Bisbenzimidazo[2,1-b:1',2'-j]benzo[lmn][3,8]phenanthroline-6,9-dione

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

HsdHan: WIST rats

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Department of Safety Assessment, Advinus Therapeutics Limited, Bengaluru 560 058, India
- Age at study initiation: 14-15 weeks

- Weight at study initiation: Males: 368 to 477 g, Females: 224 to 268 g

G1 (m/f): 424.69 ± 36.43 / 249.78 ± 12.87 g
G2 (m/f): 426.05 ± 35.33 / 256.37 ± 07.83 g
G3 (m/f): 426.61 ± 30.16 / 254.46 ± 09.92 g
G4 (m/f): 426.05 ± 28.00 / 255.30 ± 09.74 g

G1R (m/f): 407.82 ± 21.48 / 249.51 ± 05.16 g
G4R (m/f): 408.20 ± 15.55 / 247.96 ± 11.19 g

- Fasting period before study: non
- Housing: During mating, one male and one female were housed in standard polysulfone cages with stainless steel top grill. having facilities for pelleted food. Mated pairs were separated. Males were housed with their former cage mates while females were housed individually in polysulfone cages. During the study period, animals were housed in a single experimental room of T4 Barrier Area.

- Diet (e.g. ad libitum): Teklad Certified (2014C) Global 14 % Protein Rodent Maintenance Diet - Pellet manufactured by Harlan Laboratories (Envigo), P.O. Box 44220, Madison, Wi 53744-4420 was provided ad libitum to the animals.
- Water (e.g. ad libitum): Deep bore-well water passed through activated charcoal filter and exposed to UV rays was provided ad libitum to rats in polycarbonate bottles with stainless steel sipper tubes.
- Acclimation period: five days before start

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23
- Humidity (%): 65-67
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % (w/v) Na CMC (medium viscosity) in Milli-Q® water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared daily prior to start of the treatment or prepared on need basis and used within the stability period.
Homogeneity of the dose formulations during sampling/gavaging was maintained by constant stirring using a magnetic stirrer.

VEHICLE:
- Justification for use and choice of vehicle (if other than water): Based on the solubility and suspensibility test.
- Concentration in vehicle: 0 / 10 / 30 / 100 mg//mL
- Amount of vehicle (if gavage): 10 mL/kg Bwt/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For homogeneity and concentration analysis, samples were sampled in duplicate on Day 1 and once during Week 4 of the treatment and analysed. For each set, duplicate samples were drawn from top, middle and bottom layers of each preparation and in case of control, duplicate samples from only middle layer was drawn.

The analysis was done as per the method validated. One set of samples were analyzed for concentration analysis and other set (second set) of samples were stored at ambient conditions for re-analysis purpose as a backup.

Formulations were considered acceptable as mean results are within ± 15% of the theoretical concentration and the relative standard deviation (RSD) was less than 10 %.

Duration of treatment / exposure:

Males: The dose formulation was administered at least for 28 days to the rats of the specific groups once daily at approximately the same time each day (varying by ± 3 hours) for 2 weeks prior to mating and the treatment was continued during the mating period and until sacrifice.

Females: The dose formulations were administered to the specific group of rats once daily at approximately the same time each day (varying by ± 3 hours) throughout the treatment period. Treatment was done 2 weeks prior to the mating period and continued through mating, pregnancy and up to LD 13, after which, pups were sacrificed on LD 13 and parental females (dams) were sacrificed on LD 14 after overnight fasting (water allowed).

The dose formulations were administered to the high dose recovery group of rats once daily at approximately the same time each day (varying by ± 3 hours).
The volume of dose administered was 10 mL/kg Bwt/day throughout the study. The dose volume was adjusted based on the most recent body weight of individual rat.

Similarly, vehicle was administered to rats in the vehicle control and vehicle control recovery groups at 10 mL/kg Bwt/day.

The vehicle and the test item was not administered for vehicle control recovery and high dose recovery groups, respectively for 14 days following the treatment period.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

G1 Vehicle Control

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

G2 Low dose

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

G3 Mid dose

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

G4 High dose

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

G1R Vehicle Control recovery

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

G4R High dose recovery

No. of animals per sex per dose:

10 Males and 10 Females for each main groups. 5 Males and 5 Females for each recovery group.

Control animals:

yes

Details on study design:

- Dose selection rationale: Dose levels were selected in order to induce toxic effects at the highest dose, any dosage related response and no adverse effects at the mid and lowest dose.
- Grouping: Grouping was done by the method of body weight stratification and distribution.
- Rationale for selecting satellite groups: Satellit groups were were used in the control and the top dose group for observation of reversibility, persistence or delayed occurrence of systemic toxic effects.
- Post-exposure recovery period in satellite groups: 14 day no treatment period

Positive control:

non

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
All rats were observed for morbidity and mortalities twice daily i.e., once in the morning and once in the afternoon. As there were no clinical signs, the observation for morbidity and mortality was carried out once in the morning during holidays. All rats were observed for clinical signs once daily during the treatment and recovery periods.

DETAILED CLINICAL OBSERVATIONS:
Detailed clinical examination was done prior to the treatment on Day 1 and at weekly intervals thereafter during treatment and recovery periods.
During detailed clinical examination, all rats were observed for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling or bizarre behaviour like self-mutilation and walking backwards.
On the days of detailed clinical examination, general clinical signs were not performed except on treatment Day 1

BODY WEIGHT:
i) Individual body weights of males were recorded initially and at weekly intervals thereafter. Individual body weights of females were recorded initially and at weekly intervals thereafter till cohabitation (till mating confirmation) with males.
ii) All dams were weighed on GD 0, 7, 14 and 20 and on LD 0, 4 and 13.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Cagewise food consumption was calculated by using the food consumed at weekly interval per cage and dividing by the number of rats per cage and the number of days in the intervening period to determine the food intake/rat/day.
Food consumption was not measured during the cohabitation period.
Food consumption of pregnant dams was recorded on GD 7, 14 and 20 and on Day 4 and 13 of lactation period.

ESTROUS CYCLICITY:
Vaginal smear was examined and the stage of oestrous cycle was recorded daily for two weeks before start of the treatment to select for the study females with regular 4-5 days cyclicity. The vaginal smear was also examined daily from the beginning of the treatment period until evidence of mating period to determine the Day 0 of pregnancy/treatment-related effects on mating or pre-coital time. The time interval (in days) from the diestrus of an oestrous cycle to the next diestrus was considered as the oestrous cycle length of an animal. The overall pattern of each female was characterized as regular cycling (having recurring 4–5 day cycles) and irregularly cycling (having cycles with a period of diestrus longer than 3 days or a period of estrus more or less than 2 days). Incomplete cycles (having prolonged periods of either estrus or diestrus) were not included in calculating the mean cycle length.
Vaginal smears were also examined on the day of necropsy to determine the stage of the oestrous cycle.

Sacrifice and pathology:

The animals were subjected to detailed necropsy at sacrifice after overnight fasting (water allowed) and study plan specified tissues were collected. Tissues/organs collected from randomly selected 5 males and 5 females in the control and high dose groups (including reproductive organs) were examined microscopically for histopathological changes. Histopathological examination of testes also included a qualitative assessment of stages of spermatogenesis and interstitial testicular cell structure. The thyroid gland was collected from all pups on LD 13 and examined microscopically. All gross lesions were examined from all the groups.

Statistics:

ProvantisTM: Parameters of laboratory Investigations - Haematology (Coagulation tests PT and APTT data was entered retrospectively in ProvantisTM) and Clinical Chemistry data was analysed using Provantis built-in statistical tests.

The statistical analysis of the experimental data was carried out using the validated package in Excel and also using licensed copies of SYSTAT Statistical package ver.12.0. All quantitative variables like neurological observations (neuromuscular observation/body temperature/body weights), body weight, food consumption, oestrous cycle length, hormone levels, anogenital distance, organ weights and organ weight ratios were tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene’s test) within the group before performing a one-factor analysis of variance (ANOVA) modeling by treatment groups. Non-optimal (non-normal or heteroschedastic) data was transformed, before ANOVA was performed. Comparison of means between treatment groups and cont ol group was done using Dunnett’s test when the overall treatment, ‘F’ test is found to be significant.

Post-implantation loss (%), number of nipples/areolae in male pups, no. of implantations, pre-coital interval, mean litter size, sex ratio and gestation length (Days) was analysed after suitable transformation (√ x + ½) of the data. Oneway ANOVA was carried out for the transformed data. Dunnett’s pair-wise comparison of the treated means with the control mean was done when the group differences are found significant.

Z test was performed for testing the differences in proportions for mating and fertility indices.

Clinical signs:

no effects observed

Description (incidence and severity):

The orange coloured fecal pellets observed were due to the physical appearance of the test item, not related to the toxicity.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

eye examination: normal

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopically, pigmented macrophages were observed in lungs at 1000 mg/kg Bwt/day in both sexes and considered as test item-related non-adverse finding. This change showed complete recovery at the end of recovery period.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: highest dose applied

Key result

Critical effects observed:

no

Conclusions:

Daily oral (gavage) administration of Pigment Orange 43 to male and female Wistar rats at the dose levels of 100, 300 and 1000 mg/kg Bwt/day for 2 weeks prior to mating, during mating, and 2 weeks post mating (males) or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery did not induce any adverse effects.

The No Observed Adverse Effect Level (NOAEL) of Pigment Orange 43 for general toxicity (repeated dose toxicity) in males and females is considered to be 1000 mg/kg Bwt/day.

Therefore, the test item has not to be classified as subacutely toxic (STOT RD) by the oral route according to Regulation (EC) No 1272/2008.

Executive summary:

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar Rats was to determine the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. Further, this study was used to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition.

The test item Hostaperm-Orange GR was suspended in vehicle {0.5 % (w/v) Na CMC in Milli-Q® water} and administered by oral gavage at the dose levels of 100, 300 and 1000 mg/kg Bwt/day to low (G2), mid (G3) and high dose (G4)/ high dose recovery (G4R) group rats, respectively. A concurrent vehicle control (G1) and a vehicle control recovery group (G1R) of rats received the vehicle alone. The dose volume administered was 10 mL/kg Bwt/day. The main groups i.e., G1 to G4 consisted of 10 male and 10 female rats per group and recovery groups consisted of 5 male and 5 female rats per group. The dose formulations were administered once daily to specific group of rats prior to mating, during mating and post-mating periods (for males), during pregnancy and up to Lactation Day (LD) 13 (for females). In the control and high dose recovery groups, the treatment period was followed by a 14 day no treatment (recovery) period. The recovery period of the study was started from the first scheduled kill of dams. Animals in the recovery groups were not mated and consequently not used for assessment of reproduction/developmental toxicity.

The identity of Hostaperm-Orange GR was provided by the study Sponsor by a Certificate of Analysis (CoA). The stability and homogeneity of test item in the vehicle was carried out under Advinus Study No.G11233. Based on the results, the test item was found to be stable for up to 8 days at 1 and 100 mg/mL concentrations, when stored at room temperature. The dose formulations were analysed for active ingredient (a.i.) concentration on Day 1 and during Week 4 of the treatment period. The results indicated that the analysed concentrations were within ± 15 % of variations from the nominal concentrations.

All rats were observed for clinical signs once daily. Body weight was recorded at the beginning of the treatment, weekly thereafter and at termination. Food consumption was recorded weekly except during the cohabitation period. Female rats were also weighed on Gestation Day (GD) 0, 7, 14 and 20 and on Lactation Day (LD) 0, 4 and 13. Food consumption was recorded on GD 7, 14 and 20 and on LD 4 and 13. The number, weight, survival and mortality of pups were observed during the lactation period. The ano-genital distance of each pup was measured on LD 0. All the survived male pups were examined for the appearance nipples/areolae on post-natal day (PND) 13. The functional observation battery was done shortly before sacrifice for randomly selected 5 males and 5 females from each group. For recovery groups, functional observation battery was done towards the end of recovery period. Laboratory investigations such as hematology, coagulation, clinical chemistry and urinalysis were performed in randomly selected 5 males and 5 females from each group at the end of the pre-mating period for main groups and at the end of recovery period from all animals of recovery groups. Thyroxine 4 (T4) and Thyroid Stimulating Hormone (TSH) analysis were performed in all males at termination, all dams at termination on LD 13 and two pups per litter on LD 4 and 13. The animals were subjected to detailed necropsy at sacrifice after overnight fasting (water allowed) and study plan specified tissues were collected. Tissues/organs collected from randomly selected 5 males and 5 females in the control and high dose groups (including reproductive organs) were examined microscopically for histopathological changes. Histopathological examination of testes also included a qualitative assessment of stages of spermatogenesis and interstitial testicular cell structure. The thyroid gland was collected from all pups on LD 13 and examined microscopically. All gross lesions were examined from all the groups.

Under the experimental conditions employed, the following results were obtained:

• There were no treatment-related clinical signs or mortality observed at any of the doses tested.

• No treatment-related neurological abnormalities/dysfunctions were observed at all the doses tested.

• The body weights and food consumption were unaffected by the treatment at all the doses tested.

• The maternal body weight and food consumption during gestation and lactation periods were unaffected by the treatment at all the doses.

• Treatment had no effect on pre-coital time or gestation length, oestrous cycle length at all the tested doses

• No treatment-related changes were observed in the fertility indices of sires and dams at all the doses tested.

• The survival indices were not altered by the treatment at all the doses tested.

• There were no treatment-related effects on the uterine/implantation data, mean litter size and mean viable litter size.

• There were no external abnormalities in live or dead pups in any of the groups.

• No treatment-related changes in the ano-genital distance and ano-genital ratio were observed at any of the doses tested when compared to the vehicle control group.

• The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested.

• The test item administration did not reveal any treatment related changes in the hematology, coagulation and clinical chemistry parameters of both males and females.

• There were no test item related changes in the terminal body weights, organ weights and organs weight ratios in both males and females.

• Gross pathological examination of pups on LD 13 did not reveal any gross changes.

• There were no test item related gross and microscopic changes in both males and females. Microscopically in lungs, pigmented macrophages were observed at 1000 mg/kg Bwt/day in both sexes and considered as test item related non-adverse finding. This change showed complete recovery at the end of recovery period.

• The thyroid stimulating hormone (TSH) and thyroxine (T4) levels in adult rats and pups remained unaffected by test item-administration.

• No test item related changes were observed in organ weights, gross and histopathology of thyroid gland of parental rats and pups.

The No Observed Adverse Effect Level (NOAEL) of Hostaperm-Orange GR for general toxicity (repeated dose toxicity) in males and females is considered to be 1000 mg/kg Bwt/day.

Therefore, the test item has not to be classified as subacutely toxic (STOT RD) by the oral route according to Regulation (EC) No 1272/2008.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Bisbenzimidazo[2,1-b:2',1'-i]benzo[lmn][3,8]phenanthroline-8,17-dione

EC Number:

224-597-4

EC Name:

Bisbenzimidazo[2,1-b:2',1'-i]benzo[lmn][3,8]phenanthroline-8,17-dione

Cas Number:

4424-06-0

Molecular formula:

C26H12N4O2

IUPAC Name:

bisbenzimidazo[2,1-b:2',1'-i]benzo[lmn][3,8]phenanthroline-8,17-dione

Test material form:

solid: nanoform

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS

- Source: Department of Safety Assessment, Advinus Therapeutics Limited, Bengaluru 560 058, India

- Age at study initiation: 14-15 weeks

- Weight at study initiation: Males: 368 to 477 g, Females: 224 to 268 g

G1 (m/f): 424.69 ± 36.43 / 249.78 ± 12.87 g
G2 (m/f): 426.05 ± 35.33 / 256.37 ± 07.83 g
G3 (m/f): 426.61 ± 30.16 / 254.46 ± 09.92 g
G4 (m/f): 426.05 ± 28.00 / 255.30 ± 09.74 g

G1R (m/f): 407.82 ± 21.48 / 249.51 ± 05.16 g
G4R (m/f): 408.20 ± 15.55 / 247.96 ± 11.19 g

- Fasting period before study: non

- Housing: During mating, one male and one female were housed in standard polysulfone cages with stainless steel top grill. having facilities for pelleted food. Mated pairs were separated. Males were housed with their former cage mates while females were housed individually in polysulfone cages. During the study period, animals were housed in a single experimental room of T4 Barrier Area.

- Diet (e.g. ad libitum): Teklad Certified (2014C) Global 14 % Protein Rodent Maintenance Diet - Pellet manufactured by Harlan Laboratories (Envigo), P.O. Box 44220, Madison, Wi 53744-4420 was provided

- Water (e.g. ad libitum): Deep bore-well water passed through activated charcoal filter and exposed to UV rays was provided ad libitum to rats in polycarbonate bottles with stainless steel sipper tubes.

- Acclimation period: five days before start

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % (w/v) Na CMC in Milli-Q® water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared daily prior to start of the treatment or prepared on need basis and used within the stability period.
Homogeneity of the dose formulations during sampling/gavaging was maintained by constant stirring using a magnetic stirrer.

VEHICLE:
- Justification for use and choice of vehicle (if other than water): Based on the solubility and suspensibility test.
- Concentration in vehicle: 0 / 10 / 30 / 100 mg//mL
- Amount of vehicle (if gavage): 10 mL/kg Bwt/day

Details on mating procedure:

One female was placed with one male from the same group in a 1:1 ratio. Cohabitation was continued until evidence of sperms in the vaginal smear and /or vaginal plug. Subsequently, pregnant females were housed individually until LD 14.

The day of confirmed mating was designated as Gestation Day 0 (GD 0). The pre-coital time was calculated for each female.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For homogeneity and concentration analysis, samples were sampled in duplicate on Day 1 and once duri ng Week 4 of the treatment and analysed. For each set, duplicate samples were drawn from top, middle and bottom layers of each preparation and in case of control, duplicate samples from only middle layer was drawn.

The analysis was done as per the method validated. One set of samples were analyzed for concentration analysis and other set (second set) of samples were stored at ambient conditions for re-analysis purpose as a backup.

Formulations were considered acceptable as mean results are within ± 15% of the theoretical concentration and the relative standard deviation (RSD) was less than 10 %.

Duration of treatment / exposure:

Males: The dose formulation was administered at least for 28 days to the rats of the specific groups once daily at approximately the same time each day (varying by ± 3 hours) for 2 weeks prior to mating and the treatment was continued during the mating period and until sacrifice.

Females: The dose formulations were administered to the specific group of rats once daily at approximately the same time each day (varying by ± 3 hours) throughout the treatment period. Treatment was done 2 weeks prior to the mating period and continued through mating, pregnancy and up to LD 13, after which, pups were sacrificed on LD 13 and parental females (dams) were sacrificed on LD 14 after overnight fasting (water allowed).

The dose formulations were administered to the high dose recovery group of rats once daily at approximately the same time each day (varying by ± 3 hours).
The volume of dose administered was 10 mL/kg Bwt/day throughout the study. The dose volume was adjusted based on the most recent body weight of individual rat.

Similarly, vehicle was administered to rats in the vehicle control and vehicle control recovery groups at 10 mL/kg Bwt/day.

The vehicle and the test item was not administered for vehicle control recovery and high dose recovery groups, respectively for 14 days following the treatment period.

Frequency of treatment:

Daily

Details on study schedule:

Study initiation date: 08 March 2016

Experimental starting date: 10 March 2016

Acclimatization: Start: 10 March 2016; End: 14 March 2016

Pre-treatment period: Start: 15 March 2016; End: 28 March 2016

Treatment: Start: 29 March 2016; End: 11 June 2016

Recovery period: 20 May 2016 to 02 June 2016

Experiment completion: 7 July 2016

Submission of Draft report: 29 August 2016

Study completion: 21 November 2016

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

G1 Vehicle Control

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

G2 Low dose

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

G3 Mid dose

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

G4 High dose

Doses / concentrations 5

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

G1R Vehicle Control recovery

Doses / concentrations 6

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

G4R High dose recovery

No. of animals per sex per dose:

10 Males and 10 Females for each main groups. 5 Males and 5 Females for each recovery group.

Control animals:

yes

Details on study design:

- Dose selection rationale: Dose levels were selected in order to induce toxic effects at the highest dose, any dosage related response and no adverse effects at the mid and lowest dose.

- Grouping: Grouping was done by the method of body weight stratification and distribution.

- Rationale for selecting satellite groups: Satellit groups were were used in the control and the top dose group for observation of reversibility, persistence or delayed occurrence of systemic toxic effects.

- Post-exposure recovery period in satellite groups: 14 day no treatment period

Positive control:

non

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS:
All rats were observed for morbidity and mortalities twice daily i.e., once in the morning and once in the afternoon. As there were no clinical signs, the observation for morbidity and mortality was carried out once in the morning during holidays. All rats were observed for clinical signs once daily during the treatment and recovery periods.

DETAILED CLINICAL OBSERVATIONS:
Detailed clinical examination was done prior to the treatment on Day 1 and at weekly intervals thereafter during treatment and recovery periods.
During detailed clinical examination, all rats were observed for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling or bizarre behaviour like self-mutilation and walking backwards.
On the days of detailed clinical examination, general clinical signs were not performed except on treatment Day 1

BODY WEIGHT:
i) Individual body weights of males were recorded initially and at weekly intervals thereafter. Individual body weights of females were recorded initially and at weekly intervals thereafter till cohabitation (till mating confirmation) with males.
ii) All dams were weighed on GD 0, 7, 14 and 20 and on LD 0, 4 and 13.


FOOD CONSUMPTION AND COMPOUND INTAKE:
Cagewise food consumption was calculated by using the food consumed at weekly interval per cage and dividing by the number of rats per cage and the number of days in the intervening period to determine the food intake/rat/day.
Food consumption was not measured during the cohabitation period.
Food consumption of pregnant dams was recorded on GD 7, 14 and 20 and on Day 4 and 13 of lactation period.

Oestrous cyclicity (parental animals):

Vaginal smear was examined and the stage of oestrous cycle was recorded daily for two weeks before start of the treatment to select for the study females with regular 4-5 days cyclicity. The vaginal smear was also examined daily from the beginning of the treatment period until evidence of mating period to determine the Day 0 of pregnancy/treatment-related effects on mating or pre-coital time. The time interval (in days) from the diestrus of an oestrous cycle to the next diestrus was considered as the oestrous cycle length of an animal. The overall pattern of each female was characterized as regular cycling (having recurring 4–5 day cycles) and irregularly cycling (having cycles with a period of diestrus longer than 3 days or a period of estrus more or less than 2 days). Incomplete cycles (having prolonged periods of either estrus or diestrus) were not included in calculating the mean cycle length.
Vaginal smears were also examined on the day of necropsy to determine the stage of the oestrous cycle.

Sperm parameters (parental animals):

Histopathological examination of testes included a qualitative assessment of stages of spermatogenesis and interstitial testicular cell structure.

Litter observations:

a. At birth, all the pups (both dead and alive) in a litter from each dam were observed for any external deformities and recorded.

b. The number of pups born (litter size), sex and individual pup body weight of male and female pups on LD 0, 4 and 13 were recorded.

c. On LD 4, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four pups per sex per litter. Blood samples were collected from two of the surplus pups, pooled, and used for determination of serum T4 levels. In case of insufficient pups in a litter to have two surplus pups, two of the pups that were typically retained was used for blood collection for serum T4 assessments. When litter size dropped below the culling target (8 pups/litter), preference was given to remove two female pups for PND 4 blood samples in order to retain more male pups for observation of nipple retention on PND 13.

d. After standardization, the individual pup body weight was recorded on Day 13 of lactation.

e. The ano-genital distance (AGD) of each pup was measured on PND 0 and pup body weight was recorded. Ano-genital distance ratio was calculated by dividing the ano-genital distance from cube root of body weight.

f. The number of nipples/areolae in male pups were counted on PND 13.

g. All the dead and sacrificed pups were examined for malformations and subjected to gross pathological examination.

h. Fertility indices for dams, sires as well as the pup survival index till LD 4 were calculated.

Postmortem examinations (parental animals):

All adult animals were subjected to detailed necropsy at sacrifice after overnight fasting (water allowed) and study plan specified tissues were collected. Tissues/organs collected from randomly selected 5 males and 5 females in the control and high dose groups (including reproductive organs) were examined microscopically for histopathological changes. Histopathological examination of testes also included a qualitative assessment of stages of spermatogenesis and interstitial testicular cell structure. The thyroid gland was collected from all pups on LD 13 and examined microscopically. All gross lesions were examined from all the groups.

Postmortem examinations (offspring):

All pups LD 13; including dead pups were examined macroscopically for any structural abnormalities/pathological changes and findings were recorded.

Statistics:

ProvantisTM: Parameters of laboratory Investigations - Haematology (Coagulation tests PT and APTT data was entered retrospectively in ProvantisTM) and Clinical Chemistry data was analysed using Provantis built-in statistical tests.

The statistical analysis of the experimental data was carried out using the validated package in Excel and also using licensed copies of SYSTAT Statistical package ver.12.0. All quantitative variables like neurological observations (neuromuscular observation/body temperature/body weights), body weight, food consumption, oestrous cycle length, hormone levels, ano-genital distance, organ weights and organ weight ratios were tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene’s test) within the group before performing a one-factor analysis of variance (ANOVA) modeling by treatment groups. Non-optimal (non-normal or heteroschedastic) data was transformed, before ANOVA was performed. Comparison of means between treatment groups and control group was done using Dunnett’s test when the overall treatment, ‘F’ test is found to be significant.

Post-implantation loss (%), number of nipples/areolae in male pups, no. of implantations, pre-coital interval, mean litter size, sex ratio and gestation length (Days) was analysed after suitable transformation (√ x + ½) of the data. One-way ANOVA was carried out for the transformed data. Dunnett’s pair-wise comparison of the treated means with the control mean was done when the group differences are found significant.

Z test was performed for testing the differences in proportions for mating and fertility indices.

Reproductive indices:

a. Male mating index (%)

Number of males with evidence of mating
= ------------------------------------------------------------------- x 100
Number of males cohabited


b. Male fertility index (%)

Number of males siring a litter
= ------------------------------------------------- x 100
Number of males cohabited


c. Female mating index (%)

Number of females mated
= ------------------------------------------------ x 100
Number of females cohabited


d. Female fertility index (%)

Number of pregnant females (confirmed at necropsy)
= ------------------------------------------------------------------------------ x 100
Number of females used for mating


e. Mean number of implantations/group

Total number of implantations
= --------------------------------------------------------
Total number of pregnant animals


f. Post implantation loss (%)

Number of implantations - Number of live pups
= ------------------------------------------------------------------------ x 100
Number of implantations

Offspring viability indices:

a. Mean litter size per group

Total Number of pups
= -------------------------------------------------
Total Number of littered animals


b. Mean viable litter size

No. of viable pups on Day 1
= -------------------------------------------------------
No. of females littered


c. Live birth index (%)

No. of viable pups born (at first observation)
= --------------------------------------------------------------------------- x 100
Total no. of pups born (at first observation)



d. Day 4 survival index (%)

Number of viable pups on lactation Day 4
= -------------------------------------------------------------------- x 100
Number of viable pups born


e. Sex Ratio (%)

No. of male pups born
= ---------------------------------------------- x 100
Total No. of pups born


f. Ano-genital Distance Ratio (mm/g1/3 )

Ano-genital distance
= ----------------------------------------------------
Cube root of body weight

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

The orange coloured fecal pellets observed were due to the physical appearance of the test item, not related to the toxicity.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

eye examination: normal

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopically in lungs, pigmented macrophages were observed at 1000 mg/kg Bwt/day in both sexes and considered as test item-related non-adverse finding. This change showed complete recovery at the end of recovery period.

Histopathological findings: neoplastic:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

11. RESULTS AND DISCUSSION

11.1 In-Life Data

11.1.1 Detailed Clinical Examination, Clinical Signs, Morbidity and Mortality
No treatment-related clinical signs/motralities of toxicity were observed at any of the doses tested. The appearance of fecal pellets observed during treatment period was slight, moderate and dark orange coloured at 100, 300 and 1000 mg/kg Bwt/day dose groups, respectively. The orange coloured fecal pellets observed were due to the physical appearance of the test item, not related to the toxicity. The clinical sign of hair thinning with hair regrowth or local alopecia observed considered spontaneous finding and not related to treatment.

11.1.2 Functional Observation Battery
Home cage and Handling observations: No treatment-related abnormalities were observed in all the tested dose groups in both sexes.

Open field observations: No treatment-related abnormalities were observed in any of the doses tested in both sexes.

Sensory observations: No treatment-related abnormalities were observed in any of the groups in both sexes.

Motor Activity: No statistically significant variations were observed in the motor activity of rats when compared to respective vehicle control group in both sexes.

However, in the 1000 mg/kg/Bwt/day dose recovery males, higher stereotypic time at interval 1was observed. This change was considered incidental as there were no changes observed in the home cage or open filed observations.


Neuromuscular observation:
Landing hind limb footsplay: No significant changes were observed at all the tested doses in both sexes.
Grip strength: No significant changes were observed at all the tested doses in both sexes. However, in the 1000 mg/kg/Bwt/day dose recovery males and females, hindlimb grip strength was significantly lower 9.4% and 6.3% respectively and considered incidental as there were no changes in the muscle tone observed.

Physiological observation:
Body temperature: In males, the body temperature was significantly reduced at 300 and 1000 mg/kg Bwt/day with the reduction of 2.5 and 1.6% respectively.
In the 1000 mg/kg/Bwt/day dose recovery females, the body temperature was significantly lower (1.8%) when compared to control recovery group.
The above changes were considered incidental as change were minimal in magnitude.

The body weights recorded at the end of functional tests was comparable with the control group.

11.1.3 Body Weights
Males: The mean body weights were unaffected by the treatment at all the doses tested when compared to vehicle control in both sexes. In the high dose recovery group, the body weights were unaffected both during the treatment and recovery period.

11.1.4 Food Consumption
The food consumption was not altered by the treatment in both the sexes at all the doses tested, when compared to vehicle control.
However an incidence of significantly higher food consumption was observed on Weeks 7 and 9 at 1000 mg/kg/Bwt/day dose in recovery males. This change was considered to be transient and not of biological significance.

11.1.5 Oestrous Cycle Evaluation
Oestrous cyclicity was evaluated for its length and normality by examining the vaginal smears daily for two weeks during treatment period (prior to cohabitation).
The calculated mean oestrous cycle length was 4.07, 4.03, 4.07 and 4.12 days in vehicle control, 100, 300 and 1000 mg/kg Bwt/day doses, respectively. The mean oestrous cycle length in the treated groups was not significantly different from the vehicle control group.

11.1.6 Maternal Body Weights and Food Intake during the Gestation Period
Treatment had no effects on the body weights and food intake during different intervals of the gestation period at all the doses tested.

11.1.7 Maternal Body Weights and Food Intake during the Lactation Period
Treatment had no effects on the body weights and food intake during different intervals of the lactation period at all the doses tested when compared to vehicle control.

11.1.12 Fertility Index
There were no treatment-related effects on the mean pre-coital time and gestation length at all the tested doses.
No treatment-related changes were observed in the fertility indices of sires and dams at all the doses tested.


11.1.13 Uterine/Implantation Data
No treatment-related effects were observed with respect to implantations, and percentage of post implantation loss when compared to control means.

11.1.14 Oestrous Cycle Stage Prior to Sacrifice
Oestrous cycle stage was observed by vaginal smear examination for all female rats prior to sacrifice. All females in all groups showed the different stages of oestrous cycle and comparable to vehicle control group.

11.2 Pathology
Oral administration of Hostaperm-Orange GR in Wistar rats did not affect the hematology, coagulation, clinical chemistry, urinalysis, terminal fasting body weights/organ weights and their ratios, gross and histopathology endpoints in adult rats of both the sexes. Further, the male and female reproductive organs did not reveal any changes.
The pups sacrificed on PND 13 did not reveal any gross abnormality.
The thyroid stimulating hormone (TSH) and thyroxine (T4) levels in adult rats and pups remained unaffected by test item-administration. No test item-related changes were observed in organ weights, gross pathology and histopathology of thyroid gland of parental rats and pups.
Microscopically, pigmented macrophages were observed in lungs at 1000 mg/kg Bwt/day in both sexes and considered as test item-related non-adverse finding. This change showed complete recovery at the end of recovery period.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: highest dose applied

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Description (incidence and severity):

Test item had no treatment-related effects on the number of dead pups at first observation.
No treatment-related changes were observed in the survival data of pups up to LD 4 at all the tested doses.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Thyroid stimulating hormone (TSH) and thyroxine (T4) levels in pups remained unaffected by test item-administration.

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not specified

Details on results (F1)

11.1.8 Number and Body Weight of Pups during the Lactation Period
The mean number and body weight of male and female (and total number) pups per litter were not affected by the treatment at all the doses tested.

11.1.9 Ano-genital Distance (AGD)
No changes attributable to test item were detected in the AGD, body weight and ratio of AGD to the cube root of body weight of either sex.
However, an incidence of significantly higher anogenital distance (mm) was observed in female pups at 100 mg/kg Bwt/day and was considered to be incidental because of the lack of any dose relationship.

11.1.10 Areolae/Nipple Retention in Pups
The male pups did not exhibit areola/nipple retention on PND 13.

11.1.11 Survival Data of Pups
Test item had no treatment-related effects on the number of pregnancies, number littered, mean litter size, and number of dead pups at first observation. No treatment-related changes were observed in the survival data of pups up to LD 4 at all the tested doses.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: highest dose applied

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Daily oral (gavage) administration of CI Pigment Orange 43 to male and female Wistar rats at the dose levels of 100, 300 and 1000 mg/kg Bwt/day for 2 weeks prior to mating, during mating, and 2 weeks post mating (males) or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after deli very did not induce any adverse effects.

The No Observed Adverse Effect Level (NOAEL) of Hostaperm-Orange GR for general toxicity (repeated dose toxicity) in males and females is considered to be 1000 mg/kg Bwt/day.
The No Observed Adverse Effect Level (NOAEL) of Hostaperm-Orange GR for reproductive and developmental toxicity is considered to be 1000 mg/kg Bwt/day.

Therefore, the test item has not to be classified as toxic to reproduction or subacutely toxic (STOT RD) by the oral route according to Regulation (EC) No 1272/2008.

Executive summary:

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar Rats was to determine the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. Further, this study was used to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition.

The test item was suspended in vehicle {0.5 % (w/v) Na CMC in Milli-Q^®water}and administered by oral gavage at the dose levels of 100, 300 and 1000 mg/kg Bwt/day to low (G2), mid (G3) and high dose (G4)/ high dose recovery (G4R) group rats, respectively. A concurrent vehicle control (G1) and a vehicle control recovery group (G1R) of rats received the vehicle alone. The dose volume administered was 10 mL/kg Bwt/day. The main groups i.e., G1 to G4 consisted of 10 male and 10 female rats per group and recovery groups consisted of 5 male and 5 female rats per group. The dose formulations were administered once daily to specific group of rats prior to mating, during mating and post-mating periods (for males), during pregnancy and up to Lactation Day (LD) 13 (for females). In the control and high dose recovery groups, the treatment period was followed by a 14 day no treatment (recovery) period. The recovery period of the study was started from the first scheduled kill of dams. Animals in the recovery groups were not mated and consequently not used for assessment of reproduction/developmental toxicity. 

The identity of the test item was provided by the study Sponsor by a Certificate of Analysis (CoA). The stability and homogeneity of test item in the vehicle was carried out under Advinus Study No.G11233. Based on the results, the test item was found to be stable for up to 8 days at 1 and 100 mg/mL concentrations, when stored at room temperature. The dose formulations were analysed for active ingredient (a.i.) concentration on Day 1 and during Week 4 of the treatment period. The results indicated that the analysed concentrations were within ± 15 % of variations from the nominal concentrations. 

All rats were observed for clinical signs once daily. Body weight was recorded at the beginning of the treatment, weekly thereafter and at termination. Food consumption was recorded weekly except during the cohabitation period. Female rats were also weighed on Gestation Day (GD) 0, 7, 14 and 20 and on Lactation Day (LD) 0, 4 and 13. Food consumption was recorded on GD 7, 14 and 20 and on LD 4 and 13. The number, weight, survival and mortality of pups were observed during the lactation period. The ano-genital distance of each pup was measured on LD 0. All the survived male pups were examined for the appearance nipples/areolae on post-natal day (PND) 13. The functional observation battery was done shortly before sacrifice for randomly selected 5 males and 5 females from each group. For recovery groups, functional observation battery was done towards the end of recovery period. Laboratory investigations such as hematology, coagulation, clinical chemistry and urinalysis were performed in randomly selected 5 males and 5 females from each group at the end of the pre-mating period for main groups and at the end of recovery period from all animals of recovery groups. Thyroxine 4 (T4) and Thyroid Stimulating Hormone (TSH) analysis were performed in all males at termination, all dams at termination on LD 13 and two pups per litter on LD 4 and 13. The animals were subjected to detailed necropsy at sacrifice after overnight fasting (water allowed) and study plan specified tissues were collected.Tissues/organs collected from randomly selected 5 males and 5 females in the control and high dose groups (including reproductive organs) were examined microscopically for histopathological changes. Histopathological examination of testes also included a qualitative assessment of stages of spermatogenesis and interstitial testicular cell structure. The thyroid gland was collected from all pups on LD 13 and examined microscopically. All gross lesions were examined from all the groups.

Under the experimental conditions employed, the following results were obtained:

• There were no treatment-related clinical signs or mortality observed at any of the doses tested.

• No treatment-related neurological abnormalities/dysfunctions were observed at all the doses tested.

• The body weights and food consumption were unaffected by the treatment at all the doses tested.

• The maternal body weight and food consumption during gestation and lactation periods were unaffected by the treatment at all the doses.

• Treatment had no effect on pre-coital time or gestation length, oestrous cycle length at all the tested doses

• No treatment-related changes were observed in the fertility indices of sires and dams at all the doses tested.

• The survival indices were not altered by the treatment at all the doses tested.

• There were no treatment-related effects on the uterine/implantation data, mean litter size and mean viable litter size.

• There were no external abnormalities in live or dead pups in any of the groups.

• No treatment-related changes in the ano-genital distance and ano-genital ratio were observed at any of the doses tested when compared to the vehicle control group.

• The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested.

• The test item administration did not reveal any treatment related changes in the hematology, coagulation and clinical chemistry parameters of both males and females.

• There were no test item related changes in the terminal body weights, organ weights and organs weight ratios in both males and females.

• Gross pathological examination of pups on LD 13 did not reveal any gross changes.

• There were no test item related gross and microscopic changes in both males and females. Microscopically in lungs, pigmented macrophages were observed at 1000 mg/kg Bwt/day in both sexes and considered as test item related non-adverse finding. This change showed complete recovery at the end of recovery period.

• The thyroid stimulating hormone (TSH) and thyroxine (T4) levels in adult rats and pups remained unaffected by test item-administration.

• No test item related changes were observed in organ weights, gross and histopathology of thyroid gland of parental rats and pups.

The No Observed Adverse Effect Level (NOAEL) of CI Pigment Orange 43 for general toxicity (repeated dose toxicity) in males and females is considered to be 1000 mg/kg Bwt/day.

The No Observed Adverse Effect Level (NOAEL) of CI Pigment Orange 43 for reproductive and developmental toxicity is considered to be 1000 mg/kg Bwt/day.

Therefore, the test item has not to be classified as toxic to reproduction or subacutely toxic (STOT RD) by the oral route according to Regulation (EC) No 1272/2008.