group using the WILCOXONtest (one-sided) for the hypothesis of equal medians

Indices:

Conception rate, preimplantation loss, postimplantation loss

Historical control data:

MATERNAL BODY WEIGHTS (RANGE) FROM 973 (Days 1 - 20) AND 974 (Day 0) ANIMALS

Day 0: 140.0 - 196.6 g
Day 1: 152.1 - 208.6
Day 3: 158.7 - 216.0
Day 6: 167.6 - 231.5
Day 8: 174.3 - 242.1
Day 10: 181.6 - 252.9
Day 13: 186.6 - 278.4
Day 15: 192.5 - 290.4
Day 17: 183.3 - 318.4
Day 19: 190.5 - 349.8
Day 20: 193.2 - 364.3

REPRODUCTION DATA

- Females mated: 999 of which pregnant: 976 (98%)
- Aborted: 0
- Premature births: 0
- Dams with viable fetuses: 973
- Dams with all resorptions: 3
- Female mortality: 0%
- Corpora lutea (range): 10.5 - 12.3
- Implantations sites (range): 9.9 - 11.8
- Preimplantation loss (range): 1.4 - 13.3%
- Postimplantation loss (range): 3.2 - 14.0%
- Dams with resorptions: 449 (46%)
- Resorptions (range): 0.3 - 1.5
- Dead fetuses: 0
- Viable fetuses: 9778
- Litter size (range): 9.3 - 11.2
- Placenta weights (range): 0.35 - 1.14 g (males); 0.32 - 1.10 g (females)
- Fetal weights (range): 2.8 - 4.5 g (males); 2.3 - 4.4 g (females)

FETAL MALFORMATIONS:

- External malformations: 13 (9778 evaluated) ; 0.1%
- External variations: 4 (9778 evaluated); 0.04%
- External unclassified findings: 3 (9778 evaluated); 0.03%
- Visceral malformations: 13 (4650 evaluated); 0.3%
- Visceral variations: 156 (4650 evaluated); 3.4%
- Visceral unclassified findings: 1 (4650 evaluated); 0.02%
- Skeletal malformations: 39 (5128 evaluated); 0.8%
- Skeletal variations: 5035 (5128 evaluated); 98.2%
- Skeletal cartilage: 3655 (5218 evaluated); 71.3%

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

In 21 out of 25 females occasionally salivation during treatment period in group 1000 mg/kg bw/day. Salivation occurred in the respective animals only shortly, i.e. within 0-2 hours, after treatment and was observed during GD 6-8 and GD 15-19. No clinical signs or changes of general behavior were detected in any female of all test groups beyond 2 hours after treatment. The occasional salivation was most probably caused by the bad taste or smell of the test substance and was not assessed as a sign of systemic toxicity.

No other effects observed

Mortality:

no mortality observed

Description (incidence):

No test substance-related or spontaneous deaths in any females.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weights and average body weight gain of treated groups comparable to control.

The statistically significantly decreased body weight gain value in test group 1 during GD 3-6 (pre-treatment period) was assessed as incidental.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Mean food consumption of treated groups comparable to control.

The statistically significantly decreased food consumption value in the mid-dose dams during GD 17-19 was not considered biologically relevant due to the lack of dose response relationship.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Mean gravid uterus weights not influenced by the test substance. Difference between treated groups and control group revealed no dose-dependency and were assessed to be without biological relevance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Three spontaneous findings were noted in three individual females of test groups 0 and 3 (0 and 1000 mg/kg bw/d). These gross findings were:
• Dilated renal pelvis (right) in dam No. 2,
• Diaphragmatic hernia in dam No. 4,
• Stomach: erosion(s) (one) in dam No. 79.
No further necropsy findings which could be attributed to the test substance were seen in any dam.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

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

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

No test substance related differences in conception rates, mean number of corpora lutea and implantation sites.
Two females in the control group and two females in the 300 mg/kg bw/day group did not get pregnant.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no maternal developmental toxicity observed

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Changes in sex ratio:

no effects observed

External malformations:

no effects observed

Description (incidence and severity):

Three external malformations were exclusively detected in the control group.

No fetal external variations were reported. No fetal external unclassified findings were recorded.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations were noted in two fetuses of the control group and in one fetus, each, of test groups 1 and 3 (100 and 1000 mg/kg bw/d). One control fetus had associated external malformations. The incidences of these malformations were neither
statistically significantly different from control nor dose-dependent. The finding bipartite basisphenoid occurred in each one fetus of test groups 1 and 3 without relation to dose. The mean value of the incidences in test group 3 was within the historical
control range. Therefore, it was not assessed as treatment-related, adverse finding.
All other malformations in test group 1 occurred in only one fetus (33-10 F) without relation to dose and were single, isolated cases which were mostly covered by the historical control data.
The body weight of this fetus (3.2 g) was lower compared to its group mean value (females of test group 1: 3.5 g). The findings in this fetus were not assessed as treatment-related and adverse.

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.

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 ribs and the sternum and did not show any relation to dosing.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Soft tissue malformations were recorded for two control, one mid-dose and three high-dose fetuses. The finding situs inversus in the fetuses of test groups 300 mg/kg bw/day and 1000 mg/kg bw/day were single events in individual fetuses and the mean values were within the historical control data. The findings absent subclavian and anophthalmia were observed in one fetus each of test group 1000 mg/kg bw/day and the mean values were within the historical control ranges.
All these findings were single cases, they were within the historical control ranges.
No ontogenetic pattern is recognizable for all these individual malformations nor was there any cluster of any of these individual malformations seen in the other offspring. Therefore, the observed malformations were not assessed as treatment-related, adverse findings.

Three soft tissue variations were detected, i.e. malpositioned subclavian origin, dilated renal pelvis and dilated ureter. These variations were neither significantly different from the concurrent control nor dose-dependently altered. All of them can be found in the historical control data at comparable incidences. Therefore, they were not assessed as treatment-related.

No fetal visceral unclassified findings were reported.

Other effects:

no effects observed

Description (incidence and severity):

Mean placental weights comparable among groups.

Details on embryotoxic / teratogenic effects:

No teratogenic effects due to treatment were recorded.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No developmental toxicity and teratogenic effects

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

RESULTS

ANALYSES

Stability analysis

The stability of the test substance in corn oil over a period of 7 days at room temperature was

demonstrated before the start of the study.

Homogeneity analysis of the test substance preparations

Given that the test substance is completely miscible with corn oil, solutions were considered to

be homogenous without further analysis.

Concentration control analysis of the test substance preparations

The results of the analysis of the test substance preparations confirmed the correctness of the

prepared concentrations of the mid- and high-dose (samples 04 and 05, respectively). These

analytical values of the samples corresponded to the expected values within the limits of the

analytical method, i.e. were always above 90% and below 110% of the nominal concentrations.

Concerning the lowest dose (100 mg/kg bw/d), the values of samples 03, 03R and 08 new

(149.8, 139 and 124%, respectively) were outside the range of 90% - 110% of the nominal

concentrations. A mean value for all three measurements of around 138% corresponds to actual

dose of 138 mg/kg bw/d for the lowest test group. This has no influence of the validity of

the study.

Food analyses

On the basis of the duration of use and the analytical findings with respect to chemical and

microbiological contaminants, the food was found to be suitable. The EPA Fed. Reg. of 09 May

1979 (Vol. 44, No. 91, p. 27354) served as a guideline for maximum tolerable chemical contaminants.

The amount of microorganisms did not exceed 1*105/g feed.

The individual results are found in the archives of Experimental Toxicology and Ecology, BASF SE, Ludwigshafen, Germany.

Drinking water analyses

On the basis of the analytical findings, the drinking water was found to be suitable. The German

Drinking Water Regulation (“Trinkwasserverordnung”) served as the guideline for maximum

tolerable contaminants.

The individual results are found in the archives of Experimental Toxicology and Ecology, BASF

SE, Ludwigshafen, Germany.

Bedding and enrichment analyses

On the basis of the analytical findings, the bedding and the enrichment were found to be suitable.

Levels given in Lab Animal, Nov-Dec 1979, pp. 24-34, served as a guideline for maximum

tolerable contaminants.

The individual results are found in the archives of Experimental Toxicology and Ecology, BASF

SE, Ludwigshafen, Germany.

Clinical examinations of the dams

Only pregnant dams were used for the calculations of mean maternal food consumption, body

weight and body weight change. Only pregnant dams with scheduled sacrifice (GD 20) were

used for the calculation of mean gravid uterine weights, corrected (net) body weight gain and

summary of reproduction data.

The following females were excluded from the above-mentioned calculations:

Test group 0 (0 mg/kg bw/d):

• Females Nos. 13, 14 – not pregnant

Test group 2 (300 mg/kg bw/d):

• Females Nos. 54, 62 – not pregnant

4.2.1.1. Mortality

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

groups (0, 100, 300 or 1000 mg/kg bw/d).

Clinical symptoms

Nearly all (21 out of 25) high-dose females (1000 mg/kg bw/d) showed occasionally salivation

during the treatment period. Salivation occurred in the respective animals only shortly, i.e.

within 0-2 hours, after treatment and was observed during GD 6-8 and GD 15-19. No clinical

signs or changes of general behavior were detected in any female of all test groups beyond 2

hours after treatment. The occasional salivation was most probably caused by the bad taste or

smell of the test substance and was not assessed as a sign of systemic toxicity.

No further clinical signs or changes of general behavior, which may be attributed to the test

substance, were detected in any female at dose levels of 100, 300 or 1000 mg/kg bw/d during

the entire study period.

Food consumption

The mean food consumption of the high-, mid- and low-dose dams (1000, 300 and 100 mg/kg

bw/d) was generally comparable to the concurrent control group throughout the entire study

period.

The statistically significantly decreased food consumption value in the mid-dose dams during

GD 17-19 was not considered biologically relevant due to the lack of dose response relationship.

Body weight data

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 statistically significantly decreased body weight gain value in test group 1 during GD 3-6

(pre-treatment period) was assessed as incidental.

Corrected (net) body weight gain

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.

Terminal examinations of the dams

Uterus weight

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.

Necropsy findings

Three spontaneous findings were noted in three individual females of test groups 0 and 3 (0

and 1000 mg/kg bw/d). These gross findings were:

• Dilated renal pelvis (right) in dam No. 2,

• Diaphragmatic hernia in dam No. 4,

• Stomach: erosion(s) (one) in dam No. 79.

No further necropsy findings which could be attributed to the test substance were seen in any

dam.

Reproduction data

The conception rate was 92% in the control and the mid-dose groups (0 and 300 mg/kg bw/d)

and 100% in the low- and high-dose groups (100 and 1000 mg/kg bw/d). With these rates, a

sufficient number of pregnant females were available for the purpose of this study (according

to the test guidelines listed in chapter 2.3.).

There were no test substance-related and/or biologically relevant differences between the different

test groups 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.

EXAMINATION OF THE FETUSES

Sex distribution of the fetuses

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.

Weight of the placentae

The mean placental weights of test groups 1-3 were comparable to the concurrent control

group.

Weight of the fetuses

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 concurrent control

group.

Fetal external malformations

Three external malformations were exclusively detected in the control group. In one case, these

external malformations were associated with skeletal malformations. No external malformations

occurred in treated animals.

Tab. 2: Individual fetal external malformations

Test group	Dam No.-Fetus No., Sex	Finding
0 (0 mg/kg bw/d)	9-03 F	umbilical hernia
	15-03 Fa)	gastroschisis, ectrodactyly
1 (100 mg/kg bw/d)	none
2 (300 mg/kg bw/d)	none
3 (1000 mg/kg bw/d)	none

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

a)fetus with additional skeletal malformations

Tab.3: Total external malformations

		Test group0 0 mg/kgbw/d	Test group 1 100 mg/kg bw/d	Test group 2 300 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
LitterFetuses	N N	23 251	25 248	23 229	25 273
Fetal incidence	N (%)	2 (0.8)	0.0	0.0	0.0
Litter incidence	N (%)	2 (8.7)	0.0	0.0	0.0
Affectedfetuses/litter	Mean%	0.8	0.0	0.0	0.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Fetal external variations

No external variations were recorded.

Fetal external unclassified observations

No external unclassified observations were recorded.

Fetal soft tissue malformations

Soft tissue malformations were recorded for two control, one mid-dose and three high-dose

fetuses

The finding situs inversus in the fetuses of test groups 2 and 3 were single events in individual

fetuses and the mean values were within the historical control data (affected fetuses per litter, mean: 0.1%, range of 0.0 - 1.4%).

The findings absent subclavian and anophthalmia were observed in one fetus each of test

group 3 and the mean values were within the historical control ranges (anopthalmia: litters,

range of 0.0 - 4.0%, absent subclavian: affected fetuses per litter, range of 0.0 - 1.1%).

All these findings were single cases, they were within the historical control ranges. No ontogenetic

pattern is recognizable for all these individual malformations nor was there any cluster of

any of these individual malformations seen in the other offspring.

Therefore, the observed malformations were not assessed as treatment-related, adverse

findings.

Tab. 4: Individual soft tissue malformations

Test group	Dam No.-Fetus No., Sex	Finding
0 (0 mg/kg bw/d)	12-02 F	interrupted spinal cord
	25-14 F	hydronephrosis, hydroureter
1 (100 mg/kg bw/d)	none
2 (300 mg/kg bw/d)	72-06 M	situs inversus
3 (1000 mg/kg bw/d)	76-08 F	situs inversus
	86-04 F	absent subclavian
	95-09 M	anophthalmia

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

Tab. 5: Total soft tissue malformations

		Test group0 0 mg/kgbw/d	Test group 1 100 mg/kg bw/d	Test group 2 300 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
LitterFetuses	N N	23 122	25 117	23 110	25 130
Fetal incidence	N (%)	2 (1.6)	0.0	1 (0.9)	3 (2.3)
Litter incidence	N (%)	2 (8.7)	0.0	1 (4.3)	3 (12)
Affectedfetuses/litter	Mean%	1.5	0.0	0.9	2.4

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Fetal soft tissue variations

Three soft tissue variations were detected, i.e. malpositioned subclavian origin, dilated renal

pelvis and dilated ureter. These variations were neither significantly different from the concurrent

control nor dose-dependently altered. All of them can be found in the historical control data

at comparable incidences. Therefore, they were not assessed as treatment-related.

TAb. 6: Total soft tissue variations

		Test group0 0 mg/kgbw/d	Test group 1 100 mg/kg bw/d	Test group 2 300 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
LitterFetuses	N N	23 122	25 117	23 110	25 130
Fetal incidence	N (%)	3 (2.5)	2 (1.7)	2 (1.8)	4 (3.1)
Litter incidence	N (%)	3 (13)	2 (8.0)	2 (8.7)	4 (16)
Affectedfetuses/litter	Mean%	2.6	1.3	2.2	3.0

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Fetal soft tissue unclassified observations

No soft tissue unclassified observations were recorded.

Fetal skeletal malformations

Skeletal malformations were noted in two fetuses of the control group and in one fetus, each,

of test groups 1 and 3 (100 and 1000 mg/kg bw/d). One control

fetus had associated external malformations. The incidences of these malformations were neither

statistically significantly different from control nor dose-dependent.

The finding bipartite basisphenoid occurred in each one fetus of test groups 1 and 3 without

relation to dose. The mean value of the incidences in test group 3 was within the historical

control range (HCD: affected fetuses per litter, range of 0.0 – 0.7%). Therefore, it was not

assessed as treatment-related, adverse finding.

All other malformations in test group 1 occurred in only one fetus (33-10 F) without relation to

dose and were single, isolated cases which were mostly covered by the historical control data.

The body weight of this fetus (3.2 g) was lower compared to its group mean value (females of

test group 1: 3.5 g). The findings in this fetus were not assessed as treatment-related and

adverse.

TAb. 7: Individual fetal skeletal malformations

Test group	Dam No.-Fetus No., Sex	Finding
0 (0 mg/kg bw/d)	11-04 F	malpositioned and bipartite sternebra
	15-03 Fa)	absent forepaw phalanx, severely malformed sternum
1 (100 mg/kg bw/d)	33-10 F	bipartite basisphenoid, misshapen presphenoidal, severely malformed vertebral column and/or ribs
2 (300 mg/kg bw/d)	none
3 (1000 mg/kg bw/d)	91-07 M	bipartite basisphenoid

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

a)fetus with additional external malformations

Tab.8: Total skeletal malformations

		Test group0 0 mg/kgbw/d	Test group 1 100 mg/kg bw/d	Test group 2 300 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
LitterFetuses	N N	23 129	25 131	23 119	25 143
Fetal incidence	N (%)	2 (1.6)	1 (0.8)	0.0	1 (0.7)
Litter incidence	N (%)	2 (8.7)	1 (4.0)	0.0	1 (4.0)
Affectedfetuses/litter	Mean%	2.0	0.8	0.0	0.7

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Fetal skeletal variations

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.

Tab. 9: Total fetal skeletal varations

		Test group0 0 mg/kgbw/d	Test group 1 100 mg/kg bw/d	Test group 2 300 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
LitterFetuses	N N	23 129	25 131	23 119	25 143
Fetal incidence	N (%)	124 (96)	126 (96)	117 (98)	140 (98)
Litter incidence	N (%)	23 (100)	25 (100)	23 (100)	25 (100)
Affectedfetuses/litter	Mean%	96.5	96.5	98.0	97.9

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

For a better overview, all skeletal variations with statistically significant differences between

the control and any treated group were compiled in the table below. All

incidences were expressed on a fetus per litter basis.

Tab. 10: Occurrence of statistically significantly increased fetal skeletal variations (expressed as mean percentage of affected fetuses/litter)

Finding	Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 300 mg/kg bw/d	Test group 3 1000 mg/kg bw/d	HCD Mean % (range)
Incomplete ossification of supraoccipital; unchanged cartilage	20.2	16.8	34.2*	18.9	32.2 (14.5 – 63.5)
Misshapen sacral vertebra	1.3	3.2	0.9	5.5*	4.1 (0.7 – 9.8)
Unossified sternebra; unchanged cartilage	2.1	2.3	3.4	9.1*	4.3 (0.0 – 9.7)

mg/kg bw/d = milligram per kilogram body weight per day; HCD = Historical control data; % = per cent

* = p ≤0.05 (Wilcoxon-test [one-sided]) ** = p ≤0.01 (Wilcoxon-test [one-sided])

The increased incidences of skeletal variations were not

related to the dose or they were clearly inside the historical control range. Therefore, they are

not considered as treatment-related and adverse.

Fetal skeletal unclassified cartilage observations

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 ribs and

the sternum and did not show any relation to dosing.

Tab. 11: Total unclassified cartilage observations

		Test group0 0 mg/kgbw/d	Test group 1 100 mg/kg bw/d	Test group 2 300 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
LitterFetuses	N N	23 129	25 131	23 119	25 143
Fetal incidence	N (%)	89 (69)	79 (60)	73 (61)	102 (71)
Litter incidence	N (%)	23 (100)	24 (96)	22 (96)	25 (100)
Affectedfetuses/litter	Mean%	70.1	60.9	59.6	70.4

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Assessment of all fetal external, soft tissue and skeletal observations

There were noted external, soft tissue and skeletal malformations in all test groups (0, 100, 300 and 1000 mg/kg bw/d).

Three fetuses carried more than one malformation. Female control fetus No. 15-03 (0 mg/kg

bw/d) had multiple external malformations (i.e. gastroschisis and ectrodactyly) associated with

multiple skeletal malformations (i.e. absent forepaw phalanx and severely malformed sternum).

For female control fetus No. 25-14 hydronephrosis and hydroureter were recorded, while female

low-dose fetus No. 33-10 (100 mg/kg bw/d) had a bipartite basisphenoid, misshapen

presphenoidal and a severely malformed vertebral column and/or ribs (in the region of thoracic

vertebrae). The body weight of this fetus (3.2 g) was lower compared to its group mean value

(females of test group 1: 3.5 g). Further malformations, i.e. umbilical hernia, situs inversus,

interrupted spinal cord, anophthalmia, absent subclavian and malpositioned and bipartite

sternebrae were observed in individual fetuses, unrelated to the dose and, except ‘interrupted

spinal cord’ (control fetus), all of them can be found in the historical control data.

All these findings were single cases, no ontogenetic pattern is recognizable for all these individual

malformations nor was there any cluster of any of these individual malformations seen

in the other offspring of these test groups. They also do neither form a pattern or syndrome

with other minor anomalies which may raise toxicological concern. There is no evidence for

any association of these scattered findings with the treatment.

Tab. 12: Total fetal malformations

		Test group0 0 mg/kgbw/d	Test group 1 100 mg/kg bw/d	Test group 2 300 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
LitterFetuses	NN	23 251	25 248	23 229	25 273
Fetalincidence	N (%)	5 (2.0)	1 (0.4)	1 (0.4)	4 (1.5)
Litter incidence	N (%)	5 (22)	1 (4.0)	1 (4.3)	4 (16)
Affectedfetuses/litter	Mean%	2.0	0.4	0.4	1.4

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

External variations did not occur in any of the fetuses in this study. Three soft tissue variations

and a range of skeletal variations were noted

in all test groups including the controls. None of the total incidences showed a relation to dose. The individual variations were equally distributed about 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.

Tab. 13: Total fetal variations

		Test group0 0 mg/kgbw/d	Test group 1 100 mg/kg bw/d	Test group 2 300 mg/kg bw/d	Test group 3 1000 mg/kg bw/d
LitterFetuses	NN	23 251	25 248	23 229	25 273
Fetalincidence	N (%)	127 (51)	128 (52)	119 (52)	144 (53)
Litterincidence	N (%)	23 (100)	25 (100)	23 (100)	25 (100)
Affectedfetuses/litter	Mean%	50.8	51.6	52.3	52.6

mg/kg bw/d = milligram per kilogram body weight per day; N= number; % = per cent

No unclassified external and unclassified soft tissue observations were recorded for 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 the highest dose tested (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 substance to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at doses as high as 1000 mg/kg bw/d caused neither evidence of maternal nor fetal developmental toxicity.
In conclusion, the no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is the highest tested 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 oily 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 (corn oil) in parallel. A standard dose volume of 4 mL/kg body weight was used for each test group.

At terminal sacrifice on GD 20, 23-25 females per group had implantation sites. The analytical values of the low-dose samples (100 mg/kg bw/d) were above the expected range of 90% to 110% of the nominal concentrations. The mean value of around 138% of the nominal concentration corresponds to an actual low-dose of 138 mg/kg bw/d. For the mid- and high dose, the correctness of the prepared concentrations was shown. This did not affect the validity of the study. In the following report, the target doses were used.

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, all surviving females were sacrificed by decapitation (under isoflurane anesthesia) and assessed by gross pathology (including weight determinations of the 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. Thereafter, one half of the fetuses of each litter were examined for soft tissue findings and the remaining fetuses for skeletal (inclusive cartilage) findings.

The stability of the test substance preparations over a period of 7 days at room temperature was demonstrated. The correctness of the prepared concentrations was shown (mid- and high-dose). Concentration control analysis of the low-dose: the three analytical samples resulted in a mean value of around 138% of the nominal concentration.

The following test substance-related adverse effects/findings were noted:

Test group 3 (1000 mg/kg bw/d):

• No test substance-related adverse effects on dams, gestational parameters or fetuses

Test group 2 (300 mg/kg bw/d):

• No test substance-related adverse effects on dams, gestational parameters or fetuses

Test group 1 (100 mg/kg bw/d):

• No test substance-related adverse effects on dams, gestational parameters or fetuses

In conclusion, the NOAEL was assessed as 1000 mg/kg bw/day for maternal and fetal developmental toxicity.