[1,3(or 1,4)-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

[1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide

EC Number:

218-664-7

EC Name:

[1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide

Cas Number:

2212-81-9

Molecular formula:

C20H34O4

IUPAC Name:

1,3-bis[1-(tert-butylperoxy)-1-methylethyl]benzene

Constituent 2

Reference substance name:

Di-tert-butyl α,α,α',α'-tetramethyl-(p-phenylenedimethylene) diperoxide

EC Number:

220-479-1

EC Name:

Di-tert-butyl α,α,α',α'-tetramethyl-(p-phenylenedimethylene) diperoxide

Cas Number:

2781-00-2

Molecular formula:

C20H34O4

IUPAC Name:

1,4-bis[1-(tert-butylperoxy)-1-methylethyl]benzene

Test material form:

solid: flakes

Test animals

Species:

rat

Strain:

other: RccHanTM: WIST(SPF)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V., Kreuzelweg 535961 NM Horst / Netherlands
- Age at D0 post coitum: 11 weeks
- Weight at Day 0 Post Coitum: 189 to 236 g
- Fasting period before study: no
- Housing: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding
- Diet (e.g. ad libitum): Pelleted standard Harlan Teklad 2914C rodent maintenance diet
- Water (e.g. ad libitum): community tap-water
- Acclimation period: >= 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared daily. Vehicle was pre-warmed to a temperature of approximately 40 °C. Luperox F was weighed into a glass beaker on a tared precision balance and approximately 80% of the warm vehicle was added (w/v). The mixture was homogenized using an electrical homogenizer until a clear to colloid-like yellowish solution was obtained. Having obtained a homogeneous mixture, remaining vehicle was added until the required final volume. Separate formulations were prepared for each concentration.
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility
- Concentration in vehicle: 25, 75 and 250 mg/ml
- Amount of vehicle (if gavage): 4 ml/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. To confirm the stability (8 days at room temperature) samples of about 2 g of each concentration were taken from the middle of each aliquot used on day 7 of the treatment. At the end of the study, before the first planed necropsies, samples were taken from the middle to confirm concentration. The samples were delivered to the analytical depart¬ment at ambient temperature and analyzed immediately or stored frozen (at -20 ± 5 °C) until analysis.

The samples were analyzed by HPLC coupled to an UV detector following an analytical proce-dure provided by the Sponsor and adapted at Harlan Laboratories. The test item was used as the analytical standard. Analyzed samples were not discarded without written consent from the study director.
The Luperox F concentrations in the dose formulations ranged from 86.7% to 101.8% with reference to the nominal concentration and were within the accepted range of ±20%.
The homogeneous distribution of Luperox F in the preparations was approved because single results found did not deviate more than 4.7% from the corresponding mean and met the specified acceptance criterion of =15%.
In addition, the test item was found to be stable in application formulations when kept eight days at room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean value.

Details on mating procedure:

After acclimatization, females were housed with sexually mature males (1:1) in special automatic mating cages i.e. with synchronized timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the different females. The females were removed and housed individually if:
- the daily vaginal smear was sperm positive, or
- a copulation plug was observed.
The day of mating was designated day 0 post coitum.
Male rats of the same source and strain were used only for mating.

Duration of treatment / exposure:

15 days (during gestation period from day 6 to 20 post coitum)

Frequency of treatment:

daily

Duration of test:

up to GD 21

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on a previous dose range-finding study in Han Wistar rats, Harlan Laboratories study no. D81444, using dose levels of 0, 100, 300 and 1000 mg/kg/day, where no adverse effects were observed up to the highest dose level.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily cage-side clinical observations (once daily,during acclimatization and up to day of necropsy).

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Daily from day 0 until day 21 post coitum.

FOOD CONSUMPTION: Yes
- For the following periods: days 0 - 3, 3 - 6, 6 - 9, 9 - 12, 12 - 15, 15 - 18 and 18 - 21 post coitum.

WATER CONSUMPTION : No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #21
- Organs examined: gross macroscopic examination of all internal organs with emphasis on the uterus, uterine contents, corpora lutea count and position of fetuses in the uterus. The uteri (and contents) of all females with live fetuses were weighed during necropsy on day 21 post coitum to enable the calculation of the corrected body weight gain. If no implantation sites were evident, the uterus was placed in an aqueous solution of ammonium sulfide to accentuate possible hemorrhagic areas of implantation sites.
At the scheduled sacrifice, placentas were trimmed from any adherent tissue, and their wet weight taken.

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 uterus by Caesarean section, sexed, weighed individually, exam-ined for gross external abnormalities, sacrificed and allocated to one of the following procedures:

1. Microdissection technique (sectioning/dissection technique). At least one half of the fetuses from each litter was fixed in Bouin's fixative (one fetus per container). They were examined by a combination of serial sections of the head and microdissection of the thorax and abdomen. This included detailed examination of the major blood vessels and sectioning of the heart and kidneys. After examination, the tissue was preserved in a solution of glycerin/ethanol (one fetus per container). Descrip-tions of any abnormalities and variations were recorded.

2. The remaining fetuses were eviscerated and with the exception of over the paws, the skin was removed and discarded. Carcasses were processed through solutions of ethanol, glacial acetic acid with Alcian blue (for cartilage staining), potassium hydroxide with Alizarin red S (for clearing and staining ossified bone) and aqueous glycerin for pres-ervation and storage. The skeletons were examined and all abnormal findings and variations were recorded. The specimens were preserved individually in small containers.

Statistics:

The following statistical methods were used to analyze food consumption, body weights, reproduction and skeletal examination data:

• Means and standard deviations of various data were calculated and included in the report.
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Indices:

Pre- and post-implantation losses, embryonic and fetal deaths, live and dead fetuses, abnormal fetuses, fetal sex ratios and fetal body weights.

Historical control data:

See Attached backgroung material

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs were observed at any dose level during the study.

Mortality:

no mortality observed

Description (incidence):

All females survived until the scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

(See attached figures)
Mean body weight gain from day 6 to 21 post coitum was 48%, 49%, 46% and 41% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively. Mean corrected body weight gain (body weight gain at termination corrected for the gravid uterus weight) was 10.8%, 10.1%, 8.1% and 4.8% in order of ascending dose levels.
Treatment with the test item caused a statistically significant reduction in body weights, body weight gain and corrected body weight gain at the dose level of 1000 mg/kg bw/day. The statistical significance of the effect was observed from day 11 of the gestation period onwards for the body weights and from day 8 of the gestation period onwards for the body weight gain.
A reversible reduction in body weight gain was observed at the dose level of 300 mg/kg bw/day. This effect was statistically significant day 11 to 16 of the gestation period. No statistically significant changes were observed in absolute body weights or in corrected body weight gain.
At the dose level of 100 mg/kg bw/day, body weights, body weight gain and corrected body weight gain were similar to the respective values in the control group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food consumption from day 6 to 21 post coitum was 22.3, 22.2, 21.9 and 20.9 g/animal/day in order of ascending dose levels.
Treatment with the test item caused a reversible reduction in food consumption at the dose level of 1000 bw/day. The reduction was statistically significant from day 9 to 15 post coitum. Afterwards food consumption recovered and was similar to the control values towards the end of the study.
At the dose levels of 100 and 300 mg/kg bw/day, food consumption was similar to the control values during the entire treatment period.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment-related findings were noted during macroscopical examination of females at any dose level.
In group 4, a watery fluid was observed in one non-pregnant female (no. 75). Due to isolated occurrence, this finding was considered not to be related to the treatment.
No further findings were noted during necropsy in any group.

Maternal developmental toxicity

Details on maternal toxic effects:

(See Summary Table 2)
The relevant reproduction data (post implantation loss and number of fetuses per dam) were not affected by treatment with the test item. Mean number of implantations lost was 4.5, 3.3, 5.5 and 4.1 per dam whereas mean number of live fetuses was 12.6, 12.6, 12.0 and 11.8 per dam at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day.
The slightly lower mean number of fetuses in the high-dose group was due to the lower number of implantation sites. Because implantation process is considered to be completed before the treatment start, this observation was not related to the treatment.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Fetal body weights (TABLE 4) were not affected by the treatment with the test item at any dose level. Mean fetal body weights for male and female fetuses calculated on a litter basis were: 5.0 g, 5.1 g, 5.2 g and 5.1 g whereas calculated on an individual basis, they were 5.0 g, 5.1 g, 5.2 g and 5.1 g, both cited in order of ascending dose level.
Mean body weights of live fetuses calculated on individual basis were statistically significantly higher in mid-dose group and calculated on an individual basis were statistically significantly higher in all dose groups compared to the respective control values. The differences were however only minor and did not follow a dose-dependency. Therefore this observation was considered not to be related to the treatment.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No test item-related differences in the sex ratio (TABLE 2) of the fetuses were observed in any group.
The proportion of male fetuses was 47.5%, 53.3%, 46.4% and 49.8% in order of ascending dose level.

External malformations:

no effects observed

Description (incidence and severity):

No findings were noted during external examination of the fetuses in any group.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Ossification and Supernumerary Ribs
The corresponding historical control data are in an attached background material.
No test item-related effects on ossification stage or incidence of supernumerary ribs were observed at any dose levels.
Incidentally, slightly but statistically significantly lower incidence of non-ossified limb bones was observed in the mid-dose group. Due to the lack of a dose dependency, this finding was considered not to be related to the treatment.

Additional Cartilage Variations
The corresponding historical control data are in an attached background material.
No test item-related additional cartilage variations were observed at any dose levels.
Incidentally, slightly but statistically significantly higher incidence of interrupted costal cartilage was observed in the mid-dose group. Due to the lack of a dose dependency, this finding was considered not to be related to the treatment.
Further, statistically significantly higher number of fetuses with branched xiphoid cartilage was observed in all dose groups when calculated on a fetus basis. The differences were only minor and they were not statistically significant when calculated on a litter basis. Further, values were distributed without dose-dependency. For these reasons this observation was considered not be related to the treatment with the test item.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The corresponding historical control data are in an attached background material.
During visceral examination of the fetuses, findings were noted in:
36% examined fetuses (in 96% litters) in the control group
34% examined fetuses (in 96% litters) at the dose level of 100 mg/kg bw/day
35% examined fetuses (in 100% litters) at the dose level of 300 mg/kg bw/day
41% examined fetuses (in 100% litters) at the dose level of 1000 mg/kg bw/day
Fetuses with anophthalmia (unilateral) were found in all dose groups; one fetus (in litter no. 42), one fetus (in litter no. 72) and two fetuses (in litter nos. 76 and 95) with this finding were observed at the dose levels of 100, 300 and 1000 mg/kg bw/day, respectively. No fetuses with this observation were found in the control group. In addition to the unilateral anophthalmia found in fetuses in groups 2 and 3 and one of the fetuses in group 4 (from litter nos. 42, 72 and 76, respectively), reduced body weights but no other visceral findings were recorded. The second affected fetus in group 4 (from litter no. 95) had one eye missing, one eye small, reduced body weight as well as multiple abnormalities of other organs (absent kidneys and ureter and malpositioned ovaries). Based on this observation, a different mechanism causing the developmental disturbance may be concluded for the two fetuses in group 4. Considering the type and distribution of fetuses with the eye finding among groups, there were no apparent dose dependency. Although anophthalmia was not recorded in the most recent historical controls, this abnormality is known to occur spontaneously in the rat strain used in the study and therefore the observation in this study was considered to be incidental and not related to the treatment.

Further abnormalities occurred in single fetuses and therefore were considered not to be related to the treatment; a situs inversus found in one fetus in litter no. 27 at the dose level of 100 mg/kg bw/day as well as severely thin diaphragm tendinous region in one fetus in litter no. 93, severely dilated renal pelvis in one fetus in litter no. 87, and absent kidney and ureter together with severely malpositioned ovary adjacent to adrenal in one fetus in litter no. 95 at the dose level of 1000 mg/kg bw/day.
Type and distribution of variations recorded during the visceral examination did not give any indication of a test item-related effect. The variations were distributed among groups without dose dependency and their frequency remained within the historical control range.

Description (incidence and severity):

Placenta Weights
Placenta weights (TABLE 3) were not affected by the treatment with the test item at any dose level. Mean placenta weights calculated on a litter basis were: 0.515 g, 0.530 g, 0.549 g and 0.538 g whereas calculated on an individual basis, they were 0.511 g, 0.527 g, 0.542 g and 0.539 g, both cited in order of ascending dose level.
Mean values calculated on an individual basis were statistically significantly higher in all dose groups if compared to the control. The differences were only minor and not dose-dependent. Therefore this observation was considered not to be related to the treatment.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: sign of minor delay in development, probably secondary to the maternal toxicity

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

Table 1: Summary of Performance of Mated Females

Group Dose (mg/kg)	1 (0)	2 (100)	3 (300)	4 (1000)
Female numbers	1 - 24	25 - 48	49 - 72	73 - 96
Number of mated females	24	24	24	24
Not pregnant (A)	0	1	1	4
Number of females with live fetuses at termination (B)	24	23	23	20

 (A)  Female nos. 36, 56, 75, 83, 85 and 88.

(B)   Only dams with at least one live fetus at Caesarean section were used for the calculations of food consumption, body weight gain and corrected body weight gain data

TABLE 2: REPRODUCTION DATA SUMMARY
PARENTAL GENERATION - POST COITUM

		GROUP 1 0 MG/KG	GROUP 2 100 MG/KG		GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
	NUMBER OF DAMS	24	23		23	20
	CORPORA LUTEA	320	308		297	254
	MEAN (+)	13.3	13.4		12.9	12.7
	ST.DEV.	1.6	2.0		1.9	1.9
	PRE-IMPLANTATION LOSS	3	9		5	9
	% OF CORP. LUTEA (#)	0.9	2.9		1.7	3.5 #
	MEAN (+)	0.1	0.4		0.2	0.5
	ST.DEV.	0.4	0.7		0.6	1.4
	NUMBER OF DAMS AFFECTED	2	6		3	3
	IMPLANTATION SITES	317	299		292	245
	% OF CORP. LUTEA (#)	99.1	97.1		98.3	96.5 #
	MEAN (+)	13.2	13.0		12.7	12.3
	ST.DEV.	1.6	2.0		1.8	1.8
	POST-IMPLANTATION LOSS	14	10		16	10
	% OF IMPL. SITES (#)	4.4	3.3		5.5	4.1
	MEAN (+)	0.6	0.4		0.7	0.5
	ST.DEV.	1.1	0.7		0.8	0.8
	NUMBER OF DAMS AFFECTED	7	7		12	7
	IMPLANTATION SITE SCARS	0	0		0	0
	EMBRYONIC/FETAL DEATHS TOTAL	14	10		16	10
	EMBRYONIC RESORPTIONS	14	10		16	10
	FETAL RESORPTIONS	0	0		0	0
	TOTAL FETUSES	303	289		276		235
	% OF IMPL. SITES (#)	95.6	96.7		94.5		95.9
	MEAN (+)	12.6	12.6		12.0		11.8
	ST.DEV.	2.2	2.1		2.2		1.7
	LIVE FETUSES	303	289		276		235
	DEAD FETUSES	0	0		0		0
	ABNORMAL FETUSES	0	0		0		0
SEX OF FETUSES TOTAL MALES		144	154	128			117
% OF FETUSES (#)		47.5	53.3	46.4			49.8
MEAN (+)		6.0	6.7	5.6			5.9
ST.DEV.		2.4	2.6	2.1			2.0
TOTAL FEMALES		159	135	148			118
% OF FETUSES (#)		52.5	46.7	53.6			50.2
MEAN (+)		6.6	5.9	6.4			5.9
ST.DEV.		2.6	2.1	2.3			2.1

*/** : Dunnett-Test based on pooled variance significant at level 5% (*) or 1% (**)

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

+ : Steel Test significant at level 5%

 

TABLE 3. PLACENTA WEIGHTS SUMMARY

PARENTAL GENERATION - POST COITUM

 

	GROUP 1 0 MG/KG	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
PLACENTA WEIGHTS (G) (LITTER BASIS)
TOTAL FETUSES N (LITTERS)	24	23	23	20
MEAN (*)	0.515	0.530	0.549	0.538
ST.DEV.	0.043	0.052	0.061	0.052
PLACENTA WEIGHTS (G) (INDIVIDUAL BASIS)
TOTAL FETUSES N (FETUSES)	303	289	276	235
MEAN (*)	0.511	0.527 *	0.542 **	0.539 **
ST.DEV.	0.074	0.080	0.085	0.081

 

TABLE 4. REPRODUCTION DATA SUMMARY

PARENTAL GENERATION - POST COITUM

		GROUP 1 0 MG/KG		GROUP 2 100 MG/KG	GROUP 3 300 MG/KG		GROUP 4 1000 MG/KG
NUMBER OF DAMS		24		23	23		20
SEX OF FETUSES (CONT.)
LIVE MALES		144		154	128		117
LIVE FEMALES		159		135	148		118
WEIGHTS OF LIVE FETUSES (G) (LITTER BASIS)
	TOTAL FETUSES
	N (LITTERS)	24	23			23		20
	MEAN (*)	5.0	5.1			5.2+		5.1
	ST.DEV.	0.2	0.3			0.3		0.4
	MALES
	N (LITTERS)	24	23			23		20
	MEAN (*)	5.1	5.2			5.3*		5.2
	ST.DEV.	0.2	0.3			0.3		0.3
	FEMALES
	N (LITTERS)	24	23			23		20
	MEAN (*)	4.9	4.9			5.1		4.9
	ST.DEV.	0.2	0.3			0.3		0.3
	WEIGHTS OF LIVE FETUSES (G) (INDIVIDUAL BASIS)
	TOTAL FETUSES
	N (FETUSES)	303	289					276
	MEAN (*)	5.0	5.1**			5.2**		5.1*
	ST.DEV.	0.4	0.4			0.4		0.4
	MALES
	N (FETUSES)	144	154			128		117
	MEAN (*)	5.1	5.2**			5.3**		5.2**
	ST.DEV.	0.3	0.4			0.4		0.5
	FEMALES
	N (FETUSES)	159	135			148		118
	MEAN (*)	4.8	4.9			5.0**		4.9
	ST.DEV.	0.3	0.4			0.4		0.4

 

*/** : Dunnett-Test based on pooled variance significant at level 5% (*) or 1% (**)

#/## : Fisher's Exact Test significant

+ : Steel Test significant at level 5%

Fetal Examination: External Abnormalities and Variations: see Attached background material

Fetal Examination: Visceral Abnormalities and Variations

see Attached background material

Fetal Examination: Bone and Cartilage Abnormalities and Variations:

see Attached background material

Applicant's summary and conclusion

Conclusions:

The purpose of this study was to detect effects on the pregnant rat and development of the embryo and fetus consequent to exposure of the female to Luperox F from day 6 post coitum (implantation) to day 20 post coitum (the day prior to Caesarean section). Luperox F was administered orally by gavage once daily at dose levels of 0, 100, 300 and 1000 mg/kg bw/day.
All females survived until the scheduled necropsy. No clinical signs were recorded in any group.
Sins of general toxicity were observed in females at the dose levels of 1000 mg/kg bw/day. A reduction in food consumption was observed in this group together with reduced body weights, reduced body weigh gain and reduced corrected body weight gain. Effect on food consumption was reversible and at the end of the gestation it was similar to the control values despite continued treatment and was therefore considered not to be adverse. Different from the changes in food consumption, effects on body weight and body weight development remained statistically significant until the completion of the study and therefore they were considered to be adverse.
Reduction in body weight gain was observed also at the dose level of 300 mg/kg bw/day. This effect was reversible and did not result in any significant changes in absolute body weights. For these reasons it was considered not to be adverse.
Relevant reproduction data (post-implantation loss and number of fetuses per dam) were not affected by the treatment with the test item at any dose level.
No test item-related effects on placenta weights were observed at any dose level.
During visceral examination, anophthalmia was found in one fetus in each group 2 and 3 and two fetuses in group 4. In one of the fetuses at the high dose level, the eye finding was accompanied by small other eye and abnormalities in other organs indicating possible another mechanism leading to the developmental disturbance than that observed in the remaining three fetuses, in which anophthalmia was the only identified visceral change. Due to the type and distribution of this finding, which did not clearly follow dose dependency, it was considered to be incidental and not related to the treatment. Also the type and distribution of the remaining visceral findings gave no indication of a test item-related effect.
No adverse effects on fetal skeleton development occurred in the study at any dose level. A slightly higher number of fetuses with fused zygomatic arch and slightly higher number of fetuses with malpositioned pelvic girdle were found at the dose level of 1000 mg/kg bw/day. The differences to the control values were only minor and frequency of these findings was close to the historical control range. Therefore these findings were considered to be a sign of minor delay in development, probably secondary to the maternal toxicity.
Based on these results, the NOAEL (No Observed Adverse Effect Level) for maternal toxicity was considered to be 300 mg/kg bw/day whereas NOEL (No Observed Effect Level) was 100 mg/kg bw/day. For prenatal developmental toxicity, the NOEL was considered to be 300 mg/kg bw/day and the NOAEL was considered to be 1000 mg/kg bw/day.

Executive summary:

The possible toxic effects of Luperox F on the pregnant rat and development of the embryo and fetus was evaluated by continuous oral administration to female rat from day 6post coitum(implantation) to day 20post coitum(the day prior to Caesarean section). This study was performed following the OECD test guideline no. 414.

Four groups of 24 mated females per group were treated by gavage with Luperox F once daily at dose levels of 0, 100, 300 and 1000 mg/kg body weight/day (groups 1, 2, 3 and 4, respectively). A standard dose volume of 4 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (corn oil). All females were sacrificed on day 21post coitumand the fetuses were removed by Caesarean section.

All females survived until the scheduled necropsy. No clinical symptoms or signs were observed at any dose level during the study. Treatment with the Luperox F caused a reversible reduction in food consumption at the dose level of 1000 bw/day. This reduction was considered not to be adverse. Food consumption was not affected by the treatment at the dose levels of 100 and 300 mg/kg bw/day. Treatment with the Luperox F caused a statistically significant reduction in body weights, body weight gain and corrected body weight gain (body weight gain corrected for the gravid uterus weight) at the dose level of 1000 mg/kg bw/day. This effect was considered to be adverse. A reversible reduction in body weight gain in the absence of significant effects on absolute body weights or corrected body weight gain was recorded at the dose level of 300 mg/kg bw/day. This effect was considered not to be adverse. No effects on body weight gain or body weights were observed at the dose level of 100 mg/kg bw/day.The relevant reproduction data (post implantation loss and number of fetuses per dam) were not affected by treatment with the Luperox F. No Luperox F-related findings were observed during the macroscopical examination at any dose level.

No effects on placenta weights were observed at any dose level. No findings were observed during the external examination of the fetuses. No Luperox F-related effects on the sex ratio of the fetuses were noted in any group. No Luperox F-related effects on fetal body weights were noted.

Type and distribution of findings recorded during visceral examination of fetuses gave no indication of a test item-related effect.

A slight increase in the incidence of fused zygomatic arch and malpositioned pelvic girdle was observed at the dose level of 1000 mg/kg bw/day. This finding was considered not to be adverse. No effects on ossification stage or incidence of supernumerary ribs were observed at any dose level. No Luperox F-related additional cartilage variations were observed at any dose level.

Based on these results, the NOAEL (No Observed Adverse Effect Level) for maternal toxicity was considered to be 300 mg/kg bw/day whereas NOEL (No Observed Effect Level) was 100 mg/kg bw/day. For prenatal developmental toxicity, the NOEL was considered to be 300 mg/kg bw/day and the NOAEL was considered to be 1000 mg/kg bw/day.