Registration Dossier

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:
other: OECD Guideline 421 - Reproduction/Developmental toxicity screening test
Principles of method if other than guideline:
The toxicity of the test item Macrolex Rot E2G (CAS-No. 6829-22-7) on reproduction and/or development of the male and female rat was investigated in a reproduction/developmental toxicity screening test according to OECD guideline 421.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Denomination: Macrolex Rot E2G
Chemical name: 14H-Benz[4,5]isoquino[2,1 -a]perimidin-14-one.
Molecular formula: C22 H12 N2 O.
Molecular mass: 320.3 g/mol.
CAS number: 6829-22-7.
Appearance: Red powder.
Purity: 99.9 %
Specific details on test material used for the study:
Purity: 99.9%

Test animals

Species:
rat
Strain:
other: Wistar rats: Crl: W1 (Han)
Details on test animals and environmental conditions:
Test system
Species/strain: Wistar rats: Crl: WI (Han).
Supplier: Charles River Laboratories France, Domaine des Oncins, 69210 Saint-Germain-Nuelles, France.
Number of animals in the study: 80 (40 males and 40 females).
The virgin males were 11 weeks old at the start of treatment (body weight range 319 to 354 g).
The virgin females were 9 weeks old at the start of treatment (body weight range: 179 to 218 g).
Justification: One of the rodent species acceptable to the regulatory agencies. Historical control data for the strain are available at the Test Facility.

Animal husbandry
Housing: One air-conditioned room in a barrier protected unit (building K2).
Temperature: 22 + 3 °C (target range).
Relative humidity: 35 to 70 % (target range).
Air changes: At least 10 air changes per hour.
Lighting cycle: 12 hours light (artificial)/12 hours dark (except when required for technical acts).

Environmental conditions were within the targets throughout the study.
Caging: All animals were housed in plastic cages meeting European directive 2010/63/EU requirements as follows:
- Males and females: in groups of 5 of the same dose group before mating then singly during cohabitation (one male and one female per cage).
- Females: singly during gestation and lactation (one female with its litter). Tissue paper was added to the bedding towards the end of gestation.
Bedding: Dust-free sawdust (SDS/Dietex, Argenteuil, France) made from spruce tree wood, analysed at least twice a year for chemical and bacterial contaminants. Shredded paper (SDS/Dietex) was provided as enrichment. From day 20 of gestation, cellulose bedding (SERLAB, ALPHA-dri), analysed for chemical and bacterial contaminants was used.
Diet: Rat pelleted complete diet ad libitum (Diet reference A04C-10) sterilised by irradiation and analysed for a predefined list of chemical and bacteriological contaminants. Each batch of diet is supplied with a certificate of analysis which is verified and authorized for release by a veterinarian.
Water: Softened and filtered (0.2 μm) mains drinking water was available ad libitum (via an automatic watering system or bottles). Water is analysed twice a year for chemical and bacterial contaminants by Laboratoire Santé Environnement Hygiène de Lyon, France.
Contaminants: No known contaminants were present in the bedding, diet or water at levels which might have interfered with achieving the objectives of the study.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1 % (w/v) Carboxymethylcellulose, 400-800 centipoises
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Vehicle
Denomination: 1 % (w/v) Carboxymethylcellulose 400-800 centipoises.
Supplier: Sigma-Aldrich.
Storage: At room temperature (between +15 and +25 °C).
Water for injection:
Supplier: Laboratoire Aguettant
Storage: At room temperature (between +15 and +25 °C).
Frequency of preparation: At least once every 8 days.
Storage: At room temperature (between +15 and +25 °C).
Test item preparation
Preparation: The test item was prepared as a suspension in the vehicle at concentrations of 10, 30 and 100 mg/mL.
Correction factor for active ingredient: None.
Homogeneity of the test item in the vehicle: Suspensions at concentrations of 1 to 100 mg/mL have been shown to be homogeneous, according to AB20658 study.
Stability of the test item in the vehicle: 24 hours and 8 days at room temperature (+15 to +25 °C), protected from light or refrigerated (+2 and +8 °C) according to AB20658 study.
Frequency of preparation: At least once every 8 days.
Storage of formulations: At room temperature (between +15 and +25 °C).

Analyses
Analysis of preparations: Four samples of approximately 1 g were taken from the middle only for each formulation, including the control, used on the first day of treatment and on suitable day during the last common week of treatment for males and females (i.e. week 4).
One set of samples (2 x 1 g) was analysed at the Test Facility within the stability period using the validated method according to study AB20658. The duplicate samples (2 x 1 g) which were stored refrigerated (between +2 and +8 °C) will be destroyed at the end of the defined stability period if not required.
Details on mating procedure:
Animals were paired on the basis of one male and one female from the same group for a maximum of 14 days.
Vaginal smears were taken daily from the females during cohabitation.
The day of mating was confirmed by the presence of sperm in a vaginal smear and was recorded and taken as day 0 of gestation (G0). Mated females were separated from the males once mating had been confirmed and smearing ceased.
Duration of treatment / exposure:
Males: from 14 days before mating, throughout mating and up to the day before necropsy (28 days of exposure).
Females: from 14 days before mating, throughout mating and gestation and up to day 3 of lactation.
Frequency of treatment:
Once daily
Duration of test:
First day of treatment (day 0): 27 October 2014.
Mating: from 10 November 2014.
Necropsy of males: 24 November 2014,
Necropsy of females with their litter (day 4 of lactation): from 07 December 2014.
Experimental completion date (last necropsy): 15 December 2014.
No. of animals per sex per dose:
10 male and 10 female rats/dose
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for the dose selection: the dose levels were chosen based on the results of a 5-day dose range-finding study (AB20659) and a 4-week repeat dose toxicity study (AB20660) in the rat at doses up to 1000 mg/kg/day. In the absence of any toxicologically significant finding, the limit dose of 1000 mg/kg/day was selected at the high dose in the current study. The low and intermediate doses selected were 100 and 300 mg/kg/day, respectively.

Examinations

Maternal examinations:
Morbidity/mortality, clinical signs, body weight, food consumption
Ovaries and uterine content:
For each litter the following data were recorded:
− number of male and female pups born (live and dead)
− external abnormalities of the pups
− number, weight and sex of pups alive on postnatal days 1 and 4.
Fetal examinations:
All animals, including PND 4 pups, were submitted to a macroscopic examination for structural or pathological changes.
Statistics:
The following parameters were analysed statistically using the Provantis data acquisition system on each occasion for males and females separately:
− body weights and body weight gains
− food consumption
− copulation and fertility indices
− terminal body weights, absolute and relative organ weights.
Indices:
Pre-coital interval (in days), mating index (in %), female fertility index (in %), gestation index (in %), live birth index (in %), viability index (in %), sex ratio (proportion of male pups in %).
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):
There was no treatment-related clinical sign observed during the study at any dose level.
Dermal irritation (if dermal study):
no effects observed
Mortality:
no mortality observed
Description (incidence):
There was no unscheduled death during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Despite some intergroup variations, there was no adverse effect of the test item on mean body weight change of the males throughout the study or for the females during the pre-mating, gestation and lactation phases.
Mean terminal body weight was consequently comparable in all groups for both sexes.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There was no effect of test item on mean food consumption for either sex during the pre-mating period or for the females during gestation and lactation.
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:
no effects observed
Description (incidence and severity):
No organ weight, macroscopic or microscopic changes were noted to suggest a toxicological effect of Macrolex Rot E2G on the ovaries, testes and epididymides of treated animals.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No organ weight, macroscopic or microscopic changes were noted to suggest a toxicological effect of Macrolex Rot E2G on the ovaries, testes and epididymides of treated animals.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No organ weight, macroscopic or microscopic changes were noted to suggest a toxicological effect of Macrolex Rot E2G on the ovaries, testes and epididymides of treated animals.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Details on results:
There were no adverse effects of Macrolex Rot E2G on the reproduction and/or development of the male and female rat up to the limit dose of 1000 mg/kg/day inclusive.

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
There was no influence of the test item on pre-birth loss in any group since the mean numbers of implantation sites and delivered pups were similar to those in the control group.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There was no influence of the test item on pre-birth loss in any group since the mean numbers of implantation sites and delivered pups were similar to those in the control group.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There was no influence of the test item on pre-birth loss in any group since the mean numbers of implantation sites and delivered pups were similar to those in the control group.
Early or late resorptions:
no effects observed
Description (incidence and severity):
There was no influence of the test item on pre-birth loss in any group since the mean numbers of implantation sites and delivered pups were similar to those in the control group.
Dead fetuses:
no effects observed
Description (incidence and severity):
No stillborn pups were observed in any group.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
The mean duration of gestation was normal (approximately 22 days) in each group.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): The mean duration of gestation was normal (approximately 22 days) in each group.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
There was no adverse effect of exposure on fertility in any group. There was a single inseminated female in the high dose group that failed to become pregnant. This isolated finding was considered to be incidental.
Other effects:
no effects observed
Details on maternal toxic effects:
There were no adverse effects of Macrolex Rot E2G on the reproduction and/or development of the male and female rat up to the limit dose of 1000 mg/kg/day inclusive.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: other:

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
There was no influence of the test item on mean pup weight at birth and postnatal day 4 for either sex in any group.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): There was no influence of the test item on mean pup weight at birth and postnatal day 4 for either sex in any group.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The mean percentage of males per litter was normal (approximately 50 %) in all treated groups. An incidentally greater proportion of males (approximately 64 %) was noted in the control.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
There was no influence of the test item on subsequent postnatal pup survival in any group. Two or one pups were noted to be weak in the 100 and 1000 mg/kg/day groups and one was thin in the 1000 mg/kg/day group. These sporadic findings were considered to be incidental.
Seven pups were de ad or missing in the 100 mg/kg/day group (due to female nos. 32, 33, 35 and 37 that each lost one or more pup(s) between days 1 and 4 of lactation) and one in the 1000 mg/kg/day group (from female no. 79) compared with none in the control and 300 mg/kg/day groups. In the absence of any dose-relationship, these sporadic pup deaths were considered to be incidental.
External malformations:
no effects observed
Description (incidence and severity):
There were no macroscopic findings at necropsy of the F1 pups associated with maternal test item exposure. Autolysis was noted in one dead pup.
Skeletal malformations:
no effects observed
Description (incidence and severity):
There were no macroscopic findings at necropsy of the F1 pups associated with maternal test item exposure. Autolysis was noted in one dead pup.
Visceral malformations:
no effects observed
Description (incidence and severity):
There was no evidence of toxicological treatment-related findings associated with oral (gavage) administration of Macrolex Rot E2G.
Other effects:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no adverse effects of Macrolex Rot E2G on the reproduction and/or development of the male and female rat up to the limit dose of 1000 mg/kg/day inclusive.
Remarks on result:
other: 1000 mg/kg bw/day was the highest applied dose

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

There were no unscheduled deaths or systemic clinical signs associated with the test item.

There was no effect of the test item on mean body weight change or food consumption for either sex throughout the study.

There was no adverse effect of the test item on mating Performance or fertility. The mean duration of gestation was normal (approximately 22 days) in all groups. There was no influence of treatment with the test item on pre- or postnatal pup survival of either sex.

Mean pup weight at birth and postnatal day 4 was comparable with that in the experimental control.

No organ weight, macroscopic or microscopic changes were noted to suggest a toxicological effect of Macrolex Rot E2G on the ovaries, testes and epididymides of treated animals.

Applicant's summary and conclusion

Conclusions:
There were no adverse effects of Macrolex Rot E2G on the reproduction and/or development of the male and female rat up to the limit dose of 1000 mg/kg/day inclusive.
Executive summary:

Three groups of 10 male and 10 female Wistar Han rats were given the test item, Macrolex Rot E2G, by daily oral (gavage) administration at dose levels of 100, 300 and 1000 mg/kg/day from 14 days before mating then for up to 4 weeks for males and throughout mating, gestation and through day 3 of lactation for females. A control group of 10 male and 10 female rats received a similar volume (lOmL/kg) of the vehicle [1 % (w/v) Carboxymethylcellulose]. Clinical condition, body weight and food consumption ofthe animals were monitored throughout the study. After two weeks of treatment, one male and one female of the same group were paired for a maximum of 14 days. The females were retained throughout gestation, allowed to litter and rear their young through to postnatal day (PND) 4 and then necropsied. Vaginal smears were taken daily for each female from the first day of pairing to verify positive copulation. Litter parameters, including the number of pups bom, pup survival, sex and pup weights were recorded up to PND 4.

The dams with their pups were necropsied on day 4 of lactation, where applicable. All animals, including PND 4 pups, were submitted to a macroscopic examination. The numbers of uterine implantations were determined and the ovaries were weighed for each female. The males were necropsied after completion of the mating period and the testes and epididymides were weighed.

Selected tissue samples were fixed and preserved from all animals. Selected organs/tissues from group 1 and 4 animals were examined histopathologically.

There were no unscheduled deaths or systemic clinical signs associated with the test item.

There was no effect of the test item on mean body weight change or food consumption for either sex throughout the study.

There was no adverse effect of the test item on mating performance or fertility. The mean duration of gestation was normal (approximately 22 days) in all groups. There was no influence of treatment with the test item on pre- or postnatal pup survival of either sex.

Mean pup weight at birth and postnatal day 4 was comparable with that in the experimental control.

No organ weight, macroscopic or microscopic changes were noted to suggest a toxicological effect of Macrolex Rot E2G on the ovaries, testes and epididymides of treated animals.