Hexahydro-4-methylphthalic anhydride

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-01-24 to 2018-07-04

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:

22 January 2001

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

30 May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OECD Guidance No. 43 on Mammalian Reproductive Toxicity Testing and Assessment

Version / remarks:

24 July 2008

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Hexahydro-4-methylphthalic anhydride

EC Number:

243-072-0

EC Name:

Hexahydro-4-methylphthalic anhydride

Cas Number:

19438-60-9

Molecular formula:

C9H12O3

IUPAC Name:

5-methyl-octahydro-2-benzofuran-1,3-dione

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Polynt Lot No. T404217186
- Manufacturing date: 05 Jul 2017
- Expiration date of the lot/batch: 04 Jul 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Keep container tightly closed. Keep in a dry, cool and well ventilated place.

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Hsd. Han

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt., 1103 Budapest Cserkesz u. 90. Hungary
- Age at study initiation: Females: young adult and nulliparous females, ca. 9 weeks of age at start of the mating period; Males: experienced males, ca. 25-37 weeks of age at start of the mating period
- Weight at study initiation: The group averages of the body weight of the females were as similar as possible on the first day of gestation.
- Fasting period before study: none
- Housing: Before mating: 1-3 females per cage 1-2 males per cage; During mating: 1 male and 1-3 females / cage; During gestation: 2 sperm positive females per cage, if not possible 1 sperm positive female per cage; in Type II polypropylene/polycarbonate cages, Bedding: Lignocel Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (D-73494 Rosenberg, Holzmühle 1, Germany
- Diet: ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany ad libitum
- Water: tap water from municipal supply, as for human consumption from 500 mL bottle, ad libitum
- Acclimation period: 13 days for females, ca. 11 weeks for males

ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 25 °C
- Humidity: 35 - 57 %
- Air changes: above 10 air exchanges/hour by central aircondition system
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

vegetable oil

Remarks:

sunflower oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in the vehicle (sunflower oil) in concentrations of 115 mg/mL, 60 mg/mL and 20 mg/mL. Formulations were prepared in the formulation laboratory of the Test Facility from daily to every three days and stored in the refrigerator.

VEHICLE
- Justification for use and choice of vehicle: Sunflower oil was used as vehicle, because the substance is hydrolytically unstable in water.
- Lot/batch no.: 1710-4753
- Concentration in vehicle: 20, 60 and 115 mg/mL
- Amount of vehicle: 4 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical control of dosing solutions (control of test item concentration and homogeneity) was performed in the Analytical Laboratory of Test Facility two times during the study. Formulation samples prepared in sunflower oil were diluted with n-hexane and then analysed by a GC – FID method.
Five samples from different places were taken from each concentration for analysis of concentration and homogeneity on two occasions. Similarly, five samples were taken from the vehicle and analyzed. The suitability of the chosen vehicle for the test item was analytically proven. Recovery was between 97 and 99 % of nominal concentrations at 1 and 280 mg/g in sunflower oil, respectively. Test item proved to be stable in the formulations for one day at room temperature and for three days in refrigerator (5 ± 3°C). A separate analytical report provided these results.

Details on mating procedure:

- Impregnation procedure: cohoused
- The males were paired to females in the mornings for two to four hours (one male: one to three females) until the number of sperm positive females per group achieved at least six.
- M/F ratio per cage: 1 male and 1-3 females per cage
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

The sperm positive females were treated from gestational day 5 to 19.

Frequency of treatment:

daily

Duration of test:

The animals were sacrificed on gestation day 20.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

80 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

240 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

460 mg/kg bw/day (nominal)

No. of animals per sex per dose:

26 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose setting was based on no findings (OECD 421) and limited toxic findings (OECD 407) obtained with 450 mg/kg bw/day in previous studies, and the dose range finding prenatal developmental toxicity study with the test material in the rat.
The 1000 mg/kg bw/day dose in the dose range finding study caused death in 2/7 females. Therefore the dose was reduced to 460 mg/kg bw/day. A further female died subsequently to dose reduction after having received two times 1000 mg/kg bw/day and one time 460 mg/kg bw/day. Furthermore, severe clinical symptoms, as reduced activity, hunched position, piloerection, low muscle tone and diarrhea, were observed in two females after the reduction to 460 mg/kg bw/day. In the OECD 407 study mild squamous hyperplasia of the forestomach mucosa was seen at 450 mg/kg bw/day. Based on these results, 460 mg/kg bw/day was selected as the maximum applicable dose for the OECD 414 main study neither causing maternal death nor severe toxicity. The low dose was chosen to induce no toxic effect. The mid dose was interpolated geometrically.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily after dosing, when signs of toxicity were detected, animals were observed more frequently.
- Individual observation included the check of behavior and general condition.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of the male animals was not measured. Body weights of sperm positive females were measured on gestation days 0, 3, 5, 8, 11, 14, 17 and 20 (accuracy of 1 g). The corrected body weight was calculated for the 20th day of pregnancy (body weight on day 20 minus the weight of the gravid uterus).

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption was measured between gestation days 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17 and 17 to 20 by re-weighing the non-consumed diet (accuracy: 1 g).

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20 by decapitation after anesthetizing by an overdose Release® Pentobarbitat-sodium solution administered by an intraperitoneal injection.
- Organs examined: The abdomen was opened, the uterus with cervix and the left ovary were removed and weighed. The right ovary was placed into a Petri dish after removal. After removing the uterus gross pathology of dams' viscera was performed. The number of corpora lutea in each ovary and implantation sites in each uterine horn, live fetuses, early and late embryonic death and fetal death were counted.

OTHER:
Examination for Sign of Implantation:
On gestation days 13 the sperm positive females were checked for the presence of vaginal bleeding which indicated the implantation of conceptuses. If negative on day 13, the examination was repeated on day 14 of gestation.

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

Fetal examinations:

Fetuses were removed from the opened uterus. Euthanasia of the fetuses was performed by hypothermia. The fetuses were sunk in a Petri-dish filled up with water. Spontaneous movement of fetuses was observed as a viability assessment. The fetuses were washed with tap water and randomly laid on a filter paper with written ordinal numbering. Bleeding from the umbilical cord after it is cut was observed also as a sign of viability. Each live fetus and its placenta was weighed individually (fetuses accuracy 0.01 g, placentas accuracy 0.001 g). The gender of the fetuses was determined according to the anogenital distance.

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

Statistics:

The statistical evaluation of data was performed with the program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test.
Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan’s Multiple Range test was used to assess the significance of intergroup differences. If significance was the result of the Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test.

Indices:

Besides the data mentioned above the pre-implantation losses, post-implantation losses, sex distribution, the percentages of fetuses with external abnormalities, visceral abnormalities, and skeletal abnormalities were calculated.

Historical control data:

Historical control data of Hannover Wistar rats are available. The historical control study on teratology in Hannover Wistar rats was performed on 61 sperm positive females at the test facility in 2008 (Study no. 000.197.1437).

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related clinical signs observed in the females of all dose groups. Piloerection, reduced activity and hunched position were recorded for one dam on gestation day 18 and 19 in the control group.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

None of the females died in the course of the study

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight, body weight gain in the different periods, corrected body weight of the females was similar in all groups.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Minimal but statistically significant decreases of the food consumption were observed in the test item treated groups if compared to the control. This was indicated before the treatment period (from gestation day 3 to 5) and in the treatment period (from gestation day 11 to 17) in the 80 mg/kg bw/day dose group and from gestation day 5 to 11 in all groups. There was no dose response seen and these differences were considered as biologically not relevant and unrelated to the treatment.

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:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related necropsy findings were observed.
Reddish mottled lungs were recorded for 6, 9, 11 and 4 does respectively in the control, 80, 240 and 460 mg/kg bw/day group without a dose response. Pinhead sized haemorrhages were recorded in the lungs in single cases (one dam each in the test item treated groups). Both observations were not attributed to the test item. There were no macroscopic alterations recorded for the dams during necropsy in the experimental groups. Blood was observed in the uterus for one control dam.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There was no increase indicated in the mean percent of pre- and postimplantation loss (early embryonic, late- and fetal death). Moreover the CH square test indicated a statistically significant reduction (p<0.05) in the postimplantation loss in the low and high dose group.

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:

not examined

Other effects:

not examined

Details on maternal toxic effects:

The mean number of implantations, the sex distribution of the fetuses as well as in the mean number of viable fetuses was similar in all dose groups.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

460 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: No adverse effects observed.

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no treatment related differences in the body weight of the fetuses and placental weight.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The mean number of viable fetuses was similar in all dose groups.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

not examined

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no malformations found in the test item treated groups. Two fetuses in the control group had retrognathia, neck edema or whole body edema and mal-rotated forelimb/s.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The number of the affected litters was 1 in each experimental group.
A split sternebral cartilage in the 240 mg/kg bw/day group, a bipartite cartilage of the thoracic centrum, a hemicentric and cartilageously dumb-bell shaped lumbar centrum both in the low dose group and one multiply malformed fetus in the control and high dose group were found. The control fetus had a short mandible, a misshapen (wider) and split sternum, abnormal articulation of sternoclavicular joint, misshapen clavicle and scapula, bipartite cartilage of cervical VII vertebral centrum, hemicentric ossification of thoracic V vertebral centrum, a dumb-bell shaped or/and bipartite cartilage of thoracic IX and XII centrum respectively. The fetus in the high dose had a misshapen xiphoid cartilage with a hole, bipartite cartilage and hemi-centric ossification of thoracic III centrum as well as a bent left scapula and bentness of both humeri. The malformations found occurred sporadically with low incidence or/and without a dose response. Furthermore most of these changes occurred in control fetuses without a relationship to the treatment according to the historical control data. Therefore these malformations were judged to be unrelated to an effect of the test item.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The number of the affected litters was 1 each in the test item treated groups.
Microphthalmia was found in one fetus in the mid dose and one in the high dose. Situs inversus totalis was observed in one fetus in the low dose group. Microphthalmia as well as situs inversus totalis occur sporadically with low incidence in control fetuses according to the experience of this laboratory which is in line with the historical control data. Considering also the low incidence, these malformations were judged as incidental, non-treatment related.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Placentas, amnion:
Edema and paleness, both for two fetuses in the control group and latter for one in the high dose group was recorded.
Visceral variations:
Hydroureter, observed in control and 240 mg/kg bw/day groups, without a dose response, and slightly rounded apex cordis in one fetus in the 460 mg/kg bw/day were evaluated as variations. Considering the low incidences and/or the lack of dose response these variations were not attributed to the treatment.
Skeletal variations:
There was a statistical significant increase (p<0.05) in the occurrence of litters with bipartite or/and asymmetric thoracic centrum (evaluated as variations during the skeletal examination) at 240 mg/kg bw/day but no dose response was indicated.

Details on embryotoxic / teratogenic effects:

There were no test item related adverse effects on the fetal weight, external and visceral development of fetuses. There were no test item related malformations and variations found.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

460 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed.

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 1: PREGNANCY DATA OF FEMALES, MORTALITY, MALFORMATIONS

Dose groups	Control		80 mg/kg bw/day		240 mg/kg bw/day		460 mg/kg bw/day
Number of sperm positive females	26		26		26		26
Number of females with no implantation and no corpora lutea	3		6		4		6
Number of females with no implantation but corpora lutea	2		1		1		2
Number and percent of females with implantation (pregnants)	21	80%	19	73%	21	80%	18	69%
Number of females with total post-implantation loss	0		0		0		0
Number of pregnant females died due to toxicity	0		0		0		0
Number of pregnant females excluded due to a technical error (determination of mating day)	1		0		0		1
Number of females with live fetuses (number of litters)	20		19		21		17
Number and percent of litters with malformed fetuses	2	10%	2	10.5%	2	9.5%	2	11.8%

 

Table 2: SUMMARY OF CLINICAL SIGNS AND NECROPSY FINDINGS OF DAMS

	control	80 mg/kg bw/d	240 mg/kg bw/d	460 mg/kg bw/d
No of animals	20	19	21	17
Clinical Symptoms
none	19	19	21	17
piloerection	1 *	0	0	0
Reduced activity	1 *	0	0	0
Hunched posture	1 *	0	0	0
Necropsy findings
No macroscopic alterations	13	9	9	12
Reddish mottled lungs	6	9	11	4
Pinhead-sized haemorrhages in the lungs	0	1	1	10
blood in uterus	1 *	0	0	0

* the same animal

 

Table 3: SUMMARY OF GRAVID UTERINE WEIGHT, CORRECTED BODY WEIGHT AND CORRECTED BODY WEIGHT GAIN OF DAMS

		Control	80 mg/kg bw/day	240 mg/kg bw/day	460 mg/kg bw/day
Gravid uterine weight(g)	MEAN	62.5	65.4	62.5	67.9
	SD	12.06	7.90	14.48	10.60
	n	20	19	21	17 NS
Corrected body weight(g)	MEAN	262.0	258.5	259.2	255.5
	SD	26.92	16.83	12.43	16.53
	n	20	19	21	17 NS
Corrected	MEAN	50.0	47.5	46.1	44.8
body weight gain(g)	SD	26.37	11.76	9.49	12.84
	n	20	19	21	17 NS

NS = Not Significant

 

 

Table 4: PREGNANCY DATA OF FEMALES, MORTALITY, MALFORMATIONS

Dose groups	Control		80 mg/kg bw/day		240 mg/kg bw/day		460 mg/kg bw/day
Number of sperm positive females	26		26		26		26
Number of females with no implantation and no corpora lutea	3		6		4		6
Number of females with no implantation but corpora lutea	2		1		1		2
Number and percent of females with implantation (pregnants)	21	80%	19	73%	21	80%	18	69%
Number of females with total post-implantation loss	0		0		0		0
Number of pregnant females died due to toxicity	0		0		0		0
Number of pregnant females excluded due to a technical error (determination of mating day)	1		0		0		1
Number of females with live fetuses (number of litters)	20		19		21		17
Number and percent of litters with malformed fetuses	2	10%	2	10.5%	2	9.5%	2	11.8%

 

Table 5: RESULTS OF EXTERNAL, VISCERAL AND SKELETAL EXAMINATIONS

(percentile litter means and SD)

Dose groups		Control	80 mg/kg bw/day	240 mg/kg bw/day		460 mg/kg bw/day
EXTERNAL EXAMINATION
Litters examined	N	20	19	21		17
Fetuses examined	N	215	217	226		205
Fetuses with abnormalities	Mean	6.2	1.8	8.5		5.8
	SD	22.41	3.59	22.40		11.90
Variation	Mean	5.4	1.8	8.5		5.8
	SD	22.34	3.59	22.40		11.90
Malformation	Mean	0.8	0.0	0.0		0.0
	SD	3.44	0.00	0.00		0.00
Retarded in body weight	Mean	5.8	1.8	8.5		5.8
	SD	22.44	3.59	22.40		11.90
VISCERAL EXAMINATION
Litters examined	N	20	19		21	17
Fetuses examined	N	109	109		115	104
Fetuses with abnormalities	Mean	0.8	0.9		3.5	1.7
	SD	3.73	3.82		9.54	6.93
Variation	Mean	0.8	0.0		2.9	0.8
	SD	3.73	0.00		9.34	3.46
Malformation	Mean	0.0	0.9		0.6	0.8
	SD	0.00	3.82		2.73	3.46
SKELETAL EXAMINATION
Litters examined	N	20	19		21	17
Fetuses examined	N	106	108		111	101
Fetuses with abnormalities	Mean	16.8	9.5		16.7	11.4
	SD	26.02	15.02		23.38	15.80
Variation	Mean	16.0	7.8		15.6	10.2
	SD	24.56	11.58		23.58	14.19
Malformation	Mean	0.8	1.8		1.2	1.2
SD	3.73	7.65	5.46		4.85	NS

No significant findings.

Remarks:

* = p < 0.05

** = p < 0.01

U = Mann-Whitney U - test Versus Control DN = Duncan's multiple range test

 

Table 6: RESULTS OF EXTERNAL, VISCERAL AND SKELETAL EXAMINATIONS

(sum, %)

		Control	80 mg/kg bw/day	240 mg/kg bw/day	460 mg/kg bw/day
EXTERNAL EXAMINATION
Litters examined	N	20	19	21	17
Fetuses examined	N	215	217	226	205
Fetuses with abnormalities	N	7	4	12	12
	%	3	2	5	6
Variation	N	5	4	12	12
	%	2	2	5	6
Litters	N	2	4	7	4
	%	10	21	33	24
Malformation	N	2	0	0	0
	%	1	0	0	0
Litters	N	1	0	0	0
	%	5	0	0	0
Retarded in body weight	N	6	4	12	12
	%	3	2	5	6
Litters	N	2	4	7	4
	%	10	21	33	24
VISCERAL EXAMINATION
Litters examined	N	20	19	21	17
Fetuses examined	N	109	109	115	104
Fetuses with abnormalities	N	1	1	5	2
	%	1	1	4	2
Litters	N	1	1	3	1
	%	5	5	14	6
Variation	N	1	0	4	1
	%	1	0	3	1
Litters	N	1	0	2	1
	%	5	0	10	6
Malformation	N	0	1	1	1
	%	0	1	1	1
Litters	N	0	1	1	1
	%	0	5	5	6
SKELETAL EXAMINATION
Litters examined	N	20	19	21	17
Fetuses examined	N	106	108	111	101
Fetuses with abnormalities	N	15	10	14	12
	%	14	9	13	12
Litters	N	10	7	12	7
	%	50	37	57	41
Variation	N	14	8	13	11
	%	13	7	12	11
Litters	N	10	7	11	7
	%	50	37	52	41
Malformation	N	1	2	1	1
	%	1	2	1	1
Litters	N	1	1	1	1
	%	5	5	5	6

Remarks:

* = p < 0.05; CH2

** = p < 0.01; CH2

 

Applicant's summary and conclusion

Conclusions:

Oral treatment of pregnant Hsd. Han: Wistar rats from gestation day 5 up to day 19 with the test item at the dose levels of 80, 240 and 460 mg/kg bw/day did not cause maternal toxicity or signs of developmental toxicity. The No Observed Adverse Effect Level (NOAEL) for both maternal and developmental toxicity (including teratogenicity) was 460 mg/kg bw/day.

Executive summary:

The test item was examined for its possible prenatal developmental toxicity in a GLP compliant study according to OECD 414. Groups of 26 sperm-positive female Hsd. Han: Wistar rats were treated with the test item by oral administration daily at three dose levels of 80, 240 and 460 mg/kg bw/day respectively from day 5 up to and including day 19 post coitum. A control group of 26 sperm positive females was included and the animals were given the vehicle sunflower oil. The treatment volume was 4 mL/kg bw. Concentrations of the test item in the dosing formulations varied in the acceptable range between 99 and 109 % of nominal concentrations at both analytical occasions confirming proper dosing. During the study, mortality was checked and clinical observations were performed. The body weights and food consumption of the dams were also recorded. The day when sperm was detected in the vaginal smear was regarded as day 0 of gestation. Caesarean section and gross pathology were performed on gestational day 20. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and gross external abnormalities. The placentas were weighed and examined externally. About half of each litter was preserved for visceral examination and the other half of the litters were preserved for skeletal evaluation. At visceral examination the bodies were micro dissected by means of a dissecting microscope. The heads were examined by Wilson's free-hand razor blade method. After cartilage-bone staining the skeletons were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded.In total, on gestation day 20 there were 20, 19, 21 and 17 evaluated litters in the control, 80, 240 and 460 mg/kg bw/day group respectively.None of the females died before scheduled necropsy and there were no test item related clinical signs recorded in all dose groups. No treatment related necropsy findings were observed. There were no treatment related changes in food consumption or body weights indicated. The number of implantations, intrauterine mortality and sex distribution of the fetuses were not influenced by the treatment. There were no test item related adverse effects on the fetal weight, external and visceral development of fetuses. There were no test item related malformations found. Based on these observations the No Observed Adverse Effect Level (NOAEL) for both maternal and developmental toxicity (including teratogenicity) was 460 mg/kg bw/day.