Pentasodium bis[5-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-4-hydroxy-3-[(2-hydroxy-5-nitrophenyl)azo]naphthalene-2,7-disulphonato(4-)]chromate(5-)

Developmental toxicity / teratogenicity

Some information in this page has been claimed confidential.

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 421

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Test System
Species/strain: Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous.
Age at the start of the treatment period: approx. 10-11 weeks old
Body weight at the allocation of the animals to the experimental groups:
males: 264 - 314 g (mean: 295.53 g, ± 20% = 236.42 – 354.63 g)
females: 183 - 217 g (mean: 199.89 g, ± 20% = 159.91 – 239.86 g)
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals were bred for experimental purposes.

Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 +/- 3°C
- Relative humidity: 55 +/- 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 0702)
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control,
microbiological controls at regular intervals)
- The animals were kept individually in IVC cages (except during the mating period when one female will be paired with one male),
type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 190612)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

Preparation of the Animals
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made.
Before the first administration all animals used for the study were weighed and assigned to the experimental groups
with achieving a most homogenous variation in body weight throughout the groups of males and females.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The test item was weighed into a tarred plastic vial on a suitable precision balance and the vehicle (sterile water) was added to give the appropriate final concentration of the test item.
The vehicle was selected as suggested by the sponsor and on the basis of the test item’s characteristics. The test item formulation was prepared freshly on each administration day before the administration procedure. The time of preparation was recorded for all dosing formulations.
Homogeneity of the test item in the vehicle was maintained by vortexing the prepared suspension thoroughly before every dose administration.
The vehicle was also used as control item.

The following doses were evaluated:
Control: 0 mg/kg body weight
Low Dose: 100 mg/kg body weight
Medium Dose: 300 mg/kg body weight
High Dose: 1000 mg/kg body weight

The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering.
Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and NOAEL.
The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle
using the same dose volume.

Dose volumes were adjusted individually based on weekly body weight measurements. The administration volume was 5 mL/kg body weight.

Details on analytical verification of doses or concentrations:

Each dosing concentration were analysed with respect to the target nominal concentration. Stability and homogeneity of the test item in the
vehicle were analysed for the low and high dose concentrations.
Samples for the nominal concentration verification were taken in study week 1 (first week of pre-mating period), 3 (first week of mating), 5 (gestation) and 7 (gestation/lactation) from all groups (16 samples).
Samples for homogeneity analysis were taken from the top, middle and bottom of the high dose and the low dose formulation in study
week 1 and 5 (12 samples).
Samples for stability analysis were taken in the first week of the study, 0 hours after the preparation and another sample 6 hours after the preparation (at room temperature), from high and low dose formulations (4 samples).
All samples of dosing formulations were sent to Analytic department of BSL BIOSERVICE Scientific Laboratories GmbH on particular day of sampling.
Formulation analysis was performed in accordance with GLP and the procedures followed for the determination of test item concentration in the dosing formulations and control formulation were described in a separate study phase plan (BSL phase study No. 122442) issued by the Principal Investigator which was amended to study plan.

Details on mating procedure:

Mating was performed using a ratio of 1:1 (male to female). The vaginal smear of the females was checked every morning after the start of the mating period to confirm the copulation. The day of the vaginal plug and/or sperm was considered as day 0 of gestation. The cages were arranged in such a way that possible effects due to cage placement were minimised. In case of unsuccessful mating, re-mating of females with proven males of the same group was considered.

Duration of treatment / exposure:

The female animals were treated with the test item formulation or vehicle on 7 days per week basis for approximately 54 days, i.e. during 14 days of pre-mating and 14 days of mating in both males and females, during the gestation period and up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

Frequency of treatment:

7 days/wk

Duration of test:

Study Initiation Date: 17 September 2012
1st Amendment to Study Plan: 14 December 2012
Experimental Starting Date: 26 September 2012
Experimental Completion Date: 18 November 2012
Completion Date of
Delegated Phase (Histopathology): 13 February 2013

Completion Date of
Delegated Phase
(Formulation Analysis): 18 February 2013

Date of Draft Report (BSL): 20 March 2013

Doses / concentrations

Remarks:

Doses / Concentrations:
0; 100; 300; 1000 mg/kg body weight and day
Basis:
actual ingested

No. of animals per sex per dose:

80 animals (40 males and 40 females) were included in the study (10 male and 10 female animals per group).
This included the control group animals shared with BSL study No. 122429.

Control animals:

yes, concurrent vehicle

Details on study design:

Mating
Mating was performed using a ratio of 1:1 (male to female). The vaginal smear of the females was checked every morning after the start of the mating period to confirm the pregnancy. If the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the estrus cycle on that day was documented. The day of the vaginal plug and/or sperm was considered as day 0 of gestation. Females with unsuccessful mating will be allowed to mate with other male of the same group.
The cages were arranged in such a way that possible effects due to cage placement were minimised.

Body Weight and Food Consumption
The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period as well as at the end of the study.
During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum) as well as day 4 post-partum along with pups. Any animals prematurely sacrificed were weighed prior to the sacrifice.
Food consumption was measured weekly on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.

Clinical Observations
General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
Females showing signs of abortion or premature delivery prior to the scheduled scarification of the animals were sacrificed and subjected to a thorough macroscopic examination.

Litter Observations
The duration of the gestation was recorded and was calculated from day 0 of the pregnancy. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.
Live pups were counted and sexed and litters weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum. Live pups were identified by writing numbers on the back with the help of a permanent marker or by tattooing. In addition to the observations of parent animals, any abnormal behaviour of the offspring was recorded.

Examinations

Fetal examinations:

The duration of the gestation was recorded and was calculated from day 0 of the pregnancy. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities.
Live pups were counted and sexed and litters weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum. Live pups were identified by writing numbers on the back with the help of a permanent marker or by tattooing. In addition to the observations of parent animals, any abnormal behaviour of the offspring was recorded.

Statistics:

A statistical assessment of the results of the body weight, food consumption, parameters of absolute and relative organ weights were performed for each gender by comparing values of dosed with control animals of the main groups using a one-way ANOVA and a post-hoc Dunnett Test. These statistics were performed with GraphPad Prism 5.01 software (p<0.05 was considered as statistically significant).

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Details on maternal toxic effects:
Mortality
No mortalities were observed at any dose levels of male and female animals.

Clinical Observations
No test item related clinical signs were observed in male and female animals during the entire study period. However, few specific findings observed in few male and female animals of treatment groups were slight to severe piloerection, bradykinesia, abnormal breathing, moving the bedding, alopecia on various body parts and salivation. As these clinical signs were observed on few days of the treatment period, in very few animals and in absence of any effect on parameters of general health like body weight and food consumption in treatment groups, these findings were considered to be non adverse.

Body Weight Development
In male and females, no statistically significant effect on body weight and body weight gain was observed throughout the study period in treatment groups except significant increase during premating day 7-14 in HD males and significant decrease during gestation day 0-7 in LD and MD group females was observed when compared with controls. Since it was increase in body weight gain in males and due to lack of dose dependency in decrease in body weight gain during gestation day 0-7 in females, this significant effect was considered to be toxicologically irrelevant.

Food Consumption
In males, statistical analysis of food consumption data revealed no statistically significant effect in treatment groups when compared with controls.
In females, statistical analysis of food consumption data revealed significant decrease during gestation day 0-7 and 7-14 in LD and MD group females when compared with controls. As no test item related effect on group mean body weight was observed during that period and due to lack of dose dependency in decrease in food consumption, this significant effect on food consumption was considered to be non adverse.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
Litter Data
No treatment related effect was observed on total number of pups born, number of males, number of females, sex ratio, live pups, still birth and runt on PND 0 and number of males, number of females, total number of pups and sex ratio on PND 4.
All group mean and individual values for various litter data parameters from treatment groups were comparable with the controls.

Litter Weight Data
Statistical analysis of litter weight data revealed no treatment related effect on group mean litter weight, total litter weight, male litter weight and female litter weight on PND 0 and PND 4 when compared with corresponding controls.

Precoital Interval and Duration of Gestation
No treatment related effect was observed on precoital interval and duration of gestation and values were comparable between the groups. All pregnancies resulted in normal births.
All females in control and treatment groups showed evidence of copulation during 14 days mating period except one female from MD group (No. 127) did not show evidence of mating through vaginal smear. However, animal was pregnant and littered normally.
Successful mating resulted in 9, 9, 10 and 7 pregnancies in the control, low, mid and high dose respectively.

Pre- and Post-Natal Data
Pre and post natal data like group mean number of corpora lutea, number of implantation sites, number of pups born on PND 0, percent preimplantation and post implantation loss remained unaffected due to treatment when compared with controls.

Reproductive Indices
No treatment related effect on copulation index, delivery index and viability index was observed when compared with controls.
Reduced fertility index (No. of pregnant females/No. of copulated females X 100) was observed in HD (70 %) dose group as compared to C (90%), LD (90%) and MD groups (100 %).

Pup Survival Data
No significant effect on survival of the pups from PND 0 to PND 4 was observed in any treatment group when compared with controls.
Survival of the pups from PND 0 to PND 4 remained unaffected due to the treatment in all treatment groups and no pup mortality was observed in this study except one pup from HD group female 133 (pup No. 4) was found dead on PND 2. Since this pup mortalitiy was observed in one female and just one pup was dead, this incidence was not considered to be test item related.

Pup External Findings
No treatment related gross external findings were observed in pups from any of the treated groups on PND 0 and 4. However, following few external findings were observed:
In control females, one pup from female 45 (pup No. 3) was observed for hematoma at head on PND 0.
In LD females, one pup from female 116 (pup No. 2) was observed for redness on the nose on PND 0.
In HD females, one pup from female 140 (pup No. 2) was observed for dark spot at the neck on PND 0.
All these findings were considered to be spontaneous in nature and not related to the treatment with test item.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

Pathology

At necropsy of male (after minimum total dosing of 28 days - on day 29) and females (on post-natal day 4) by using a high dose of Ketamine/Xylazine (3:1), macroscopic examination of the animals revealed no test item related macroscopic findings in males and females. Few spontaneous gross pathological findings observed in MD and HD male animals were yellow spots on epididymides, dark discolouration of organs like kidneys and mesenteric lymph nodes. These findings related to discolouration in males were attributed to the colour of the test item and as such not a systemic effect due to the test item administration.

Organ Weight

In males, statistical analysis of organ weight data revealed, statistically significant decrease in absolute prostate (with seminal vesicles and coagulating gland) weight in MD group and relative prostate weight in HD group when compared with the control. Since the difference in group mean absolute and relative prostate weight was marginal and in the light of absence of histopathological findings, this significant effect on prostate weight was considered to be non adverse.

In females, no statistically significant effect on absolute and relative (to body weight) organ weights was observed in any treatment group when compared with the controls.

Histopathology

Dark discoloration of kidney and/or mesenteric lymph node was observed in all males treated at 1000 mg/kg/day and a proportion of males treated at 300 mg/kg/day. At 100 mg/kg/day, mesenteric lymph node of one single male was discolored dark. These color changes were considered to be caused by the color of the test item itself, but were not evaluated histologically according to the study plan.

Histologically, no test item-related findings were noted in the male and female reproductive organs. Reproductive organs of most females showed typical post-partum histomorphology. One control female, one female treated at 100 mg/kg/day and three females treated at 1000 mg/kg/day did not show any indication of recent pregnancy at terminal sacrifice. This was considered to be unrelated to treatment, as histomorphology of their reproductive organs indicated physiological sexual cycling.

As a conclusion, no test item-related pathological lesions were noted in male and female reproductive organs in this study. The NOAEL (No Observed Adverse Effect Level) for pathology of reproductive organs is therefore considered to be 1000 mg/kg/day under the conditions of this study.

Macroscopic colour changes of kidney and mesenteric lymph node were considered to be directly caused by the test item, but were not evaluated histologically.

Dose Formulation Analysis

Concentration analysis of formulation samples was determined in study week 1, 3, 5 and 7 for all dose groups. The mean recoveries observed in LD, MD and HD groups were 109.2%, 107.5% and 90.5% of the nominal concentration, respectively.

Stability of formulation samples was investigated in study week 1 for LD and HD dose groups. After 6 hours storage at -20°C recovery compared to starting value was 112.3 and 98.9%.

Homogeneity of formulation samples was determined in study week 1 and 5 for LD and HD dose groups. The mean recovery observed for LD dose group was 113.9 and 105.8% of the nominal value, and 89.5 and 106.5% for HD dose group. The coefficients of variation of the different sampling locations (top, middle and bottom) were 3.2 and 1.7% in LD dose group, and 2.5 and 2.2% in HD dose group.

Applicant's summary and conclusion

Conclusions:

Based on the data generated from this reproduction/ developmental toxicity screening test with FAT 40821/A, the no observed adverse effect level (NOAEL) is considered to be 1000 mg/kg body weight for reproduction/ developmental toxicity screening in males and females.

Executive summary:

This study was performed with the aim to assess the possible effects of FAT 40821/A on male and female fertility and embryofetal development after repeated dose administration in Wistar rats.

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 54 days, i.e. during 14 days of pre-mating and 14 days of mating in both males and females, during the gestation period and up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days were completed. Animals of an additional control group were handled identically as the dose groups but receivedaqua ad injectionem(sterile water), the vehicle used in this study. The 4 groups comprised 10 male and 10 female Wistar rats.

During the period of administration, the animals were observed each day for signs of toxicity. Animals that died were examined macroscopically and at the conclusion of the test, surviving animals were sacrificed and observed macroscopically.

Body weight and food consumption were measured weekly, except for food consumption measurements which were not taken during the mating period in female animals and the mating and post-mating period in male animals.

After 14 days of treatment to both male and female, animals were mated (1:1) for a maximum of 14 days. The subsequent morning onwards the vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated and housed individually. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition and on day 4 post-partum.

The males were sacrificed after completion of the mating period on treatment day 30 and the females along with their pups were sacrificed on post natal day 4. Non-pregnant females were sacrificed on day 26. The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.

Pups sacrificed on postnatal day 4 and those found dead, were carefully examined for gross external abnormalities.

A full histopathological evaluation of testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) from C and HD Group animals and all organs showing gross lesions except discolorations due to the color of the test item were examined. 

Female reproductive organs were also evaluated in non-pregnant females.

The following doses were evaluated:

Control: 0  mg/kg body weight;Low Dose: 100mg/kg body weight;Medium Dose: 300 mg/kg body weight, High Dose: 1000 mg/kg body weight

The test item formulation was prepared freshly on each day of administration. The test item was dissolved inaqua ad injectionem(sterile water) and administered daily during 14 days of pre-mating and 14 days of mating in both male and female animals, during the gestation period and up to post-natal day 3 in females. Males were dosed for 28 days. Dose volumes were adjusted individually based on weekly body weight measurements. Theadministration volume was 5 mL/kg body weight.

Summary Results

Mortality

No mortality occurred in the control or any of the dose groups during the treatment period of this study.

Clinical Signs

No test item related clinical signs were observed in male and female animals during the entire study period. However, few specific findings observed in few male and female animals of treatment groups were slight to severe piloerection, bradykinesia, abnormal breathing, moving the bedding, alopecia on various body parts and salivation.

Body Weight Development

In male and females, no statistically significant effect on body weight and body weight gain was observed throughout the study period in treatment groups except significant increase during premating day 7-14 in HD males and significant decrease during gestation day 0-7 in LD and MD group females was observed when compared with controls.

Food Consumption

In males, statistical analysis of food consumption data revealed no statistically significant effect in treatment groups when compared with controls.

In females, statistical analysis of food consumption data revealed significant decrease during gestation day 0-7 and 7-14 in LD and MD group females when

compared with controls.

Litter Data

No treatment related effect was observed on total number of pups born, number of males, number of females, sex ratio, live pups, still birth and runt on PND 0

and number of males, number of females, total number of pups and sex ratio on PND 4.

Litter Weight Data

Statistical analysis of litter weight data revealed no treatment related effect on group mean litter weight, total litter weight, male litter weight and female litter weight on PND 0 and PND 4 when compared with corresponding controls.

Precoital Interval and duration of Gestation

No treatment related effect was observed on precoital interval and duration of gestation and valueswere comparable between the groups. All pregnancies resulted in normal births.

Pre and Post Natal Data

Pre and post natal data like group mean number of corpora lutea, number of implantation sites, number of pups born on PND 0, percent preimplantation and post implantation loss remained unaffected due to treatment when compared with controls.

Reproductive Indices

No treatment related effect on copulation index, delivery index and viability index was observed when compared with controls.

Reduced fertility index (No. of pregnant females/No. of copulated females X 100) was observed in HD (70 %) dose group as compared to C (90%), LD (90%)

and MD groups (100 %).

Pup Survival Data

No significant effect on survival of the pups from PND 0 to PND 4 was observed in any treatment group when compared with controls.

Survival of the pups from PND 0 to PND 4 remained unaffected due to the treatment in all treatment groups and no pup mortality was observed in this study

except one pup from HD group female 133 (pup No. 4) was found dead on PND 2.

Pup external Findings

No treatment related gross external findings were observed in pups from any of the treated groups on PND 0 and 4. However, few spontaneous findings like

hematoma at head in one pup from control group female, redness on the nose in one pup from LD group female and dark spot at the neck in one pup from HD

group was observed on PND 0.

All these findings were considered to be spontaneous in nature and not related to the treatment with test item.

Pathology

At necropsy of male and females, macroscopic examination of the animals revealed no test item related macroscopic findings in males and females.

Fewspontaneous gross pathological findings observed in MD and HD male animals wereyellow spotson epididymides, dark discolouration of organs like kidneysand mesenteric lymph nodes.

Organ Weight

In males, statistical analysis of organ weight data revealed, statistically significant decrease in absolute prostate (with seminal vesicles and coagulating gland)weight in MD group and relative prostate weight in HD group when compared with the control.

In females, no statistically significant effect on absolute and relative (to body weight) organ weights was observed in any treatment group when compared with the controls.

Histopathology

Histologically, no test item-related findings were noted in the male and female reproductive organs. Reproductive organs of most females showed typical post-partum histomorphology. One control female, one female treated at 100 mg/kg/day and three females treated at 1000 mg/kg/day did not show any indication of recent pregnancy at terminal sacrifice. This was considered to be unrelated to treatment, as histomorphology of their reproductive organs indicated physiological sexual cycling.

As a conclusion, no test item-related pathological lesions were noted in male and female reproductive organs in this study. The NOAEL (No Observed Adverse Effect Level) for pathology of reproductive organs is therefore considered to be 1000 mg/kg/day under the conditions of this study.

Macroscopic colour changes of kidney and mesenteric lymph node were considered to be directly caused by the test item, but were not evaluated histologically.

Dose Formulation Analysis

Concentration analysis of formulation samples was determined in study week 1, 3, 5 and 7 for all dose groups. The mean recoveries observed in LD, MD and HD groups were 109.2%, 107.5% and 90.5% of the nominal concentration, respectively.

Stability of formulation samples was investigated in study week 1 for LD and HD dose groups. After 6 hours storage at -20°C recovery compared to starting value

was 112.3 and 98.9%.

Homogeneity of formulation samples was determined in study week 1 and 5 for LD and HD dose groups. The mean recovery observed for LD dose group

was 113.9 and 105.8% of the nominal value, and 89.5 and 106.5% for HD dose group. The coefficients of variation of the different sampling locations

(top, middle, bottom) were 3.2 and 1.7% in LD dose group, and 2.5 and 2.2% in HD dose group. 

Conclusion

In conclusion, the repeated dose administration of FAT 40821/A in sterile water to the male (28 days) and female (maximum 54 days) Wistar rats at dosages of 100, 300 and 1000 mg/kg body weight revealed neither mortalities nor findings of toxicological relevance.

Based on the data generated from this reproduction/ developmental toxicity screening test withFAT 40821/A, the no observed adverse effect level (NOAEL) is considered to be 1000 mg/kg body weight forreproduction/ developmental toxicity screening in males and females.