Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May-June 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, no restrictions, fully adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Principles of method if other than guideline:
The study was conducted to provide data on the possible effects of PERKALINK 900 on pregnant female rats and the development of the embryo and fetus consequent to continuous oral administration in the diet given from gestation day (GD) 0 until GD 21. The test substance was given in constant concentrations of 0 (control), 800 mg/kg diet (low-dose), 1600 mg/kg diet (mid-dose), and 3200 mg/kg diet (high-dose). During the in-life phase clinical signs, maternal body weight and food consumption were recorded. At Caesarean section females and fetuses of all groups were macroscopically examined. Fetuses, placentas and reproductive organs were weighed and fetuses were further processed for fetopathological examination.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene
EC Number:
412-570-1
EC Name:
1,3-bis(3-methyl-2,5-dioxo-1H-pyrrolinylmethyl)benzene
Cas Number:
119462-56-5
Molecular formula:
Hill formula: C18 H16 N2 O4
IUPAC Name:
3-methyl-1-({3-[(3-methyl-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl]phenyl}methyl)-2,5-dihydro-1H-pyrrole-2,5-dione
Details on test material:
Test substance name : Perkalink 900
Chemical name : 1,3-bis(citraconimidomethyl) benzene
CAS-Reg. no. : 119462-56-5
Batch number : 812191/01
Purity : 92.3% (see CoA attached)
TNO dispense reference no. : 09002F
Appearance : off-white pastilles

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 12 weeks old
- Weight at study initiation: ± 205 g
- Housing: under conventional conditions in macrolon cages (Male: type 3, individually and Female: type 4, 4 per cage) with wood shavings (Lignocel Type 3/4) as bedding material and strips of paper (Enviro-dri) as environmental enrichment. No other test system was housed in the same room during the study.
- Diet: ad libitum; The rats were fed a cereal-based (closed formula) rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3) from a commercial supplier (SDS Special Diets Services, Witham, England). The feed was provided as a powder, in stainless cans, covered by a perforated steel plate that serves to prevent spillage. The feed in the feeders was refreshed about once per week.
- Water: ad libitum; Each cage was supplied with domestic mains tap-water suitable for human consumption. The water was given in polypropylene bottles, which were cleaned weekly and filled as needed.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Details on exposure:
DIET PREPARATION
Experimental diets were prepared by mixing powdered Rat & Mouse No. 3 breeding diet, RM3 with the appropriate amounts of test substance. The diets were mixed in a mechanical blender (Lödige, Paderborn, Germany). The experimental diets were stored in a freezer (<-18°C). The feed in the feeders was replaced with fresh portions once a week, and filled up when necessary.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses to determine the stability, homogeneity and content of the test substance in the diet were conducted using High Performance Liquid Chromatography (HPLC) with UV detection, after extraction of the diet with a suitable solvent.
Details on mating procedure:
At the start of mating, 2 nulliparous females were caged with one male for mating until a sperm positive vaginal smear was detected. Every consecutive morning vaginal examinations were made to ascertain copulation by detection of sperm cells in the vaginal smear.
The mated females were distributed over the four experimental groups in such a way that the animals from the same day of pregnancy were, as far as possible, equally distributed over all groups. Females mated by the same male were placed in different groups. Upon evidence of copulation, positive females were housed individually. The day a sperm-positive smear was detected was considered as GD 0.
Duration of treatment / exposure:
21 days (starting on GD 0)
Frequency of treatment:
7 days/week
Duration of test:
23 days
Doses / concentrationsopen allclose all
Dose / conc.:
800 ppm (nominal)
Remarks:
corresponds to 53 mg/kg bw/day
Dose / conc.:
1 600 ppm (nominal)
Remarks:
corresponds to 105 mg/kg bw/day
Dose / conc.:
3 200 ppm (nominal)
Remarks:
corresponds to 196 mg/kg bw/day
No. of animals per sex per dose:
24
Control animals:
yes, plain diet
Details on study design:
The study comprised 4 groups of 24 mated female rats each, viz. one control group kept on diet without test substance, and three test groups receiving different levels of the test substance in the diet. These groups were intended to provide information on the prenatal developmental toxicity of the substance and to establish a no-observed-adverse-effect level (NOAEL).

Examinations

Maternal examinations:
General clinical observations
Each animal was observed daily in the morning hours by cage-side observations. On working days, all cages were checked again in the afternoon for dead or moribund animals to minimize loss of animals from the study. On Saturdays, Sundays and public holidays only one check per day was carried out. All abnormalities, signs of ill health or reactions to treatment were recorded.

Body weight
Body weights were recorded on GD 0, 3, 7, 10, 14, 17 and 21.

Food consumption
The food consumed for each mated female was measured over the periods: GD 0-3, 3-7, 7-10, 10-14, 14-17, and 17-21 by weighing the feeders. The results were expressed in g per animal per day and g per kg body weight per day.

Intake of the test substance
The intake of the test substance per kg body weight per day was calculated from the nominal dietary concentration of the test substance, the food consumption and the mean body weight measured at the beginning and the end of the pertaining period.
Ovaries and uterine content:
The females were killed by decapitation under CO2/O2 anaesthesia on GD 21 and examined for gross abnormalities.
The uteri (including the fetuses), ovaries and placentas of all females killed on GD 21 were examined for the following parameters:
- number of corpora lutea
- number of implantation sites
- number of early and late resorptions
- number of live and dead fetuses
- sex of the fetuses
- number of grossly visible malformed fetuses and fetuses with external abnormalities
- weight of ovaries (left and right ovary weighed together)
- weight of uterus, containing placentas and fetuses
- weight of uterus, empty
- weight of live fetuses (individually)
- weight of the placentas of live fetuses
- gross evaluation of placentas
Fetal examinations:
Fetuses were sacrificed by hypothermia. Subsequently, half of the fetuses of each litter of the prenatal developmental toxicity study were fixed in Bouin's fixative, examined for soft tissue anomalies according to a method modified after Barrow and Taylor and then discarded.
The other half of the fetuses were fixed in 70% alcohol, subsequently partly eviscerated, and then cleared in potassium hydroxide and stained with Alizarin Red S modified after Dawson. They were examined for skeletal abnormalities and then retained.
During the fetopathological examination of the fetuses, the observer was unaware of the dose group of the fetuses.
Statistics:
The results were analyzed using the methods mentioned below. As a level of significance was considered: p< 0.05.
- Clinical findings were evaluated by Fisher's exact probability test.
- Body weight, body weight gain, organ weights and food consumption data were subjected to one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests.
- Fisher's exact probability test was used to evaluate the number of mated and pregnant females and females with live fetuses.
- Number of corpora lutea, implantation sites, live and dead fetuses and early and late resorptions were evaluated by Kruskal-Wallis nonparametric analysis of variance followed by the Mann Whitney U-test.
- Mortality data, data of the pathology of parent females and the fetopathological screening were evaluated by the Fisher’s exact probability test.
Indices:
For each group the following indices were recorded:
- female fecundity index = (number of pregnant females/number of females mated) x 100
- pre-implantation loss = [(number of corpora lutea - number of implantation sites)/ number of corpora lutea] x 100
- post-implantation loss = [(number of implantation sites- number of live fetuses)/number of implantation sites] x 100
- gestation index = (number of females with live fetuses/number of females pregnant) x 100
- sex ratio = (number of live male fetuses/number of live fetuses) x 100
Historical control data:
Historical control data are available.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
Daily clinical observations during the gestation period did not reveal any treatmentrelated changes in the animals’ appearance, general condition or behaviour.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A statistically significantly decreased body weight was observed in the high-dose PERKALINK 900 group. Body weight change was statistically significantly decreased in the high-dose group during the periods GD 0-7, GD 7-14, and during the total period of gestation (GD 0-21).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption of the high-dose PERKALINK 900 pregnant females was statistically significantly decreased during the periods GD 7-14 and GD 14-21.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
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:
effects observed, treatment-related
Description (incidence and severity):
Necropsy of the mated females did not reveal any differences on the gravid and empty uterus weights and ovary weights. Carcass weight of the pregnant females was statistically significantly decreased in the high-dose PERKALINK 900 group; as such a statistically significantly decreased net weight change from day 0 was observed in the high-dose PERKALINK 900 group.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic findings at necropsy did not reveal any treatment-related changes.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Details on results:
In conclusion, compared to the control group, statistically significant effects were observed on body weight, body weight change, food consumption, carcass weight and net weight change from day 0 in the animals fed with 3200 mg PERKALINK 900/kg diet. Based on these observed effects the no-observed-adverse-effect level (NOAEL) for maternal toxicity following daily oral PERKALINK 900 administration was 1600 mg/kg diet (equivalent to a mean test substance intake of 105 mg PERKALINK 900/kg body weight/day).

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY
No mortality was observed. Daily clinical observations during gestation did not reveal any treatment-related findings in the appearance, general condition or behaviour of the animals between the test groups and the control group.

BODY WEIGHT AND BODY WEIGHT CHANGE
Body weight of the females of the high-dose group was statistically significantly decreased on GD 7, 14 and 21. Body weight gain of the high-dose group females was statistically significantly decreased between GD 0-7 and 7-14. Furthermore, body weight gain during the entire gestation period (GD 0-21) was statistically significantly decreased in the high-dose group.

FOOD CONSUMPTION
Food consumption (both g/kg body weight/day and g/animal/day) was statistically significantly reduced in females of the high-dose group between GD 7-14 and GD 14-21 compared to the control group.

TEST SUBSTANCE INTAKE
The test substance intake during gestation was calculated from the nominal concentrations of the test- and control substances in the diets and the food consumption and body weight of the animals. Test substance intake via diet during different periods of gestation ranged from 49.39 to 60.28 mg/kg body weight/day (low-dose group); 97.91 to 117.87 mg/kg body weight/day (mid-dose group); and 178.93 to 221.75 mg/kg body weight/day (high-dose group). The mean test substances intake during the whole treatment period (GD 0-21) amounted to 53.21, 105.14, and 196.23 mg/kg body weight/day for the 800, 1600, and 3200 mg/kg diet PERKALINK 900 groups, respectively.

REPRODUCTION AND LITTER DATA
In each group, 24 females were mated and 23, 21, 21, and 22 females of the control, low-, mid-, and high-dose groups, respectively, appeared to be pregnant and all had viable fetuses at Ceasarean section. No differences were observed in the female fecundity index and gestation index among the groups. No differences were observed in the number of corpora lutea, implantation sites, pre- and post implantation loss, live and dead fetuses, and resorptions among the groups. In the mid-dose group, a decrease in live male fetuses and an increase in live female fetuses were observed. The statistically significantly change seen on the sex ratio in the mid-dose group (48%) was considered to be caused by the relatively high percentage (59%) of males in the control group (historical control range is 45-57%) and is therefore not considered a treatment-related effect.

PARENTAL NECROPSY
Organ weights and net weight change
A statistically significant decrease in carcass weight and net weight change from day 0 was observed in the females of the high-dose group. The mean weight of the gravid uterus, empty uterus and ovaries did not differ among the control group and the groups treated with the sustbance.

Macroscopic findings in dams at necropsy
No statistically significant differences were observed in the incidence of parental necropsy observations among the groups. The findings observed were incidental and not related to treatment.

In each group, 24 females were mated and 23, 21, 21, and 22 females of the control, low-, mid-, and high-dose PERKALINK 900 group, respectively, were pregnant at Caesarean section and all females had live fetuses. No differences were observed in the female fecundity index and gestation index among the groups. Furthermore, no treatment related differences were observed in the number of corpora lutea, implantation sites, pre- and post-implantation loss, live and dead fetuses, and sex ratio among the groups.

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
>= 105 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
>= 196 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Maternal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: see 'Description (incidence and severity).
Description (incidence and severity):
statistically significant effects on body weight, body weight change, food consumption, carcass weight and net weight change at 196 mg/kg bw/day.

Results (fetuses)

Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No treatment-related effects were observed for fetal external- and placental observations and weights.
Skeletal malformations:
no effects observed
Description (incidence and severity):
No treatment-related effects were observed at fetal visceral and skeletal examinations.
Visceral malformations:
no effects observed
Description (incidence and severity):
No treatment-related effects were observed at fetal visceral and skeletal examinations.
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
FETAL NECROPSY
Fetal external observations
Small fetuses were observed at low (fetal) incidences (1 or 2) in all groups and did not differ between the groups. Subcutaneous haemorrhagic areas on the skin were observed in the control, low-, mid-, and high-dose groups at (fetal) incidences of 1.7 % (4), 0.9% (2), 1.0% (2), and 0.4% (1), respectively. A tail tip was missing from one fetus of the low-dose group (fetus 11 of dam 81) and from one fetus of the mid-dose group (fetus 8 of dam 117). These observations are considered incidental findings not related to treatment.

Findings of the placenta
In the low-dose group an enlarged placenta with a cyst was observed and an enlarged placenta was also observed in the high-dose group. No treatment-related findings of the placenta were observed.

Fetal and placental weight
Values are calculated for all fetuses together and for male and female fetuses separately. No statistically significant differences were observed on placenta and fetal weight among the groups.

VISCERAL EXAMINATION
Visceral malformations
The only malformation observed was: situs inversus in one fetus of the mid-dose group.

Visceral anomalies
Both in the low-dose group and in the high-dose group, one fetus showed a pronounced lobular pattern in the liver. No other visceral anomalies were observed.

Visceral variations
Visceral variations included haemorrhagic areas in the nasal cavity, folded retinas, pericard and stomach filled with haemorrhagic fluid, increased renal pelvic cavitation, dilated urinary bladders, and bent and kinked ureters. None of the animals in the lowdose group showed a folded retina, which was a statistically significant decrease compared to the control group. No other statistically significant effects in incidences of visceral variations were observed among the groups.

In conclusion, no treatment-related adverse effects were observed at visceral examination of the fetuses.

SKELETAL EXAMINATIONS
Skeletal malformations
One fetus in the low-dose group was found with two fused ribs. In the mid-dose group one fetus showed kyphoscoliosis of the vertebral column. These findings are considered incidental and not related to treatment.

Skeletal anomalies
In the control, low-, and mid-dose group, wavy ribs were observed at low fetal incidences (=0.9%; 1 fetus/group). Two sternebrae were fused in two fetuses of the control group and three or more sternebrae were fused in one fetus of the low-dose group. One fetus of the control group showed one dislocated sternebrae and one fetus of the low-dose group showed separated sternebrae. These findings are considered incidental and not related to treatment.

Skeletal variations
Skeletal variations were seen in interparietal bones (supernumerary), supraoccipital bones (holes), ribs (accessory lumbar ribs), and sternebrae (irregular shape). No statistically significant differences are observed in skeletal variations among the groups.

Skeletal retardation
Statistically significant differences in skeletal retardations were:
- Decreased fetal incidence of one or two incompletely ossified caudal bodies in the high-dose group.
- Increased fetal incidence of one or two incompletely ossified cervical arches in the low-dose group.
- Decreased fetal incidence of three or more incompletely ossified caudal arches in the mid-dose group.
- Decreased fetal and litter incidence of 1-2 incompletely ossified metacarpals in the mid-dose group.
- Decreased fetal incidence of 3-6 unossified digits of the proximal front phalanges and decreased fetal and litter incidence of 7-10 unossified digits of the proximal front phalanges in the mid-dose group.
- Decreased fetal incidence of 1-2 unossified metatarsals in the mid-dose group.
- Increased fetal incidence of 5-8 incompletely ossified digits of the proximal hind phalanges in the mid-dose group.
- Decreased fetal incidence of 1-5 incompletely ossified digits of the distal hind phalanges in the mid-dose group.
- Decreased fetal incidence of 7-10 unossified digits of the proximal hind phalanges in the low-, mid-, and high-dose groups.

In conclusion, no treatment-related effects were observed on skeletal malformations, skeletal anomalies, skeletal variations, and skeletal retardations. The observed statistically significant differences in retardations of fetal skeletons were incidental, inconsistent and/or not dose-related. This kind of variation in skeletal ossification is considered as normal developmental variability. No other indications of developmental toxicity were observed. Therefore, these findings are considered as non-adverse variations.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 196 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxicity (litter data, fetal external, visceral, and skeletal examinations

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
The available data do not indicate that PERKALINK 900 causes developmental toxicity.
Executive summary:

The developmental toxicity of PERKALINK 900 was studied in a GLP-compliant OECD 414 guideline study in which female rats received 0, 800, 1600 and 3200 mg/kg diet in an oral feeding study. Animals were exposed from GD 0-21. The NOAEL for maternal toxicity was 1600 mg/kg diet (105 mg/kg bw/day) based on statistically significant effects on body weight, body weight change, food consumption, carcass weight and net weight change.

Since no effects were observed on fertility, reproductive performance, reproductive organ weights, litter data, fetal external, visceral, and skeletal examinations, the NOAEL for reproducive and developmental toxicity is at least 3200 mg/kg diet (196 mg/kg bw/day), the highest dose tested.