Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
toxicity to reproduction
Data waiving:
other justification
Justification for data waiving:
other:
Reproductive effects observed:
not specified
Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information
Short description of key information:
In accordance with REACH Annex IX column 1, section 8.7 experimental testing for reproductive toxicity was waived. The available data on sub-acute, sub-chronic and chronic administration (see section on repeated dose toxicity) indicated no adverse effects on reproductive organs or tissues.

Effects on developmental toxicity

Description of key information
A key study using Nicotinamide was carried out according to EU Method B.31, OECD Guideline 414 (Prenatal Developmental Toxicity Study) and Guideline on Detection of Toxicity to Reproduction for Medicinal Products (EC-Doc. 1111 3387/93), prepared within the International Conference on Harmonisation (lCH). Groups of 20 rabbits were administered test item per oral gavage in doses of 50, 150 or 450 mg/kg bw/day. A vehicle control group was run concurrently. Administered volumes were 10 mL/kg bw/day, with doses adjusted daily. 
Under the conditions of this study the systemic no-observed effect level (NOEL) for the dams was 50 mg/kg bw/day, treatment by gavage from the 6th to 20th day of pregnancy. Starting from 150 mg/kg bw/day onwards, reduced food intake was noted. In addition, reduced body weight gain was noted at 450 mg/kg bw/day. The no-observed-effect level (NOEL) for the fetuses was also 50 mg mg/kg bw/day.
In addition a second key study was carried out using Nicotinic acid in a read-across approach according to EU Method B.31, OECD Guideline 414 (Prenatal Developmental Toxicity Study) and U.S. Food and Drug Administration (1964) Redbook. The read-across from Nicotinic acid to Nicotinamide is justified. The study was carried out on rat as a second species.
The NOAEL for maternal toxicity derived from this study was 200 mg/kg bw/d based on effects on body weight (equivalent to 198 mg/kg bw/day for Nicotinamide). The NOAEL on reproduction toxicity and developmental toxicity is 200 mg/kg bw/day (equivalent to 198 mg/kg bw/day Nicotinamide) based on the significantly decreased placental and pup body weight (males only). No teratogenic effects were observed.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
other: Detection of Toxicity to Reproduction for Medicinal Products (EC-Doc. 1111 3387/93). Guideline prepared within the International Conference on Harmonisation (lCH).
GLP compliance:
yes (incl. certificate)
Remarks:
LPT Laboratory of Pharmacology and Toxicology KG
Limit test:
no
Species:
rabbit
Strain:
Himalayan
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 4 to 8 months
- Weight at study initiation: 2.15 - 2.90 kg
- Housing: The dams were kept separately in breeding cages with wire floors (with an area of approx. 0.2 m2)
- Diet: Ad libitum
- Water: Ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 +/- 3
- Humidity (%): 55 +/- 15
- Photoperiod (hrs dark / hrs light): 150 lux at approximately 1.5 m room height and darkened in a 12 hours dark/12 hours light cycle

IN-LIFE DATES:
- From: 15 April 2002
- To: 17 Sept. 2002
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5 % gel (METHOCEL E4M)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- The test substance was suspended in 0.5 % aqueous hydroxypropyl methylcellulose gel, the test substance-vehicle mixtures were freshly prepared daily.
- The administered volume was 10 mL/kg bw/day. The daily dose of the test substance was adjusted to the animal's actual body weight daily.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of test item in 0.5 % aqueous Methocel preparations obtained during an embryotoxicity study following oral administration to rabbits was determined based on the spectrophotometric method (HPLC/UV). The concentrations of test item were analysed directly after dilution of the substance-carrier mixtures with aqua. Samples from the aqueous test substance-carrier mixtures were withdrawn at study initiation at each dose level group for the determination of the test substance concentration and stability as well as for the determination of the homogeneity of the preparations. Further, samples were taken from the aqueous test substance-carrier mixtures on gestation day 20 at the end of the application phase before administration to the last animal of the dose level groups for the determination of the test substance concentration.
The actual concentrations of the samples taken from the aqueous test substance-carrier mixtures in group 2 were within the range of 101.3 - 104.1 % of the nominal test item concentrations indicating correctly prepared application mixtures and a sufficient stability.
The actual concentrations of the samples taken from the aqueous test substance-carrier mixtures in group 3 were within the range of 103.1 - 106.3 % of the nominal test item concentrations indicating correctly prepared application mixtures and a sufficient stability.
The actual concentrations of the samples taken from the aqueous test substance-carrier mixtures in group 4 were within the range of 103.3 - 106.3 % of the nominal test item concentrations indicating correctly prepared application mixtures and a sufficient stability.
Details on mating procedure:
- Impregnation procedure: Cohoused
- M/F ratio per cage: 1 male and 1 female animal
- Length of cohabitation: Forenoon
- After 1 day of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after unsuccessful attempt: No. Rabbits lacking signs of copulation were excluded from the analysis and replaced by additional spare animals.
- Verification of same strain and source of both sexes: Yes. Sexually mature ('proved') male rabbits of the same breed served as partners. Mating was monogamous.
- Proof of pregnancy: Observation referred to as day 0 of pregnancy. A post-mortem negative staining according to SALEWSKI was carried out in the replaced animals in order to confirm the nonpregnancy status.
- Any other deviations from standard protocol: None
Duration of treatment / exposure:
From day 6 to 20 of pregnancy
Frequency of treatment:
Once daily
Duration of test:
Start of study
Date of protocol: 2 April 2002
Start of the experimental phase: 15 April 2002
Start of the in-life phase (1 st mating day): 16 April 2002
1st administration: 22 April 2002
Termination of study
Termination of in-life phase: 17 September 2002
Termination of skeletal evaluation: 2 October 2002
Last date of the raw data: 15 October 2002
Date of final report: 20 March 2003
Remarks:
Doses / Concentrations:
50, 150 and 450 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
Four (4) groups of pregnant rabbits were established, each obtained from matings which were carried out on a daily basis. The rabbits were randomly assigned to their respective groups according to a number scheme considering their mating day i.e. in a cyclic way following the listing of successful copulation.

1 control: 20 females
2 low dose: 20 females
3 intermediate dose: 20 females
4 high dose: 20 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected in agreement with the sponsor, based on data obtained from the dose-range-finding study. In this dose-range-finding study dose levels of 10, 100, 300, 600 and 1000 mg/kg bw/day as well as an additional group at 600 mg/kg bw/day (with test material batch used in the main study) were administered from the 6th to 20th day of pregnancy at a constant administration volume of 10 mg/kg bw/day. Each dose level was tested in 2 or 3 pregnant rabbits. From 600 mg/kg bw/day onwards, a reduction was noted for the food intake. In addition, a reduction of body weight was noted at 1000 mg/kg bw/day and also at 600 mg/kg bw/day.
One of three dams in each group treated with 600 mg/kg bw/day and all three dams at 1000 mg/kg bw/day died prematurely. In the high-dosed dams prelethal symptoms were noted in form of dyspnoea, abdominal position and/or reduced motility one to a few days before exitus. Necropsy revealed pulmonal and gastric lesions in all prematurely deceased dams, an anaemic spleen was noted in three of five prematurely deceased dams. No effects were seen at 300 mg/kg bw/day or less. At the materno-toxic dose of 600 mg/kg bw/day, the postimplantation loss was increased to 42.9 % (control: 0.0 %), the incidence of skeletal retardations appeared to be slightly increased. A complete post-implantation loss of 100 % was noted in the dams treated with 1000 mg/kg bw/day and 600 mg/kg bw/day.
From 300 mg/kg bw/day onwards the incidence of accessory 13th rib(s) appeared to be slightly increased. However, only the fetuses of 2 dams were analysed.
There was no increase in the incidence of malformations at any of the tested doses, not even at materno-toxic doses.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily
- Viability: Checks were made early each working day and again in the afternoon to look for dead or moribund animals. This would have allowed post mortem examinations to be carried out during the working period of that day. On Saturdays and Sundays, a similar procedure was followed except that the final check was carried out at approximately midday.
Animals showing signs of abortion or premature delivery were sacrificed on the same day. Fetuses obtained this way were examined for abormal development whenever possible.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily
- Clinical observations: Individual animals were observed for any signs of behavioural changes, reaction to treatment or illness. Immediately after administration any signs of illness or reaction to treatment were recorded. In case of changes the animals were observed until the symptoms disappeared. In addition, animals were checked regularly throughout the working day from 7.00 a.m. to 3.45 p.m. On Saturdays and Sundays, animals were checked regularly from 7.00 a.m. to 11.00 a.m. with a final check performed at approximately 3.30 p.m. Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each rabbit was recorded on day 0 of gestation (the day of detection of a positive mating sign), followed by daily weighings - always at the same time of the day. The body weight gain was also calculated in intervals (i.e. 0-3, 3-6, 6-9, 9- 12, 12-15, 15-18, 18-21, 21-24, 24-27 and 27-29). Furthermore the net weight change from day 6 is given. These values are stated in the report. These measurements were also used for calculating the daily amount of test substance to be administered.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- The quantity of food consumed by each rabbit was recorded. Food intake per rabbit (g/rabbit/day) was calculated using the total amount of food given to and left by each rabbit in each group on completion of a treatment day.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Daily monitoring by visual appraisal of the drinking water consumption was maintained throughout the study.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day no. 29
- Dissection technique and evaluation of the animals: The ovaries and uteri were removed; the uteri (in toto) were weighed. In order to check for possible drug effects, a dissection with macroscopic examination of the internal organs of the dams was carried out on the day of scheduled laparotomy or on the day on which the animals were found dead. In case of macroscopic findings, the affected maternal tissues were preserved in 10 % buffered formalin for possible future histopathological examination.
- Organs examined: Macroscopic inspection (gross evaluation) of the placentae for example for focal indurations. The number of fetuses (alive and dead) and placentae was determined. Sex and viability of fetuses were determined. The number of fetuses alive was determined after a 6-hour and a 24-hour stay in an incubator at a temperature of + 34 °C. Number and size of resorptions were determined. Corpora lutea in the ovaries, implantations and location of fetuses in the uterus were determined. The gravid uterus weight was determined. Weights of fetuses and weight of the placentae were determined (fetuses were considered as runts if their weight was less than 70 % of the mean litter weight). Fetuses were inspected externally for damages, especially for malformations.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Corpora lutea
number per dam
absolute number per group
mean per group
- Implantations
number per dam
distribution in the uterine horns
absolute number per group
mean per group
- Resorptions
number per dam
distribution in the uterine horns
absolute number per group
mean per group
mean % per group
early resorptions < 2 g
late resorptions > 2 g
- Weight of placentae
individual data per fetus
mean per litter
mean per group
litter mean per group
litter mean per sex and group
Fetal examinations:
- Weight of fetuses
individual data per fetus
mean per litter
mean per sex and litter
litter mean per group
litter mean per sex and group
- Fetuses
number per dam (alive and dead)
number of fetuses per sex and dam
distribution in the uterine horns
absolute number of fetuses alive per group
mean number of fetuses alive per group
mean % of fetuses alive per group
- Runts
mean % per sex and group
number per dam
mean per group
- Malformed fetuses
individual data per fetus
mean per group and type of malformation
Statistics:
The following statistical methods were used:
For all numerical values, homogeneity of variances was tested using the Bartlett chisquare test. When the variances were homogeneous, the Dunnett test was used to compare the experimental groups with the control group. In case of heterogeneity of variances, the Student's t-test was carried out. For the assessment of a toxicological relevance limit of significance was p =< 0.01 in all cases. In addition, the limit of significance of p =< 0.05 is stated in the tables.
Multiple t-test based on DUNNETT, C. W. J. Amer. Statist. Assoc. 501, 1096 - 1121 (1955)
p = 0.01 delta t = 3.04 for 76 degrees of freedom
Student's unpaired t-test
D. COLQUHOUN (1971)
Lectures on Biostatistics
Clarendon Press, Oxford
p = 0.01 delta t = 2.712 for 38 degrees of freedom
For the comparison of classification measurements (for example malformation-, resorption-, retardation- and variation rate) the was employed.
FISHER's exact test (n < 100) or chisquare-test with Yates' correction for continuity (n < 100) (p =< 0.05 and p =< 0.01). All data were evaluated statistically in this manner. In tables in which individual results differ significantly from those of the control group, these data are indicated. The mean values and standard deviations were calculated to the highest possible degree of accuracy and then rounded to the reported number of decimal places. Hence, deviations to the last decimal place of up to +/- 1 may occur caused by rounding.
Indices:
- Malformation rate
per group in % = malformed fetuses/ fetuses x 100
- Fetuses with variations
individual data per fetus
mean per group and type of variation
- Variation rate
mean % = fetuses with variations / fetuses x 100
- Fetuses with retardations
individual data per fetus
mean per group and type of retardation
- Retardation rate
mean % = fetuses with retardations / fetuses x 100
- Pre-implantation loss
mean % = corpora lutea minus implants / corpora lutea x 100
- Post-implantation loss
mean % = implants minus living fetuses / implants x 100
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
BEHAVIOUR, EXTERNAL APPEARANCE, FAECES, MORTALITY
Neither 50 nor 1 50 mg/kg bw/day caused any substance-related influence on behaviour and external appearance. None of the dams died prematurely. At 450 mg/kg bw/day, one of 21 pregnant dams was sacrificed prematurely after abortion on gestation day 26 and excluded from statistics. The 20 high-dosed dams employed revealed no changes in behaviour and external appearance. The faeces of all dams were of normal consistency during the whole experiment.

BODY WEIGHT AND BODY WEIGHT CHANGE
At 50 and 150 mg/kg bw/day, body weight was within the normal range of the control. The increase in the mean body weight from the start value (day 0 of pregnancy) was plus 16 % and 13 %, respectively, at the time point of laparotomy (control: plus 14 %). At 450 mg/kg bw/day, the mean maternal body weight was slightly reduced during the last days of treatment, statistically significant reductions (at p =< 0.05) were noted on gestation days 19 to 21 compared to the control (by up to 6 %). The mean maternal body weight change was significantly decreased (at p =< 0.01) on gestation days 6 to 9. The increase in the mean body weight from the start value (day 0 of pregnancy) was plus 11 % at the time point of laparotomy (control: plus 14 %).

FOOD AND DRINKING WATER CONSUMPTION
No substance-related influence was noted on absolute and relative food consumption compared to the control at 50 mg/kg bw/day. At 150 mg/kg bw/day, absolute and relative food intake were reduced, predominantly during the first days of treatment period (up to 32 % below the control value). Food intake increased after the end of treatment. At 450 mg/kg bw/day, absolute and relative food intake were - dose-related more distinctly - reduced during the treatment period (up to 52 % below the control value). A distinctly reduced food consumption or refusal of food intake was noted in individual dams on a few administration days. Statistically significant reductions (at
p =< 0.05 and p =< 0.01) were noted on gestation days 7 to 9, 11 - 13, 18 and 19. Food intake increased after the end of treatment and was even above that of the control animals, statistically significant increases (at p =< 0.05 and p =< 0.01) were noted on gestation days 26 to 29.
Drinking water consumption showed no substance-related changes in any treated group during daily visual appraisal.

EXAMINATIONS OF THE DAMS AT TERMINATION
- Necropsy findings: No substance-related pathological findings were noted at macroscopic inspection of the internal organs and the placentae in the dams treated with either 50, 150 or 450 mg/kg bw/day. Necropsy revealed pale liver and kidneys in the high-dosed dam no. 74 (450 mg/kg bw/day) which aborted prematurely. The following macroscopic changes were noted in the control group: The uterus of control dam no. 22 was filled with light-brown liquid, all fetuses of this dam were runts while the placentae were normally developed. Furthermore, a beige focus (approx. 8 x 4 mm) was noted at one placenta (fetus no. 4) of control dam no. 24. The afore-mentioned findings were considered as spontaneous.
- Uterus weight and net body weight change: The gravid uterus weight, the carcass weight and the net weight change from day 6 onwards (carcass weight minus day 6 body weight) of the animals treated with 50 or 150 mg/kg bw/day were not influenced by the exposure to the test compound. At 450 mg/kg bw/day, the gravid uterus weight was decreased (minus 31 % below the control, statistically significant at p s 0.01). This finding was caused by the increased post-implantation loss in this test groups.
- Reproduction data of dams: There were no substance-related differences between the control group and the animals treated either with 50 or 150 mg/kg bw/day in the number of corpora lutea, implantation sites, resorptions and live fetuses or the values calculated for the pre- and post-implantation losses. In the intermediate-dosed dams (150 mg/kg bw/day) the number of total resorptions was slightly but statistically significantly increased (at p s 0.05) compared to the control, the number of fetuses was accordingly decreased. These findings were considered as incidental as they were still within the range of background data. At 450 mg/kg bw/day, one of 21 pregnant dams suffered an abortion on gestation day 26 and was excluded from statistics. A complete post-implantation loss at an early state of pregnancy was noted in 2 of 20 dams. A statistically significant increase (at p =< 0.01) was noted for the number of resorptions (early and total resorptions) and an according decrease for the number of fetuses.
Dose descriptor:
NOEL
Effect level:
50 mg/kg bw (total dose)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
EXAMINATION OF THE FETUSES
- Sex distribution of fetuses: The sex distribution of the fetuses in test groups 2, 3 and 4 (50, 150 or 450 mg/kg bw/day) was comparable to the control fetuses.
- Weight of placentae: The mean placental weights in test groups 2, 3 and 4 (50, 150 or 450 mg/kg bw/day) as compared to the control group were not influenced by the administration
of the test compound to the dams.
- Weight of fetuses: The mean fetal weights were not influenced by the administration of 50 or 1 50 mg/kg bw/day as compared to the control group. The litter of control dam no. 22 consisted of 7 runts - an incidence which may occur spontaneously. The mean fetal weight was 35.4 g in the control group and 37.0 g if the litter was excluded (control background range: 36.7 - 42.0 g). At 450 mg/kg bw/day, the mean fetal weight was statistically significantly reduced at p =< 0.05 in male fetuses (33.1 g) and total fetuses (33.9 g) compared to the control group when the litter with runts of the control group was excluded from the mean value determination (37.1 g / 37.0 g, mean values of the control group excluding runts). Mean values of the control group of 35.5 g and 35.4 g were calculated respectively including runts.
- External examination of the fetuses: External examination of the fetuses revealed no malformed fetuses at 50, 150 or 450 mg/kg bw/day. Two malformed fetuses were noted in the control group: malrotated limbs and/or malrotated fore paws were noted in one fetus each of dam no. 2 and 24. The incidences of mal rotated fore paws and mal rotated limbs are within the range of background incidences (see Appendix 3) for this rabbit strain according to number and type and, hence, they are considered as spontaneous. No substance-related macroscopically visible variations were noted at 50, 150 or 450 mg/kg bw/day. An extended stomach was noted in two fetuses of the intermediate-dosed dam no. 62 (150 mg/kg bw/day), these fetuses were found dead 6 hours after laparotomy. In the control group, all 7 fetuses of dam no. 22 were classified as runts (unclassified external observation). All fetuses died within 2 minutes after laparotomy. No runts were noted at any tested dose level. One dead fetus (dam no. 87 at 450 mg/kg bw/day) was noted at laparotomy. Mortality in both control group and high dose group was considered to be high and at the upper limit of the background range. In the low and intermediate groups the mortality was well within the expected values.
- Skeletal examination of the fetuses: No skeletal malformation was noted at any of the tested dose levels (50, 150 or 450 mg/kg bw/day). The skeletal variations observed in this study (control included) were related to the sternum (sternebra(e) bipartite, fused or misaligned to a slight degree); the ribs (accessory 13th rib(s), rib(s) fused to a slight degree, rib(s) shortened)' caudal vertebral bodies (fused) and thoracic vertebral bodies (misaligned) or thoracic vertebral arches (fused). At 50 mg/kg bw/day, the fetal incidences of accessory 13th rib(s) was marginally increased (statistically significant at p =< 0.05). However, this finding is still within the LPT background data. A dose-related significant increase (at p =< 0.05 or p =< 0.01) in the number of variations was noted from 150 mg/kg bw/day onwards: accessory 13th rib(s), rib(s) shortened and total skeletal variations. In addition, an increased fetal incidence was noted for fused sternebra(e) at 450 mg/kg bw/day only (statistically significant at p =< 0.05). However, this finding is still within the range of background data. Skeletal retardations in all groups were related to the skull (incomplete ossification of frontal, parietal, interparietal, supraoccipital region); hyoid (missing ossification); sternebra(e) (incomplete and/or missing ossification) and the caudal, cervical, thoracic, lumbar or pelvic vertebral bodies (incomplete or missing ossification, bipartite) and metacarpalia/ metatarsalia (missing ossification). At 50 mg/kg bw/day, the incidence of retardations was within the range of the control group and, hence, within the range of background data according to number and type. Correlating with the increased incidence in accessory 13th rib(s) a dose-related statistically significant increase (at p =< 0.01) was noted in the number of retardations for the incidence of missing ossification of vertebral bodies (less than 7 lumbar vertebral bodies ossified) from 150 mg/kg bw/day onwards. Hence, the fetal incidence for total retardations was increased at 450 mg/kg bw/day. Correlating with the increase in the number of fetuses with a 13th rib a significant decrease (at p =< 0.05 or p =< 0.01) was noted for missing ossification of sternebra(e) at 150 and 450 mg/kg bw/day. The following spontaneous skeletal retardations were noted for the seven runts of control dam no. 22: missing or incomplete ossification of skull, hyoid, sternum, caudal, pelvic vertebral bodies (less than 5 pelvic vertebral bodies ossified), metacarpalia and metatarsalia. Hence, the statistically significant deviations from control - noted for these parameters in all test groups - were judged as incidental.

SYNOPSIS AND ASSESSMENT OF FETAL EXTERNAL AND SKELETAL OBSERVATIONS
There are no substance-related differences between the test groups 1 (control), 2 (50 mg/kg bw/day), 3 (150 mg/kg bw/day) and 4 (450 mg/kg bw/day) concerning external variations and malformations and skeletal malformations. One dead fetus was noted at laparotomy. At all tested dose levels the viability of fetuses at 6 or 24 hours was within the normal range.
- External examination: The external examination of the fetuses revealed two malformed control fetuses: malrotated limbs and/or malrotated fore paws were noted in one fetus each of dam no. 2 and 24. The incidences of malrotated fore paws and malrotated limbs are within the range of background incidences (see Appendix 3) for this rabbit strain according to number and type and, hence, they are considered as spontaneous. No substance-related macroscopically visible variations were noted at 50, 150 or 450 mg/kg bw/day. An extended stomach was noted in two fetuses of the intermediate-dosed dam no. 62 (150 mg/kg bw/day), these fetuses were found dead 6 hours after laparotomy. In the control group, all 7 fetuses of dam no. 22 were classified as runts (unclassified external observation). All fetuses died within 2 minutes after laparotomy. No runts were noted at any tested dose level.
- Skeletal examination: The skeletal examination (according to DAWSON) revealed no malformed fetuses. A significant (p =< 0.05 or p =< 0.01) increase for the following variations was found: From 150 mg/kg bw/day onwards a dose-related increase was noted for accessory 13th rib(s), rib(s) shortened and total skeletal variations. Correlating with the increased incidence in accessory 13th rib(s) a dose-related statistically significant increase (at p =< 0.01) was noted in the number of skeletal retardations for the incidence of missing ossification of vertebral bodies (less than 7 lumbar vertebral bodies ossified) from 1 50 mg/kg bw/day onwards. Hence, the fetal incidence for total retardations was increased at 450 mg/kg bw/day. Correlating with the increase in the number of fetuses with a 13th rib a significant decrease (at p =< 0.05 or p =< 0.01) was noted for missing ossification of sternebra(e) at 150 and 450 mg/kg bw/day.
- All further prenatal changes noted at 50, 150 or 450 mg/kg bw/day are without any biological relevance.
Dose descriptor:
NOEL
Effect level:
50 mg/kg bw (total dose)
Based on:
test mat.
Basis for effect level:
other: embryotoxicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
Under the conditions of this rabbit embryotoxicity study the systemic no-observed effect level (NOEL) for the dams was 50 mg/kg bw/day, treatment by gavage from the 6th to 20th day of pregnancy. Starting from 150 mg/kg bw/day onwards, reduced food intake was noted. In addition, reduced body weight gain was noted at 450 mg/kg bw/day.
The no-observed-effect level (NOEL) for the fetuses was also 50 mg mg/kg bw/day. From the materno-toxic dose of 150 mg/kg bw/day onwards, an increased incidence was noted for skeletal variations in form of accessory 13th rib(s), rib(s) shortened and total skeletal variations) and hence for the skeletal retardations (less than 7 lumbar vertebral bodies and total retardations - only at 450 mg/kg bw/day). Correlating with the increase in the number of fetuses with a 13th rib a decreased incidence was noted for missing ossification of sternebra(e) at 150 and 450 mg/kg bw/day.
No substance-related increase was noted in the incidence of malformations at any of the tested doses, not even at materno-toxic doses.
In conclusion, the test compound possessed no teratogenic properties, not even at materno-toxic doses. Slight embryotoxicity was noted at materno-toxic doses in form of increased incidences of skeletal variations and retardations.
Executive summary:

A study was carried out according to EU Method B.31, OECD Guideline 414 (Prenatal Developmental Toxicity Study) and Guideline on Detection of Toxicity to Reproduction for Medicinal Products (EC-Doc. 1111 3387/93), prepared within the International Conference on Harmonisation (lCH). Groups of 20 rabbits were administered test item per oral gavage in doses of 50, 150 or 450 mg/kg bw/day. A vehicle control group was run concurrently. Administered volumes were 10 mL/kg bw/day, with doses adjusted daily.

INFLUENCE ON THE DAM

None of the dams treated with either 50 or 150 mg/kg bw/day died prematurely. At 450 mg/kg bw/day, one of 21 pregnant dams was sacrificed prematurely after abortion on gestation day 26. - - Clinical signs: No substance-related behavioural changes were noted at 50, 150 or 450 mg/kg bw/day.

- Body weight: No substance-related influence on body weight and net weight change (carcass weight minus day 6 body weight) was noted at 50 or 150 mg/kg bw/day. At 450 mg/kg bw/day, the mean maternal body weight was slightly reduced during the last days of treatment, statistically significant reductions (at p =< 0.05) were noted on gestation days 19 to 21 compared to the control (by up to 6 %). The mean maternal body weight change was significantly decreased (at p =< 0.01) on gestation days 6 to 9. The increase in the mean body weight from the start value (day 0 of pregnancy) was plus 11 % at the time point of laparotomy (control: plus 14 %).

Food consumption, drinking water consumption: The food consumption was not influenced at 50 mg/kg bw/day. At 150 mg/kg bw/day, absolute and relative food intake were marginally reduced, predominantly during the first days of treatment (up to 32 % below the control). A statistically significant reduction (at p =< 0.05) was noted on gestation day 7. Food intake increased after the end of treatment. At 450 mg/kg b.w./day, absolute and relative food intake were - dose-related more distinctly - reduced during the treatment period (up to 52 % below the control value). A distinctly reduced food consumption or even refusal of food intake was noted in individual dams on a few administration days. Statistically significant reductions (at p =< 0.05 and p =< 0.01) were noted on gestation days 7 to 9, 11, 13, 18 and 19. Food intake increased after the end of treatment and was even above that of the control animals, statistically significant increases (at p =< 0.05 and p =< 0.01) were noted on gestation days 26 to 29. Treatment did not influence drinking water consumption.

- Autopsy findings: No substance-related pathological findings were observed at 50, 150 or 450 mg/kg bw/day.

- Uterus and carcass weights: The gravid uterus weight and the carcass weight of the animals treated with either 50 or 150 mg/kg bw/day were not influenced by exposure to the test compound. At 450 mg/kg bw/day, the gravid uterus weight was decreased (minus 31 % below the control, statistically significant at p =< 0.01) caused by the increased post-implantation loss in this test group.

INFLUENCE ON THE FETUS

Corpora lutea/implantation sites/resorptions/ weight and number of fetuses alive/placental weights: No substance-related influence was detected on the prenatal fetal development at 50 or 150 mg/kg bw/day with respect to the number of corpora lutea, implantation sites, resorptions, fetal and placental weights, viability, sex distribution and number of live fetuses at birth as well as for the pre- and post-implantation loss. At 450 mg/kg bw/day, one of 21 pregnant dams suffered an abortion on gestation day 26 and was excluded from statistics. A complete post-implantation loss at an early state of pregnancy was noted in 2 of 20 dams. A statistically significant increase (at p =< 0.01) was noted for

the number of resorptions (early and total resorptions) and an according decrease for the number of fetuses. The mean fetal weight was reduced in male and total fetuses. One dead high-dosed fetus (450 mg/kg bw/day) was noted at laparotomy. The viability of fetuses during the incubator stay at 6 or 24 hours was within the normal range at all tested doses. No runts were noted at laparotomy.

- Malformations: No substance-related increase was noted in the incidence of external or skeletal malformations at mg/kg bw/day.

- Variations: At 50, 150 or 450 mg mg/kg bw/day, no substance- related variations were found in the

fetuses at external examination.

- Skeletal variations (examination according to DAWSON) were of a similar incidence in the

control and at 50 mg/kg bw/day. A dose-related significant increase (at p =< 0.05 or p =< 0.01) in the number of skeletal variations was noted from 150 mg/kg bw/day onwards: accessory 13th rib(s), rib(s) shortened and total skeletal variations.

- Retardations: Skeletal retardations were of a similar incidence in the control and at 50 mg/kg bw/day. Correlating with the increased incidence in accessory 13th rib(s) a dose-related statistically significant increase (at p =< 0.01) was noted in the number of retardations for the incidence of missing ossification of vertebral bodies (less than 7 lumbar vertebral bodies ossified) from 150 mg/kg bw/day onwards. Hence, the fetal incidence for total retardations was increased at 450 mg/kg bw/day. Correlating with the increase in the number of fetuses with a 13th rib a significant decrease (at p =< 0.05 or p =< 0.01) was noted for missing ossification of sternebra(e) at 150 and 450 mg/kg bw/day.

CONCLUSION

Under the conditions of this rabbit embryotoxicity study the systemic no-observed effect level (NOEL) for the dams was 50 mg/kg bw/day, treatment by gavage from the 6th to 20th day of pregnancy. Starting from 150 mg/kg bw/day onwards, reduced food intake was noted. In addition, reduced body weight gain was noted at 450 mg/kg bw/day.

The no-observed-effect level (NOEL) for the fetuses was also 50 mg mg/kg bw/day. From the materno-toxic dose of 150 mg/kg bw/day onwards, an increased incidence was noted for skeletal variations in form of accessory 13th rib(s), rib(s) shortened and total skeletal variations) and hence for the skeletal retardations (less than 7 lumbar vertebral bodies and total retardations - only at 450 mg/kg bw/day). Correlating with the increase in the number of fetuses with a 13th rib a decreased incidence was noted for missing ossification of sternebra(e) at 150 and 450 mg/kg bw/day.

No substance-related increase was noted in the incidence of malformations at any of the tested doses, not even at materno-toxic doses.

In conclusion, the test compound possessed no teratogenic properties, not even at materno-toxic doses. Slight embryotoxicity was noted at materno-toxic doses in form of increased incidences of skeletal variations and retardations.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on study results obtained the substance is not classified and labeled according to Regulation 1272/2008/EEC (CLP) and Directive 67/548/EEC (DSD).