5,12-dihydro-2,9-dimethylquino[2,3-b]acridine-7,14-dione

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Remarks:

In accordance with OECD Guideline for Testing of Chemicals, Number 443, “Extended One-Generation Reproductive Toxicity Study”, adopted on 25 June 2018.

Type of information:

experimental study

Remarks:

The Cohorts 1A and Cohort 1B (without extension to F2 generation) were selected for F1 generation assessments based on the available toxicity information of test item in consultation with the sponsor.

Adequacy of study:

key study

Study period:

14 May 2021 to 02 August 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The Cohorts 1A and Cohort 1B (without extension to F2 generation) were selected for F1 generation assessments based on the available toxicity information of test item in consultation with the sponsor

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

adopted on 25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

There was no information or data available from repeated dose/reproduction toxicity and teratology studies for the test item. However, the estimated NOAEL of a structural analogue compound, Pigment Red 282 was 1000 mg/kg body weight/day obtained from a 28-Day Repeated Dose Oral Toxicity study in Wistar Rats performed according to OECD Test Guideline 407.
(Reference:https://echa.europa.eu/registration-dossier/-/registered-dossier/15097/7/6/2/? documentUUID=8787be8c-e05e-49c5-af69-63f79a57bcf6).
Based on the above information, a dose range finding study for prenatal developmental toxicity study [Bioneeds study no. BIO-DTX 053] with the test item by oral gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 was conducted with the doses of 0, 111, 333 and 1000 mg/kg body weight/day for vehicle control, low, mid and high dose groups, respectively in consultation with the sponsor. The study included the general and maternal end point assessments such as, maternal body weights, feed consumption, corrected body weight for maternal body weight gain, gravid uterus weight, uterine content observations, implantations, and gross pathological examinations. The dose range finding study also included the fetal/prenatal developmental assessments such as, fetal weights (sex wise) per litter, fetal external examinations, and fetal gross soft tissue examinations. The results of this range finding study did not produce any indication of maternal and fetal (prenatal developmental) toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.
Hence, the same dose levels of 0, 111, 333 and 1000 mg/kg body weight/day were selected as vehicle control, low, mid, and high dose groups, respectively in consultation with the sponsor for the present “Extended One-Generation Reproductive Toxicity Study with the test item by oral gavage in Sprague Dawley Rats”.

The test item was administered continuously in graduated doses to three groups of males and females. The parental (P) generation animals were dosed for a defined
pre-mating period (10 weeks) and a two-week mating period. Treatment of the
P females was continued during pregnancy and lactation until termination following weaning of respective litters. The F1 offspring received further treatment with the test item from weaning till adulthood.
The Cohorts 1A and Cohort 1B (without extension to F2 generation) were selected for F1 generation assessments based on the available toxicity information of test item in consultation with the sponsor.

Test material

Test material information

Constituent 1

Reference substance name:

5,12-dihydro-2,9-dimethylquino[2,3-b]acridine-7,14-dione

EC Number:

213-561-3

EC Name:

5,12-dihydro-2,9-dimethylquino[2,3-b]acridine-7,14-dione

Cas Number:

980-26-7

Molecular formula:

C22H16N2O2

IUPAC Name:

2,9-dimethyl-5,7,12,14-tetrahydro-5,12-diazapentacene-7,14-dione

Test material form:

solid: nanoform

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Stability and homogeneity of the test material in the vehicle under test conditions (e.g. in the exposure medium) and during storage: The stability and homogeneity of the test item in dose formulations was established by the analytical investigation
The prepared test item formulations were stable at room temperature for 6 hours followed by 48 hours in 0.5% w/v Carboxymethyl cellulose within the mean percent recovery range of 85 to 115.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): Triturated well in a mortar (grinding)

- Final concentration of a dissolved solid: Low dose-11.1, Mid dose-33.3 and High dose-100 mg/mL

FORM AS APPLIED IN THE TEST: Suspension

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Rat is one of the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal Species: Rat (Rattus norvegicus)
Strain: Sprague Dawley
Justification for Selection of Species : Rat is one of the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.
Source of Supply : Hylasco Biotechnology India Pvt. Ltd,
Charles River Technology Licensee, CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals) Registration No.: 1808/PO/RcBt/S/15/CPCSEA

Animals were housed under standard laboratory conditions in an environmentally monitored, temperature-controlled room with adequate fresh air supply (12 to 15 air changes per hour), room temperature 19.8 to 23.1oC, relative humidity
48 to 64%, with 12 hours light and 12 hours dark cycle. The temperature and relative humidity were recorded once daily.
Animals were housed in a standard polypropylene cage (size: L 430 x B 285 x H 150 mm) with stainless steel mesh top grill having facilities for holding pelleted feed and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material.
P Animals:
i. Pre-mating
Maximum of two animals of same sex and group per cage were housed.
ii. Mating
During mating, two animals (one male and one female) of same group were housed.
iii. Post mating
After confirmation on presence of sperm in the vaginal smear (Day 0 of pregnancy), the mated pairs were separated.
Males were housed with their former cage mates while females were housed individually during gestation and lactation periods.
Sterilized paper shreds were provided as a nesting material from gestation day 20 onwards.
F1 Animals:
i. During Lactation period (postnatal period)
All F1 pups of both sexes were housed along with the dam.
ii. Post Weaning
Two animals of same sex and group per cage were housed.
Altromin Maintenance diet for rats and mice 1324 manufactured by Altromin Spezialfutter GmbH & Co. KG was provided ad libitum to the rats throughout the experimental period. The contaminant analysis test reports for the feed are presented as Annexure 2.
A sample of each batch of the diet used during the study was retained under appropriate conditions (-20°C) and discarded on the day of finalization of the report.
Water was provided ad libitum throughout the acclimatization and experimental period. Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% w/v

Details on exposure:

The test item formulations/vehicle were administered to animals through oral gavage using stainless steel intubation cannula attached to a disposable syringe once daily.
All the doses were administered in an equi-volume of 10 mL/kg with the concentration of 11.1, 33.3 and 100 mg/mL for 111, 333 and 1000 mg/kg body weight dose groups respectively. Vehicle was administered to vehicle control group at an equi-volume of 10 mL/kg body weight. The actual dose volume for each animal was calculated based on the most recent body weight.
Parental (P) Generation Animals:
-The parental (P) animals (both males and females) were treated for a period of
10 weeks during pre-mating period.
The parental (P) males were treated during mating period and continued the treatment until sacrifice [a total of 88 to 95 days of test item administration].
The parental (P) females were treated during cohabitation period until confirmation of mating, during mating, throughout gestation and lactation periods and up to weaning of F1 animals [a total of 113 to 124 days of test item administration]. .
F1 Animals:Direct dosing by oral gavage
Cohort 1A - From day of weaning (PND 21) to PND 95
Cohort 1B - From day of weaning (PND 21) to PND 102

Details on mating procedure:

Each P female was placed with a randomly selected, single and unrelated male from the same dose group (1:1 pairing) until evidence of mating. Day 0 of pregnancy was defined as the day on which mating evidence was confirmed (presence of sperm in vaginal smear).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and homogeneity of the test item in dose formulations was established by the Analytical Department of Bioneeds India Private Limited (Bioneeds study no.:
BIO-ANM 1717).
The prepared test item formulations were stable at room temperature for 6 hours followed by 48 hours in 0.5% w/v Carboxymethyl cellulose within the mean percent recovery range of 85 to 115.

Duration of treatment / exposure:

Parental (P) Generation Animals
The parental (P) animals (both males and females) were treated for a period of
10 weeks during pre-mating period.
The parental (P) males were treated during mating period and continued the treatment until sacrifice [a total of 88 to 95 days of test item administration]
The parental (P) females were treated during cohabitation period until confirmation of mating, during mating, throughout gestation and lactation periods and up to weaning of F1 animals [a total of 113 to 124 days of test item administration
F1 Animals
Cohort 1A - From day of weaning (PND 21) to PND 95
Cohort 1B - From day of weaning (PND 21) to PND 102

Frequency of treatment:

once daily

Details on study schedule:

Study Initiation Date: 14 May 2021
Experimental Starting Date: 14 May 2021
Parental (P) Generation
Acclimatization Date : Start: 14 May 2021; End: 19 May 2021
Treatment Date :
Males: Start: 20 May 2021; End: 22 August 2021
Females: Start: 20 May 2021; End: 20 September 2021
Cohabitation period: Start: 28 July 2021; End: 09 August 2021
Necropsy Date :
Males: Start: 16 August 2021; End: 23 August 2021
Females: Start: 10 September 2021; End: 21 September 2021
F1 Generation
Weaning Date: Start: 09 September 2021; End: 20 September 2021
Treatment Date :
Cohort 1A: Start: 09 September 2021; End: 03 December 2021
Cohort 1B: Start: 09 September 2021; End: 10 December 2021
Necropsy Date :
Cohort 1A: Start: 23 November 2021; End: 04 December 2021
Cohort 1B: Start: 30 November 2021; End: 11 December 2021
In-life End Date :11 December 2021
Histopathology Completion Date: 01 May 2022
Experimental Completion Date : 01 May 2022
Draft Report Submission Date : 30 May 2022
Study Completion Date: 02 August 2022

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Dose concentration = 0 mg/mL

Doses / concentrations 2

Dose / conc.:

111 mg/kg bw/day

Remarks:

Dose concentration = 11.1 mg/mL

Doses / concentrations 3

Dose / conc.:

333 mg/kg bw/day

Remarks:

Dose concentration = 33.3 mg/mL

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Dose concentration = 100 mg/mL

No. of animals per sex per dose:

A total of 200 (100 males + 100 females) Sprague Dawley rats were selected for parental (P) generation and distributed to four P generation groups. Each P generation group (G1, G2, G3 and G4) consisted of 25 males and 25 females. A total of 160 males and 160 females were selected on the day of weaning (PND21) and randomly assigned to two cohorts [Cohort 1A (80 males + 80 females) and Cohort 1B (80 males + 80 females)]. Each cohort consisted of four groups and consisted of 20 males and 20 females per group.

Control animals:

yes, concurrent no treatment

Details on study design:

There was no information or data available from repeated dose/reproduction toxicity and teratology studies for the test item. However, the estimated NOAEL of a structural analogue compound, Pigment Red 282 was 1000 mg/kg body weight/day obtained from a 28-Day Repeated Dose Oral Toxicity study in Wistar Rats performed according to OECD Test Guideline 407.
(Reference:https://echa.europa.eu/registration-dossier/-/registered-dossier/15097/7/6/2/? documentUUID=8787be8c-e05e-49c5-af69-63f79a57bcf6).
Based on the above information, a dose range finding study for prenatal developmental toxicity study [Bioneeds study no. BIO-DTX 053] with the test item by oral gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 was conducted with the doses of 0, 111, 333 and 1000 mg/kg body weight/day for vehicle control, low, mid and high dose groups, respectively in consultation with the sponsor. The study included the general and maternal end point assessments such as, maternal body weights, feed consumption, corrected body weight for maternal body weight gain, gravid uterus weight, uterine content observations, implantations, and gross pathological examinations. The dose range finding study also included the fetal/prenatal developmental assessments such as, fetal weights (sex wise) per litter, fetal external examinations, and fetal gross soft tissue examinations. The results of this range finding study did not produce any indication of maternal and fetal (prenatal developmental) toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.
Hence, the same dose levels of 0, 111, 333 and 1000 mg/kg body weight/day were selected as vehicle control, low, mid, and high dose groups, respectively in consultation with the sponsor for the present “Extended One-Generation Reproductive Toxicity Study with by oral gavage in Sprague Dawley Rats”.

Examinations

Parental animals: Observations and examinations:

Clinical Signs of Toxicity and Mortality/Morbidity
Detailed Clinical Examination
Body Weight
Feed Consumption
Oestrus Cyclicity
Reproductive Performance Evaluation
Delivery and Litter Observations
Uteri Observations
Clinical Pathology and Thyroid hormonal estimations
Thyroid Hormone Levels Estimation
Sperm Parameters
Gross Pathology
Histopathology

Oestrous cyclicity (parental animals):

Oestrus cycles were monitored daily for a period of 2-weeks during pre-mating period before initiation of cohabitation.
During cohabitation period until evidence of mating.
At termination (on the day of necropsy) to correlate with histopathology of reproductive organs.

Sperm parameters (parental animals):

Sperm motility was evaluated immediately after sacrifice. The percentage of progressively motile sperms was determined subjectively. Samples for evaluating sperm morphology was taken from the suspension used for the sperm motility. Sperm sample was examined as fixed and 200 spermatozoa per sample were classified as either normal (both head and mid-piece/tail appear normal) or abnormal. Another testis reserved for spermatid head counts was stored in -20ºC from each animal and subjected to evaluation for homogenization resistance spermatid head counts to calculate daily sperm production.

Litter observations:

The day of parturition was considered as lactation day (LD) 1 for the dam and postnatal day (PND) 1 for pups. The number of male/female pups born (live/dead/cannibalized) per litter were recorded and sex ratio (m/f) and live birth index was calculated.
The number of pups survived/dead per litter were recorded during lactation period and pup survival index (%) per litter between lactation day 1 to 4, 5 to 7 and 8 to 14 and 15 to 21 was calculated

Postmortem examinations (parental animals):

All the parental males were sacrificed after completion of mating procedure. Males were randomized throughout all dose groups and restricted to maximum of 20 animals per day for necropsy.
The females not confirmed with mating were sacrificed after 25 days from the day of termination of cohabitation process. The females confirmed with mating but not littered were sacrificed 25 days after confirmation of mating. All the littered females were sacrificed on lactation day 22. Organ Weight and Tissue Preservation
The organs such as Adrenals, Bone marrow smear, Brain Epididymis [males], Eye with optic nerve, Gastrointestinal tract, Heart, Kidneys, Liver, Lungs, Ovaries, Peripheral (sciatic) nerve, Pituitary gland, Prostate -dorsolateral and ventral [males], Seminal vesicles with coagulating glands and their fluids as a unit – [males], Skeletal muscle Skin with mammary gland [both males and females], Spleen, Spinal cord , Testes [males], Thymus, Thyroid along with parathyroid, Trachea, Urinary bladder, Uterus with cervix [females], Vagina [females] and Vas deferens [males] from all P animals were collected, weighed, and preserved.

Histopathology
Histopathological examination was conducted on all the tissues collected from the vehicle control (G1) and high dose (G4) group animals (with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure) sacrificed at termination.
Additionally, reproductive organs of all animals suspected of reduced fertility (those that failed to conceive or sire from all the dose groups) were subjected to histopathological examination.

Postmortem examinations (offspring):

Necropsy and Gross Pathology

The animals were euthanized using deep Isoflurane anaesthesia followed by exsanguination and subjected to gross pathological examination. Gross pathological examination was performed on all the animals sacrificed.
Cohort 1A and 1B Males and Females: The cohort 1A animals were sacrificed on PND 96 and the cohort 1B animals were sacrificed on PND 103.

Cohort 1A: The organs and tissues such as Adrenals, Bone marrow smear, Brain Epididymis [males], Eye with optic nerve, Gastrointestinal tract, Heart, Kidneys, Liver, Lungs, Ovaries, Peripheral (sciatic) nerve, Pituitary gland, Prostate -dorsolateral and ventral [males], Seminal vesicles with coagulating glands and their fluids as a unit – [males], Skeletal muscle Skin with mammary gland [both males and females], Spleen, Spinal cord , Testes [males], Thymus, Thyroid along with parathyroid, Trachea, Urinary bladder, Uterus with cervix [females], Vagina [females] and Vas deferens [males] of all high dose and control animals were examined for histopathology.

Cohort 1A Females (Ovarian Follicular and Corpora luteal Assessments):
Histopathological examination of ovaries including quantitative evaluation of primordial and small growing follicles, as well as corpora lutea was conducted for all control and high dose animals.

Cohort 1B: The reproductive and endocrine tissues were processed to the block stage.

Organ-typic Development:
C1A Males: Caput, corpus and cauda of the epididymis and the vas deferens were examined for appropriate organ-typic development.
C1A Females: Ovary with oviduct, uterus and vagina were examined for appropriate organ-typic development.

Statistics:

The raw data was subjected to computer statistical processing. The data was subjected to various statistical analyses using SPSS software version 27.

All analysis and comparisons were evaluated at the 95% level of confidence (P<0.05) indicated by the aforementioned tests.

The statistical analysis was followed to the parameters as mentioned below table.
Parameter
Type of Analysis - One-way ANOVA with Dunnett's post-test
• Bodyweight (weekly/gestation/lactation)
• Percent Change in body weight (weekly/gestation/lactation)
• Feed consumption (weekly/gestation/lactation)
• Copulatory interval
• Gestation length
• Mean oestrus cycle length
• Absolute/relative organ weights
• Mean pup weight per litter
• Mean pup anogenital distance ratio per litter
• Serum thyroid hormonal values
• Occurrence of postnatal developmental landmarks of F1 pups
• Responses for sensory reflexes of F1 pups
• Sexual maturation time points of F1 animals
• Splenic lymphocyte sub-populations
• Sperm motility/morphological changes/daily sperm production
• Ovarian follicular count (Independent sample T - test) Parametric -
Type of Analysis - Non-parametric - Kruskal-Wallis followed by the Mann-Whitney
• Implantations/litter
• No. of pups/litter
• Sex ratio/litter at birth and during the lactation period
• Litter size at birth and during the lactation period
• Post-implantation loss/litter
• Postnatal loss/litter
Type of Analysis - Cross Tabs - Frequency comparison Chi-square test/ Fischer's Exact Test
• Reproductive indices
• No. of dams with/without live pups
• No. of dams with/without dead pups
• No. of litters with/without resorptions

Reproductive indices:

The following reproductive performance indices were calculated for all P animals.

Mating and Fertility Index :
The male / female mating, and fertility indices were calculated as mentioned below:
Male mating index (%) = No. of males with confirmed mating/ Total no. of males cohabited X 100

Female mating index (%)= No. of sperm-positive females/Total no. of females cohabited X 100

Male fertility index (%)=No. of males impregnating a female/Total no. of males cohabited X 100

Female fertility index (%)= No. of females with evidence of implantation sites/No. of sperm-positive females X 100

Cohabitation Record and Copulatory Interval (Pre-coital Interval)

The day of initiation of mating and day of confirmation of mating were recorded for each female, and the pre-coital interval was calculated as mentioned below:

Pre-coital interval (days)=Date of confirmation of mating – Date of initiation of cohabitation

Gestation Length: The day of confirmation of mating and day of parturition were recorded for each female and the gestation length per litter was calculated as mentioned below:

Gestation length (days)=Date of parturition – Date of evidence of mating (GD 0)

Gestation Index
The gestation index (%) per group was calculated as mentioned below:
Gestation index (%)=No. of females with live born/No. of females with evidence of pregnancy X 100

Parturition Index
The parturition index (%) per group was calculated as mentioned below:
Parturition index (%)= No. of females littered/No. of females with evidence of pregnancy X 100

Female Fecundity or Pregnancy Index
The pregnancy index (%) per group was calculated as mentioned below:
Pregnancy index (%) = No. of females evident of presence of live/dead fetuses/pups /No. of females with evidence of matingX 100

Offspring viability indices:

The day of parturition was considered as lactation day (LD) 1 for the dam and postnatal day (PND) 1 for pups. The number of male/female pups born (live/dead/cannibalized) per litter were recorded and sex ratio (m/f) and live birth index was calculated (litter as a unit) as mentioned below:
Sex ratio (m/f)= No. of male offspring/Number of female offspring
Live birth index (%) per litter=No. of pups born alive/Total no. of pups bornX 100

The number of pups survived/dead per litter were recorded during lactation period and pup survival index (%) per litter between lactation day 1 to 4, 5 to 7 and 8 to 14 and 15 to 21 was calculated (litter as a unit) as mentioned below:

Pup survival index (%) on LD 4/7/14/21 =Total no. of live pups on LD 4/7/14/21/No. of pups born/4/7/14X 100

The sex ratio (m/f) on LD 4, 7, and 13 was calculated per litter, as mentioned below:
Sex ratio (m/f) on LD 4/7/14/21=No. of male offspring on LD 4/7/14/21/No. of female offspring on LD 4/7/14/21

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Dark pink coloured faeces were noted in all the tested dose group animals of both sexes. This coloration is due to coloured nature of the test item but not an adverse sign.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In group G3, statistically significant reduction in percent change in mean gestational body weight gain during GD 7 to 14 was noted when compared with vehicle control group. This noted change is considered as incidental and unrelated to treatment, as the change is a single occurrence and there were no such incidences noted during other phases of the gestation such as during GD 0 to 7 and 14 to 20 in the same dose level.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

In group G2 males, statistically significant reduction in mean feed consumption was noted during week 1 when compared with vehicle control group. This noted change is considered as incidental and unrelated to treatment, as the change is a single occurrence for mean feed consumption and there were no such incidences noted throughout the experimental period at the same dose level.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

statistically significant increase in mean haematocrit value in all the tested dose group males and statistically significant increase in mean absolute lymphocytes of group G2 males were noted when compared with vehicle control group. These differences are considered as incidental and unrelated to treatment, as the obtained mean values are within in-house historical control range of same species and strain and also similar changes were not occurred in other sex of same dose level.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

statistically significant decrease in mean total protein levels of group G4 females were noted when compared with vehicle control group.This difference is considered as incidental and unrelated to treatment, as the obtained mean value is within in-house historical control range of same species and strain and also similar changes did not occur in other sex of same dose level.

Endocrine findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The few microscopic findings observed in this study such as ultimobranchial cyst(s) in thyroid gland, epithelial cyst(s)in thymus and all other findings were considered incidental as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rats.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

A mean oestrus cycle length of 4.76, 4.91, 4.95 and 4.92 days was noted from groups G1, G2, G3 and G4 respectively. The mean length of oestrus cycle per female during treatment period was unaffected by the test item administration in any of the tested dose groups.
However, statistically significant increase in mean oestrus cycle length was noted in all the tested dose groups when compared with vehicle control group. This noted change is not considered as test item related as the obtained mean values are well within the
in-house historical control range and no irregularities in oestrus cycle were noted in any of the tested dose groups.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

The test item did not produce any systemic toxicity, reproductive toxicity and developmental toxicity at all the tested dose levels (111, 333 and 1000 mg/kg body weight/day) when administered to parental generation animals for a period of 10 weeks during pre-mating period (both P males and females), 2 weeks during mating period (both P males and P females), throughout gestation and lactation periods.

Therefore, the no-observed-adverse-effect-level (NOAEL) of the test item is considered as 1000 mg/kg body weight/day for systemic, reproduction and developmental toxicity end points.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Remarks:

1000 mg/kg body weight/day for systemic, reproduction and developmental toxicity end points

Effect level:

ca. 1 000

Based on:

test mat.

Remarks:

test item did not produce any systemic toxicity, reproductive toxicity and developmental toxicity at all the tested dose levels (111, 333 and 1000 mg/kg body weight/day) when administered to parental generation animals for a period of 10

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food efficiency

haematology

clinical biochemistry

urinalysis

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

other:

Remarks on result:

other: test item did not produce any systemic toxicity, reproductive toxicity and developmental toxicity at all the tested dose levels (111, 333 and 1000 mg/kg body weight/day) when administered to parental generation animals for a period of 10

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

The dark pink coloured faeces were noted in all the testing dose group animals of both sexes. This coloration is due to coloured nature of the test item but not any adverse signs.

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 1A (C1A)
The noted non-toxicological and statistically significant increase in mean body weight on PND 42 in group G2 females when compared with vehicle control group .

A statistically significant decrease in mean body weights on PND 63, 70, 77, 84 and 91 and statistically significant decrease in percent change in mean body weight gain with respect to postnatal day (PND) 21 on PND 63, 70, 77, 84 and 91 in groups G3 and G4 males were noted when compared with vehicle control group.
These changes are not considered as adverse as there were no other systemic toxicity effects noted in any of these tested dose groups but are considered as stress induced effects due to test item administration.
Cohort 1B (C1B)
The noted non-toxicological and statistically significant increase in mean body weights on PND 77 and 84 in group G2-C1B females; increase in mean body weights on
PND 77, 84, 91 and 98 in group G3-C1B females; increase in mean body weights on PND 84, 91 and 98 in group G4-C1B females; increase in percent change in mean
body weight gain with respect to postnatal day (PND) 21 on PND 84 in group
G3-C1B females when compared with vehicle control group are considered as incidental.

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 1A (C1A)
A statistically significant decrease in mean feed consumption during week 4 in group G3-C1A males when compared with vehicle control group is considered as incidental.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant increase in mean haemoglobin, haematocrit, and prothrombin time levels in group G3-C1A females were noted when compared with vehicle control group. These differences are considered as incidental and unrelated to treatment, as the obtained mean values are within in-house historical control range of same species and strain and similar changes were not occurred in other sex of same dose level.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant decrease in mean glucose, creatinine, cholesterol, and albumin levels of group G4 males; statistically significant decrease in mean creatinine levels in all the tested dose group females; statistically significant decrease in mean albumin levels in group G3 females was noted when compared with vehicle control group. These differences are considered as incidental and unrelated to treatment, as the obtained mean value is within in-house historical control range of same species and strain and similar changes were not occurred in other sex of same dose level.

Urinalysis findings:

no effects observed

Sexual maturation:

no effects observed

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Cohort 1A (C1A)

In group G2-C1A,
- decrease in mean absolute weight of liver and increase in relative weight of brain (males).
- increase in mean absolute and relative weight of liver and increase in absolute weight of iliac lymph nodes (females).

In group G3-C1A,
- decrease in mean terminal body weight (males).
- increase in mean relative weight of adrenals, kidneys and thyroid along with parathyroid (males).
- increase in mean relative weight of liver (females).

In group G4-C1A,
- decrease in mean terminal body weight (males).
- decrease in mean absolute weight of epididymides, heart, brain, liver (males); increase in mean relative weight of adrenals, testes, kidneys, prostate and thyroid along with parathyroid (males).
- increase in mean absolute and relative weight of thymus (females).

Cohort 1B (C1B)

In group G2-C1B,
- decrease in mean relative weight of mandibular lymph nodes (females).
In group G3-C1B,
- decrease in mean relative weight of adrenals and thyroid along with parathyroid (males).
- increase in mean absolute weight of spleen (females).
In group G4-C1B,
- increase in mean terminal body weight (females).
- decrease in mean absolute and relative weight of adrenals (males).
- decrease in mean relative weight of iliac and mandibular lymph nodes (females).

These differences are considered as incidental and unrelated to treatment, as the obtained mean values are within in-house historical control range of same species and strain and also neither gross pathological changes nor microscopic changes were noted in any of these organs in all the tested dose group animals.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In test-item administered group of animals of both the sexes, the contents of gastrointestinal tract were found to be pink in colour which could be attributed to physical appearance of the test item (pink in colour).
A single incidence of small sized testis was observed in G3-C1A group male rat and was microscopically correlated with moderate atrophy of tubules. This change was considered as an isolated spontaneous finding in rats.

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Few of the microscopic findings observed in this study such as ultimobranchial cyst(s) in thyroid gland, epithelial cyst(s)in thymus and all other findings were considered incidental as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rats.
Quantitative ovarian follicular assessment (primordial and primary follicles) in all parental and C1A females of control and high dose groups did not reveal any test item related variations. Similarly, quantitative evaluation of corpora luteal count in
C1A females of control and high dose group did not reveal any test item related variations.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Splenic Lymphocyte Sub-populations statistically significant increase in mean T cytotoxic (CD8+) cells populations in group G4-C1A males; increase in mean T cytotoxic (CD8+) cells populations in group G3-C1A females; decrease in T "helper" (CD4+) cells populations in group G3-C1A females were noted when compared with vehicle control group. These differences are considered as incidental and unrelated to treatment as the obtained mean value is within in-house historical control range of same species and strain.

Details on results (F1)

The test item did not produce any systemic toxicity, reproductive toxicity and developmental toxicity at all the tested dose levels (111, 333 and 1000 mg/kg body weight/day)
when administered to F1 generation animals from PND 21 to 95 (for C1A animals) and PND 21 to
102 (for C1B animals) under experimental conditions employed.

Therefore, the no-observed-adverse-effect-level (NOAEL) of the test item is considered as 1000 mg/kg body weight/day for systemic, reproduction and developmental toxicity end points.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

1000 mg/kg body weight/day for systemic, reproduction and developmental toxicity end points

Generation:

other: cohort 1A and cohort 1B

Effect level:

>= 1 000

Based on:

test mat.

Remarks:

the test item did not produce any systemic toxicity, reproductive toxicity and developmental toxicity at all the tested dose levels (111, 333 and 1000 mg/kg body weight/day) when administered to parental generation animals for a period o

Sex:

male/female

Basis for effect level:

viability

sexual maturation

clinical signs

mortality

body weight and weight gain

food efficiency

haematology

clinical biochemistry

urinalysis

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

other: Splenic Lymphocyte Subpopulation Analysis

Remarks on result:

other: the test item did not produce any systemic toxicity, reproductive toxicity and developmental toxicity at all the tested dose levels (111, 333 and 1000 mg/kg body weight/day) when administered to parental generation animals for a period o

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

In conclusion, the test item did not produce any systemic toxicity, reproductive toxicity and developmental toxicity at all the tested dose levels (111, 333 and 1000 mg/kg body weight/day) when administered to parental generation animals for a period of 10 weeks during pre-mating period (both P males and females), 2 weeks during mating period (both P males and P females), throughout gestation and lactation periods up to weaning of F1 animals (P females) as well as when administered to F1 generation animals from PND 21 to 95 (for C1A animals) and PND 21 to 102 (for C1B animals) under experimental conditions employed.
Therefore, the no-observed-adverse-effect-level (NOAEL) of the test item is considered as 1000 mg/kg body weight/day for systemic, reproduction and developmental toxicity end points.

Executive summary:

The objective of this Extended One-Generation Reproductive Toxicity (EOGRT) Study in Sprague Dawley Rats was to evaluate the effects of test item as a result of pre and postnatal exposure on development as well as a thorough evaluation of systemic toxicity in pregnant and lactating females, young and adult offspring.
The study was conducted to serve as a test for reproductive endpoints that required the interaction of males with females, females with conceptus, females with offspring and the F1 generation before and after sexual maturity. This study was also conducted to evaluate the spermatogenesis for males and oestrous cycles, follicle counts/oocyte maturation and ovarian integrity (histopathology) for females.

This EOGRT study was also conducted to evaluate gonadal function, the oestrus cycle, epididymal sperm maturation, mating behavior, conception, pregnancy, parturition and lactation. Furthermore, the present EOGRT study was conducted to provide detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, and physical and functional development until adulthood, and was expected to identify specific target organs in the offspring. In addition, this study provided and/or confirmed information about the effects of Hostaperm Pink E on the integrity and performance of the adult male and female reproductive systems.

A total of 200 (100 males + 100 females) Sprague Dawley rats were selected for parental (P) generation and distributed to four P generation groups. Each P generation group (G1, G2, G3 and G4) consisted of 25 males and 25 females. A total of 160 males and 160 females were selected on the day of weaning (PND21) and randomly assigned to two cohorts [Cohort 1A (80 males + 80 females) and Cohort 1B (80 males
+ 80 females)]. Each cohort consisted of four groups and consisted of 20 males and
20 females per group. The animals in G1 (P generation) and G1-C1A/G1-C1B
(F1 generation) groups were administered with vehicle [0.5% w/v Carboxymethyl Cellulose], the animals in G2 (P generation) and G2-C1A/G2-C1B (F1 generation),
G3 (P generation) and G3-C1A/G3-C1B (F1 generation), and G4 (P generation) and G4-C1A/G4-C1B (F1 generation) groups were administered with Hostaperm Pink E at the dose levels of 111, 333 and 1000 mg/kg body weight/day respectively. The vehicle and test item formulations were administered orally by gavage at the dose volume of 10 mL/kg body weight.

The stability and homogeneity of test item in dose formulations were established before initiation of the treatment. The test item formulations were stable at room temperature for 6 hours followed by 48 hours in 0.5% w/v Carboxymethyl cellulose within the mean percent recovery range of 85 to 115 at the concentrations of 10.0 mg/mL and
100.0 mg/mL. Homogeneity and dose formulation analysis for dose concentration verification was performed during week 1, 8, 16 and 26 of the treatment periods and the mean results were within the range of 85 to 115% recovery to the nominal concentration and the relative standard deviation (% RSD) was less than 10%.

All the P generation animals were observed once daily for clinical signs, twice daily for mortality/morbidity and weekly once for detailed clinical examination. The body weights (throughout the experimental period) and feed consumption (throughout the experimental period, except during cohabitation period) was recorded once weekly for all the P animals. The assessment for haematology, clinical chemistry, urinalysis, and thyroid hormone (Thyroxine Hormone and Thyroid Stimulating Hormone) levels was conducted for randomly selected males and females from each P generation group at termination. Sperm parameters (motility, morphology, and sperm concentration/daily sperm production) were estimated for all P males. Oestrus cyclicity evaluation was determined during pre-mating and cohabitation period for all P females.
The body weights and feed consumption were recorded during gestation (Gestation Day 0, 7, 14 and 20) and during lactation (Lactation Day 1, 4, 7, 14 and 21) for all the
P females. The gross pathology and organ weighing was performed on the day of termination for all the P animals. Histopathological examination was conducted on all the tissues collected from the P generation vehicle control and high dose group animals. All the P males and females were evaluated for reproductive performance or indices such as, mating and fertility index (for P males and females) and pre-coital interval, gestation length, fecundity index, gestation index, post-implantation loss and postnatal loss (for females). All P females were observed for birth parameters (number of live/dead pups born, litter size, sex ratio, and live birth index per litter) and for litter observations (number of live/dead pups during lactation period, sex ratio and pup survival index per litter).

All the P males and females did not reveal any test item related changes in systemic and reproductive endpoints in any of the tested dose groups. However, in test-item administered group animals of both the sexes, the faecal matter was noted with ‘pink’ in colour which could be attributed to physical appearance of the test item (pink in colour). There were no test item-related histopathological findings noted in high dose parental animals of both sexes. Some of the noted microscopic findings observed in this study such as ultimobranchial cyst(s) in thyroid gland, epithelial cyst(s)in thymus and all other findings were considered incidental as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rats of this strain and age.

The F1 generation pups were observed once daily for external examinations and
twice daily for mortalities till termination, weighed individually on postnatal day (PND) 1, 4, 7, 14 and 21, observed for occurrences of postnatal developmental landmarks, observed for responses towards to sensory reflexes during postnatal period, measured for anogenital distance on PND 4, observed for retention of any nipples/areolae in male pups on PND 13, observed for gross pathological observations at termination. The assessment for serum Thyroxine (T4) levels was conducted for PND 4 pups and assessment for serum Thyroxine (T4) and Thyroid Stimulating Hormone levels was conducted for PND 21 pups.

There were no test item related changes in growth parameters, postnatal developmental landmarks, sensory reflexes, thyroid hormone levels in F1 generation pups. No gross pathological changes noted in of the any of the F1 pups of both sexes from all the tested dose group litters.

All the Cohort 1A (F1 generation) animals were observed once daily for clinical signs, twice daily for mortality/morbidity and weekly once for detailed clinical examination. The body weights and feed consumption were recorded once weekly. All the
C1A animals were evaluated for occurrence of sexual maturation (for balanopreputial separation in C1A males and for vaginal patency in C1A females). All C1A females were evaluated for mean occurrence of first cornified cells and time interval between vaginal patency and occurrence of first cornified cells. All C1A females were evaluated for oestrus cyclicity from PND 75 to until sacrifice. The assessment for haematology, clinical chemistry, urinalysis and thyroid hormone (Thyroxine Hormone and Thyroid Stimulating Hormone) levels was conducted for randomly selected males and females from each C1A group at termination. Sperm parameters (motility, morphology and daily sperm production) were estimated for all C1A males. The gross pathology and organ weighing were performed on the day of termination for all the C1A animals. Histopathological examination was conducted on all the tissues collected from the
C1A vehicle control and high dose group animals. The flow cytometric analysis of splenic samples for assessment of splenic lymphocyte sub-populations of T "helper" (CD4+) cells, T cytotoxic (CD8+) cells, natural killer (NK) cells and B lymphocytes was conducted for randomly selected males and females from each C1A group.

All the C1A males and females did not reveal any test item related changes in systemic and reproductive endpoints in any of the tested dose groups when compared with vehicle control group, except slight reduction in mean body weight and percent change in mean body weight gain in all the tested dose group males. There were no changes noted in sub-populations of splenic lymphocytes such as, T "helper" (CD4+) cells,
T cytotoxic (CD8+) cells, natural killer (NK) cells and B lymphocytes at any of the tested dose groups when compared with vehicle control group. There were no test item-related histopathological findings noted in high dose C1A animals of both sexes. Some of the noted microscopic findings observed in this study such as ultimobranchial cyst(s) in thyroid gland, epithelial cyst(s)in thymus and all other findings were considered incidental as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rats.

All the Cohort 1B (F1 generation) animals were observed once daily for clinical signs, twice daily for mortality/morbidity and weekly once for detailed clinical examination. The body weights and feed consumption were recorded once weekly. All the
C1B animals were evaluated for occurrence of sexual maturation (for balano-preputial separation in C1B males and for vaginal patency in C1B females). The gross pathology and organ weighing were performed on the day of termination for all the C1B animals.

All the C1B males and females did not reveal any test item related changes in systemic and reproductive endpoints, except reduction in mean body weight and percent change in mean body weight gain in all the tested dose group of both sexes.