Ferrate(4-), hexakis(cyano-C)-, Et 2-[6-(ethylamino)-3-(ethylimino)-2,7-dimethyl-3H-xanthen-9-yl]benzoate copper(2+) salts

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 30 June 2022 to 03 January 2023

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

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 (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Ferrate(4-), hexakis(cyano-C)-, Et 2-[6-(ethylamino)-3-(ethylimino)-2,7-dimethyl-3H-xanthen-9-yl]benzoate copper(2+) salts

EC Number:

235-469-2

EC Name:

Ferrate(4-), hexakis(cyano-C)-, Et 2-[6-(ethylamino)-3-(ethylimino)-2,7-dimethyl-3H-xanthen-9-yl]benzoate copper(2+) salts

Cas Number:

12237-63-7

Molecular formula:

C34H31N8O3Cu3Fe

IUPAC Name:

ferrate(4-), hexakis(cyano-C)-, Et 2-[6-(ethylamino)-3-(ethylimino)-2,7-dimethyl-3H-xanthen-9-yl]benzoate copper(2+) salts

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 is also recommended by various regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Total number of animals: 116 (58 Males + 58 Females)

A total of 68 females were received and evaluated pre-exposure for estrous cyclicity. The females that failed to exhibit typical 4 to 5-day cycles were excluded from the experiment and sacrificed after initiation of the treatment by CO2 asphyxiation.
Finally, 58 females were selected for the study.
Females used were nulliparous and non-pregnant.

Age at Receipt: 9 to 10 weeks

Body Weight at Receipt:
Males: 271.57 to 321.66 g
Females: 200.03 to 230.80 g

Animals were housed under standard laboratory conditions in an environmentally monitored, air-conditioned room with adequate fresh air supply (12 to 15 air changes per hour), room temperature 19.3 to 24.1℃ and relative humidity 45 to 65% with 12 hours fluorescent light and 12 hours dark cycle.
The temperature and relative humidity were recorded once daily.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

as it is the probable route of exposure to humans

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

The prepared test item formulations and vehicle were administered to the animals by oral (gavage) route once daily within the established stability conditions.
Main group males: The males were treated for a period of two weeks (14-days) during pre-mating, during cohabitation period and continued up to the day before scheduled sacrifice (total of 36 days of treatment).

Main group females:
-All the females were treated for a period of two weeks (14-days) during pre-mating period, during cohabitation period and until confirmation of mating.
-The pregnant females were continued with treatment during pregnancy (gestation) and up to lactation day 13 (ranging from a total period of 48 to 69 days).
-The not littered females/non-pregnant females were continued with treatment further 26 days from the day of confirmation of mating i.e., presumed GD 0 and until one day prior to scheduled sacrifice (group G1 - one female until experimental day 41; group G2 - one female until experimental day 41, another female until experimental day 42; group G3 one female until experimental day 41 and other female until experimental day 52; group G4, one female until experimental day 43, second female until experimental day 54 and another female until experimental day 60, the female which was found dead due to dystocia was until experimental day 37).
Recovery group animals (both sexes): The recovery group animals were treated until the first scheduled sacrifice of dams (total of 49 days of administration) and kept without treatment for further 21-days of the recovery period.
The test item formulations/vehicle were administered by oral (gavage) route using stainless steel intubation cannula attached to a disposable syringe. All the doses were administered in an equivolume of 10 mL/kg with the concentration of 10, 30 and 100 mg/mL of test item for low, mid, and high dose groups, respectively. Vehicle control was administered to vehicle control group at an equivolume of 10 mL/kg body weight.
The actual dose volume for each animal was calculated based on the most recent body weight.
The prepared formulations were maintained under stirring conditions using magnetic stirrer during administration to maintain homogeneity.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation analysis for dose concentration verification was analyzed for homogeneity and dose concentration formulation. The results were found within the acceptable range of ±15% (i.e., 85% to 115%) to the nominal concentration and relative standard deviation (%RSD) was ˂ 10%.

Duration of treatment / exposure:

The prepared test item formulations and vehicle were administered to the animals by oral (gavage) route once daily within the established stability conditions.
Main group males: The males were treated for a period of two weeks (14-days) during pre-mating, during cohabitation period and continued up to the day before scheduled sacrifice (total of 36 days of treatment).

Main group females:
-All the females were treated for a period of two weeks (14-days) during pre-mating period, during cohabitation period and until confirmation of mating.
-The pregnant females were continued with treatment during pregnancy (gestation) and up to lactation day 13 (ranging from a total period of 48 to 69 days).
-The not littered females/non-pregnant females were continued with treatment further 26 days from the day of confirmation of mating i.e., presumed GD 0 and until one day prior to scheduled sacrifice (group G1 - one female until experimental day 41; group G2 - one female until experimental day 41, another female until experimental day 42; group G3 one female until experimental day 41 and other female until experimental day 52; group G4, one female until experimental day 43, second female until experimental day 54 and another female until experimental day 60, the female which was found dead due to dystocia was until experimental day 37).
Recovery group animals (both sexes): The recovery group animals were treated until the first scheduled sacrifice of dams (total of 49 days of administration) and kept without treatment for further 21-days of the recovery period.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle Control/
Vehicle Control Recovery

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Low Dose

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Mid Dose

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High Dose/
High Dose Recovery

No. of animals per sex per dose:

Vehicle Control/Low Dose/Mid Dose/High Dose: 12
Vehicle Control Recovery/High Dose Recovery: 5
A total of 116 (58 males + 58 females) Sprague Dawley rats were selected for the study and distributed to four main (G1, G2, G3 and G4) and two recovery groups (G1R and G4R). Each main group consisted of 12 males and 12 females, and each recovery group consisted of 5 males and 5 females.

Control animals:

yes, concurrent vehicle

Details on study design:

The animals in group G1/G1R were administered with vehicle [0.5% w/v Carboxymethyl cellulose], animals in groups G2, G3 and G4/G4R were administered with test item at the dose levels of 100, 300 and 1000 mg/kg body weight/day for low, mid and high dose levels, respectively. Vehicle and test item formulations were administered with the dose volume of 10 mL/kg body weight/day once daily at similar times on each day.
All the main group males were treated during pre-mating (14-days), during mating and continued during post-mating period (total of 36 days) until one day prior to termination. The pregnant females from main groups were treated during pre-mating (14-days), during mating, pregnancy and up to lactation day 13 (total of 48 to 69 days). The non-pregnant females from main groups were treated during pre-mating (14-days), during mating until confirmed as mated and further 26 days from the day of confirmation of mating (total of 41 to 60 days). All the recovery group animals were treated for a period of 49 days followed by a 21-day recovery period without treatment.

Results and discussion

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: systemic toxicity

2

Dose descriptor:

LOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: systemic toxicity

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

other: reproductive system of both sexes

Organ:

gonad

Treatment related:

yes

Any other information on results incl. tables

For systemic toxicity assessment, all the main group animals were observed once daily for clinical signs of toxicity, twice daily for mortality/morbidity and once in a week for detailed clinical examination. Body weight of all the animals were recorded once in a week until termination and on gestation day (GD) 0, 7, 14 and 20 and on lactation day (LD) 1, 4, 7, 13 and 14 for pregnant females. Feed consumption (gram/animal/day) was recorded once in a week during pre-mating for all animals and during
GD 0 to 7, 7 to 14 and 14 to 20 and during LD 1 to 4, 4 to 7 and 7 to 13 for pregnant females. Ophthalmological (for all animals) and neurological/functional observations (for 5 randomly selected males and 5 randomly selected females from each group) were conducted towards end of the dosing period. The investigations of clinical pathology (haematology, clinical chemistry and urinalysis for five randomly selected males and haematology and clinical chemistry for five randomly selected females from each group) was conducted at termination. Serum thyroxine hormone (T4) levels were estimated for all males by ELISA method. The organs were collected and weighed and organ weight relative to terminal body weight was calculated. All the animals were observed for both external and internal gross pathological changes during conduct of necropsy. The histopathological examination was conducted on all the tissues collected from the vehicle control and high dose group animals.
For reproductive toxicity assessment, all the main group animals were evaluated for reproductive performances such as mating and fertility indices. All the females were evaluated for gestation index, parturition index and for pre-coital interval and gestation length. The females were evaluated for oestrus cyclicity starting from treatment until evidence of mating and at termination. All the litters were observed for at birth (delivery) parameters such as no. of live/dead pups born, litter size, sex ratio (m/f), live birth index, no. of survived or dead pups and pup survival index. The total number of implantation sites was recorded for each litter during necropsy and the post-implantation and postnatal losses per litter was calculated.
For developmental toxicity assessment, all survived pups from each litter were observed once daily for external behavioural changes and twice daily for mortality until termination i.e., on postnatal day (PND) 13. All the pups from each litter were weighed individually at birth and on PND 4, 7 and 13. All the survived pups were measured for anogenital distance on PND 4 and all male pups were observed for retention of nipples on PND 13. All the pups were subjected for both external and internal gross pathological changes during conduct of necropsy. The collected serum from PND 13 pups was subjected (liter as a unit) to estimate the serum thyroxine hormone (T4) levels using ELISA method.
For systemic toxicity assessment and reversibility, persistence, or delayed occurrence of systemic toxic effects of test item, all the recovery group animals were observed once daily for clinical signs of toxicity, twice daily for mortality/morbidity and once in a week for detailed clinical examination. Body weight and feed consumption of all the animals were recorded once in a week until termination. Ophthalmological and neurological/functional observations were performed for all animals towards end of the recovery period. The investigations for clinical pathology were conducted at termination. The organs were collected and weighed and organ weight relative to terminal body weight was calculated. All the animals were observed for both external and internal gross pathological changes during conduct of necropsy.
In groups G2 and G3, the animals were noted with pink colored fecal matter and urine during treatment period. These observations were due to colored nature of the test item, but not adverse treatment related effects. No test item related changes were noted in mean body weight, percent change in mean body weight gain and mean feed consumption. The ophthalmological examination and neurological/functional examinations did not reveal any test item-related changes. The obtained clinical pathology levels, serum T4 levels and the absolute and relative organ weights did not reveal any test item-related changes. The gross pathological examination didn’t reveal any changes during necropsy.
In group G4/G4R, the animals were noted with pink colored fecal matter, urine and stain over the fur. Along with these, 2 (out of 12) males and 3 (out of 12) females from group G4 were noted with lethargy and/or perinasal staining from treatment day 22 and continued until termination. In group G4, 1 (out of 12) female was noted with dystocia and found dead on GD 20. In group G4R, 1 (out of 5) female was noted with lethargy and found dead on treatment day 43. Test item related reduction in mean body weight, percent change in mean body weight gain and feed consumption was noted throughout the experimental period at this dose level. The ophthalmological examination and neurological/functional examinations did not reveal any test item-related changes. The obtained clinical pathology and serum T4 levels did not reveal any adverse test item related changes, except some of the higher or lower values which could be secondary effects caused due to other systemic effects noted at this dose level. The noted changes in organ weights at this dose level could be secondary effects but not direct test item-related effects. The gross pathological examination didn’t reveal any changes during necropsy in all survived animals.
In groups G2 and G3, there were no effects noted on reproductive performance of both sexes. In group G4, the mating and fertility performance (both sexes), gestation and parturition index of females were reduced, and this could be due to stress induced by the test item exposure. At this dose level, higher incidences of post-implantation loss were noted. There were no test item-related irregularities observed in oestrus cyclicity of females from all the tested dose groups. No changes were noted in ‘at birth’ (delivery) parameters and/or ‘litter observations’ in all the tested dose groups. The live birth index and pup survival index was unaffected by test item exposure in all the tested dose groups.
In all the tested dose groups (G2, G3 and G4), there were no test item-related developmental or external behavioural changes and no mortalities noted in any of pups per litter. The mean pup weight, mean pup anogenital distance and its ratio in either sex per litter were un-affected by the test item exposure. There were no incidences of retention of nipples in male pups examined on PND 13 from all the litters. The estimated serum T4 levels of PND 13 pups did not reveal any changes. There were no gross pathological changes noted in any of the pups during scheduled sacrifice.
There were no test item-related histopathological changes noted in high dose group animals of both sexes.

Applicant's summary and conclusion

Conclusions:

Based on the obtained results from the study conducted according to the OECD test guideline 422, the following conclusions are made:
-The dose level of 100 mg/kg body weight/day did not produce any systemic toxicity effects, except with noted pink colored fecal matter and urine (mild) during treatment period, which could be due to colored nature of the test item. There was no indication of reproductive and developmental toxicity noted at this dose level.
-The dose level of 300 mg/kg body weight/day did not produce any systemic toxicity effects, except with noted pink colored fecal matter and urine (moderate) during treatment period, which was due to colored nature of the test item. There was no indication of reproductive and developmental toxicity noted at this dose level.
-The dose level of 1000 mg/kg body weight/day produced systemic toxicity such as clinical signs of toxicity, mortalities [1 (out of 12) from group G4 females and 1 (out of 5) from group G4R females], reduced body weights and its gain, effects on clinical pathological end points. This dose level also imposed slight effects on reproductive performance of both sexes which could be secondary effects caused due to systemic effects. However, there was no indication of developmental toxicity at this dose level.
Therefore, the dose level of 300 mg/kg body weight/day is estimated as NOAEL and the dose level of 1000 mg/kg body weight/day is estimated as LOAEL for systemic and reproduction toxicity end points. The dose level of 1000 mg/kg body weight/day is estimated as NOAEL for developmental toxicity end points.