[1,3-dihydro-5,6-bis[[(2-hydroxy-1-naphthyl)methylene]amino]-2H-benzimidazol-2-onato(2-)-N5,N6,O5,O6]nickel

Endpoint summary

Description of key information

Oral repeated dose toxicity:

An OECD guideline Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test was performed with the test item (C.I. Pigment Orange 68).

 

The test item was administered to the main groups (10 male and 10 female) by oral gavage at dose levels of 0, 110, 330 and 1000 mg/kg bw/day. Recovery groups (5 male and 5 females) received dose levels of 0 and 1000 mg/kg bw/day. The dose formulations were administered prior to mating, during mating and post-mating periods for males, and prior to mating, during mating, during pregnancy and up to Lactation Day 13 for females. In the control and high dose recovery groups, the treatment period was followed by a 14 day no treatment (recovery) period. Animals in the recovery groups were not mated.

 

No toxicologically significant changes were noted in any of the parameters investigated in this study (such as clinical signs, functional observations, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination). A No Observed Adverse Effect Level (NOAEL) for the test item for repeated dose toxicity of 1000 mg/kg bw/day was established.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

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 (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

HanTac: WH

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Vivo Bio Tech Ltd., Sy. #349/A, Pregnapur-502311, Gajwel Mandal, Medak District, Telangana
- Age at study initiation: 14-15 weeks

- Weight at study initiation: Males: 365 to 425 g, Females: 208 to 246 g

G1 (m/f): 391.33 ± 16.05 / 226.33 ± 09.83 g
G2 (m/f): 392.30 ± 14.45 / 226.51 ± 09.72 g
G3 (m/f): 390.10 ± 13.41 / 225.05 ± 09.66 g
G4 (m/f): 391.73 ± 14.84 / 223.27 ± 11.59 g

G1R (m/f): 379.56 ± 04.69 / 221.71 ± 01.52 g
G4R (m/f):381.16 ± 04.30 / 218.58 ± 03.11 g

- Fasting period before study: no
- Housing:
Pre-mating: Two rats of same sex were housed per cage except for the last animal in the recovery group which was housed singly in sterilized standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles with stainless steel sipper.
Mating and post-mating: During mating, two rats (one male and one female) were housed in standard polysulfone cages with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles. After confirming the presence of sperm in the vaginal smear or vaginal plugs (Day ‘0’ pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually in polysulfone cages. The sterilised nesting material (paper shreds) was provided near-term.
- Enrichment: Polycarbonate Rat huts were provided to the animals as environmental enrichment objects in the cages that either provide shelter or exercising opportunities to minimize animal stress and promote overall well-being. This was changed along with cage twice a week. The enrichment was provided both during pre-mating and post mating period for males and pre-mating period for females. For recovery groups enrichment was provided throughout the experimental period.

- Diet: 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: 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


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 25°C
- Humidity (%): 65 - 68
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 0.1 % (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 conducted for study no N3335 with the same test item.
- Concentration in vehicle: 0 / 11 / 33 / 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:

The test item was determined quantitatively in dose formulations by UV/Vis spectrometry at 433 nm.
Calibration: linear regression line in the range from 5 to 30 µg/mL in DMSO.
Sample preparation: samples for analysis of active ingredient content and homogeneity were taken and diluted with the diluent to get the final concentration of about 15 µg/mL of the test item.

For homogeneity and active ingredient (a.i.) concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 of the treatment period and during 2nd month (day 34) of treatment and were analysed in-house.
For each set, duplicate samples were drawn from top, middle and bottom layers of each preparation and in case of control duplicate samples from the middle layer were drawn.
The analysis was done as per the method validated under Advinus Study No. G13350. One set of samples was analysed for concentration (a.i) analysis.
Formulations were considered acceptable as mean results are within ± 15 % of the theoretical concentration and the relative standard deviation (RSD) was less than 10 %.
The unused back up samples were disposed as analysis results of the first set of samples were within the acceptable limits

Duration of treatment / exposure:

Males: The dose formulation was administered for a period of 49 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 range of treatment duration was 52 to 56 days. 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) for a period of 52 days.
The dose volume administered for each rat was 10 mL/kg body weight 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.

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 once

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

G1 Vehicle Control

Dose / conc.:

110 mg/kg bw/day (nominal)

Remarks:

G2 Low dose

Dose / conc.:

330 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, concurrent vehicle

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 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:

no

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.
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. The reproductive organs of any animals failing to mate were examined in all the dose 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):

There were no clinical signs and mortalities observed both during the treatment and recovery periods at all the treated groups in both sexes.
However, the light to dark orange coloured faeces were observed in the treated group rats. The coloured faeces were attributed to the nature of the test item and not an adverse finding.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weights and net weight gains were unaffected by the treatment at all the doses tested in both sexes when compared to vehicle control.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The food consumption was not altered by the treatment in both the sexes at all the doses tested, when compared to vehicle control group.

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:

effects observed, treatment-related

Description (incidence and severity):

There were no indication for test item-related clinical and anatomic pathology changes in adult males and females at all the doses tested except for presence of light orange colored stomach/intestinal contests in all treated males and in one low dose female. This change in color of stomach/intestinal contents did not result in any gross/microscopic changes in respective tissues.

Neuropathological findings:

no effects observed

Description (incidence and severity):

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

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

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 the test item to male and female Wistar rats at the dose levels of 110, 330 and 1000 mg/kg bw/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 the test item for general toxicity (repeated dose toxicity) in males and females is considered to be 1000 mg/kg bw/day.

Therefore, the test item has not to be classified as subacutely toxic (STOT RE) 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 (0.1 % (w/v) Na CMC in Milli-Q® water} and administered by oral gavage at the dose levels of 110, 330 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 bw/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 G13350. Based on the results, the test item was found to be stable for up to 6 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 the test item 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

reliable

System:

other: no system affected at the highest dose tested

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Repeated dose toxicity (STOT RE)

The NOAEL for repeated dose toxicity is above the threshold for classification.

Therefore, the test item has not to be classified for repeated dose toxicity (STOT RE) according to Regulation (EC) No 1272/2008.