N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-oxo-2-[[2-(trifluoromethyl)phenyl]azo]butyramide

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

This study according to OOECD TG 421 is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item to rats. The test item was administered in water as vehicle at dosages of 100, 300, and 1000 mg/kg body weight/day, and controls received the vehicle only. The test item was administered to male rats for 14 days prior to pairing and for 28 days in total, and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

Test item-related effects were restricted to secondar effects only (discolored feces) and there were no findings of toxicological relevance. Body weight and food consumption of parent animals were unaffected, and no differences in organ weights were noted. There were no macroscopical or microscopical findings related to the treatment with the test item in parental animals nor offspring.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 05 Jan 2012 to 31 Jul 2012

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 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Animals: Rat, RccHanTM: WIST(SPF)
- Rationale: Recognized by international guidelines as a recommended test system.
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Number of Animals: 44 males (11 per group) and 44 females (11 per group)
- Age (at delivery): 10 weeks
- Body Weight Range (at Start of Treatment): 301 to 344 g (males) and 191 to 228 g (females)
- Identification: Cage card and individual animal number (ear tattoo).
- Randomization: Computer-generated random algorithm. In addition body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
- Accommodation: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J.Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland; batch/lot no. 02105111001) with paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey / UK), batch/lot no. 55/T-6060). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet: Pelleted standard Harlan Teklad 2914C (batch no. 73/11) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum. Results of representative analyses for contaminants were included in the study report.
- Water: Community tap-water from Itingen was available ad libitum in water bottles. Results of bacteriological assay, chemical and contaminant analyses of representative samples were included in the study report.
- Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study. At least 5 days.

ENVIRONMENTAL CONDITIONS
Standard laboratory conditions, continuously monitored.
- Temperature (°C): 22 +/- 3
- Humidity (%): 30 to 70
- Air changes (per hr): . Air-conditioned with 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12 (with at least eight hours music during the light period)

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

DOSE FORMULATIONS
The dose formulations were not corrected for purity and were prepared as supplied by the Sponsor. The dose formulations were prepared weekly as indicated by the results of stability analyses in the non-GLP dose range-finding study. The test item was weighed into a glass beaker on a tared Mettler balance and the vehicle was added. The mixtures were stirred using a magnetic stirrer and stored at room temperature (20 °C +/- 5 °C). Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

STORAGE OF DOSE FORMULATIONS
The dose formulations were stored at room temperature (20 +/- 5 °C) in glass beakers. They were stable for at least 8 days, based upon the results of stability analyses performed during the non-GLP dose range-finding study.

TREATMENT
- Method: Oral, by gavage
- Rationale for Method: Administration by gavage is a common and accepted route of exposure for this type of studies.
- Frequency of Administration: Once daily
- Daily Target Dose Level: 0 mg/kg/day (control group), 100 mg/kg/day (group 2), 300 mg/kg/day (group 3), 1000 mg/kg/day (group 4)
- Rationale for Dose Level Selection: The dose levels were selected based on a previous dose range-finding toxicity study in Wistar rats, Harlan Laboratories study (non-GLP).
- Dose Volume: 10 mL/kg body weight
- Dose Concentrations: 0 mg/mL/day (control group), 10 mg/mL/day (group 2), 30 mg/mL/day (group 3), 100 mg/mL/day (group 4)

Details on mating procedure:

During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed. The females were removed and housed individually if the daily vaginal smear was sperm positive, or a copulation plug was observed.

The day on which a positive mating was determined (copulation plug or sperm) was designated day 0 post coitum.

If a female did not mate during the 14-day pairing period, a second pairing of this female with a male in the same group, which had already mated successfully, was considered. If mating was not recorded during this additional pairing period of a maximum of 14 days, the female was sacrificed and, if indicated, the reproductive organs examined histopathologically in order to ascertain the reason for the infertility.

All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

METHOD
After experimental start, a sample from the control group as well as three samples (top, middle and bottom) of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples were also taken to confirm stability (4 hours and 8 days at room temperature). During week 3 of treatment, samples were also taken to confirm homogeneity and concentration. The aliquots for analysis of dose formulations were stored at -20 +/- 5°C until analysis. The test item was used as the analytical standard.

RESULTS
The linearity of the analytical systems used for sample analyses was demonstrated with a good relationship between absorbance measured and working standard concentrations. All calibration points met the acceptance limit of +/- 20% variation from the calibration curve derived by linear regression analysis. The regression coefficients calculated were found to be better than 0.99.

Blank samples showed no significant absorbance and, therefore, it was confirmed that only bidistilled water was applied within the control experiment.

The application formulations investigated during the study were found to comprise test material in the range of 93.8% to 108.3% and, thus, the required content limit of +/-20% with reference to the nominal content was met. The homogeneous distribution of the test item in the preparations was approved because single results found did not deviate more than 3% (acceptance criterion: <15%) from the corresponding mean.

In addition, the test item was found to be stable in application formulations when kept four hours or eight days at room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean.

In conclusion, the results indicate the accurate use of the test item and bidistilled water as vehicle during this study. Application formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.

Duration of treatment / exposure:

Minimum 4 weeks (males) and approximately 7 weeks (females)

Frequency of treatment:

Once daily

Details on study schedule:

MALES
- Acclimatization: 5 days minimum
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum
- Treatment Ends: On day before sacrifice
- Necropsy: After a minimum of 28 days treatment

FEMALES
- Acclimatization: 5 days minimum
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum
- Gestation: Approximately 21 days
- Treatment Ends: On day 3 post partum
- Necropsy: On day 4 post partum

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

11

Control animals:

yes, concurrent vehicle

Details on study design:

The purpose of this study was to generate preliminary information concerning the effects of the test item on male and female reproductive performance such as gonadal function, mating behavior, conception and parturition and to assess major effects on pup development.

Positive control:

No

Parental animals: Observations and examinations:

VIABILITY/MORTALITY: Twice daily

CLINICAL SIGNS: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

FOOD CONSUMPTION
Males: Weekly during pre-pairing and after pairing periods.
Females: Pre-pairing period days 1 - 8 and 8 - 14, gestation period days 0 - 7, 7 - 14 and 14 - 21 post coitum and lactation period days 1 - 4 post partum.
No food consumption was recorded during the pairing period.

BODY WEIGHTS
Recorded daily from treatment start to day of necropsy.

Oestrous cyclicity (parental animals):

Not examined

Sperm parameters (parental animals):

Organ weight:
Testes and epididymides of all parental males were weighed

Histopathology:
Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.

Detailed examination of the testes (including sperm staging) was performed on control males and males at 1000 mg/kg/day. The stages were checked for completeness of cell populations, completeness of stages and degenerative changes.

Litter observations:

The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum.

Postmortem examinations (parental animals):

TERMINATION AND NECROPSY
Males were sacrificed after they had been treated for at least 28 days. Dams were sacrificed on day 4 post partum. If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.
All animals sacrificed or found dead were subjected to a detailed macroscopic examination to establish, if possible, the cause of death. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution. At the scheduled sacrifice, all animals were killed by an injection of sodium pentobarbital. All P generation animals were exsanguinated.
All parent animals were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.
For the parent animals, special attention was directed at the organs of the reproductive system. The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

ORGAN WEIGHTS
At the scheduled sacrifice, the testes and epididymides of all parental males were weighed separately.

TISSUE PRESERVATION
The ovaries from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution. The testes and epididymides from all parental males were preserved in Bouin’s fixative. The prostate and seminal vesicles from all males were fixed in neutral phosphate buffered 4% formaldehyde solution.

HISTOTECHNIQUE
All organ and tissue samples to be examined by the principal investigator for histopathology were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis was stained by PAS-hematoxylin. Special stains were used at the discretion of the principal investigator for histopathology.

HISTOPATHOLGOLGY
Slides of all organs and tissues collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the principal investigator for histopathology. The same applied to all occurring gross lesions and to all animals, which died spontaneously or had to be terminated in extremis.
Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.
Histological examination of ovaries was carried out on any female that did not give birth. In addition, microscopic examination of the reproductive organs of all infertile males was made, if necessary.
A histopathology peer review was performed.

Postmortem examinations (offspring):

Pups were sacrificed on day 4 post partum. At the scheduled sacrifice, all animals were killed by an injection of sodium pentobarbital. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution. Dead pups, except those excessively cannibalized, were examined macroscopically.

Statistics:

The following statistical methods were used to analyze food consumption, body weights and reproduction data:
- Means and standard deviations of various data were calculated.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Reproductive indices:

From the on-line recorded reproduction data, the following parameters were calculated:
fertility indices, mean precoital time, post-implantation losses, mean litter size, pup sex ratios and viability indices.

Offspring viability indices:

From the on-line recorded reproduction data, the following parameters were calculated: dead/live pups at first litter check, pups sex ratio

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Yellow discoloration of feces in males and females of dose groups

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

1. IN-LIFE DATA
VIABILITY / MORTALITY
All animals survived the scheduled study period.

CLINICAL SIGNS OR OBSERVATIONS
There were no findings of toxicological relevance at any dose level.
In males, yellow discoloration of the feces was noted from treatment day 5 of the pre-pairing period and continued throughout the pairing period. The severity of this finding was generally dose-dependent, but was considered to be a typical passive effect that results from oral administration of a dyestuff and was of no toxicological relevance.
In females, a similar finding was noted from day 5 of the pre-pairing period and continued throughout the subsequent pairing, gestation and lactation periods. As in males, the severity of this finding was generally dose-dependent, and was ascribed the same lack of toxicological relevance as in the males. Slight hair loss was noted in a single female during the gestation period, but in view of the late onset and low incidence, was considered to be without toxicological implications.

FOOD CONSUMPTION OF MALES
Pre-pairing Period
In males, although the mean daily food consumption values at 300 mg/kg/day and 1000 mg/kg/ day were significantly lower (p<0.05) than that of the control males during days 8 - 14, these differences were not dose-related and were considered to be a result of an incidentally elevated control value rather than to the treatment with the test item.

FOOD CONSUMPTION OF FEMALES
Pre-pairing, Gestation and Lactation Periods
The mean daily food consumption values of the test item-treated females compared favorably with the control females throughout the pre-pairing, gestation and lactation periods.

BODY WEIGHTS OF MALES
Pre-Pairing and Pairing Periods
There were no differences of toxicological relevance between the mean body weights of test item-treated or control males. The mean body weight gain of all groups compared favorably.

BODY WEIGHTS OF FEMALES
Pre-Pairing, Pairing, Gestation and Lactation Periods
No test item-related effects on body weights and body weight gain of females were observed at any dose level.
In females treated with 1000 mg/kg/day, a significant increase (p<0.05) in mean body weight was noted on day 4 of the gestation period. This isolated finding was considered to be incidental. The body weights recorded during all other periods were similar to those of the control females.
At 300 mg/kg/day, all body weights were similar throughout the various phases.
At 100 mg/kg/day, significantly elevated mean body weights (p<0.05) were noted on days 0 - 5 of the gestation period only. In the absence of a dose-response relationship, these differences were considered to be incidental. The body weights recorded during all other periods were similar to the controls.

2. REPRODUCTION AND BREEDING
MATING PERFORMANCE AND FERTILITY
The mean and median precoital times for all groups were similar.
The respective parameters of the mating performance were unaffected as all dose levels and similar to those of the control values.
Mating of female no. 84 was not detected. Three females were not pregnant: no. 55 (control group) and nos. 81 ad 83 (1000 mg/kg/day).

DURATION OF GESTATION
No effects on duration of gestation were observed at any dose level.

CORPORA LUTEA COUNT
No effects on corpora lutea count were observed at any dose level.

IMPLANTATION RATE AND POST-IMPLANTATION LOSS
The implantation rate was unaffected in all groups.
Although the mean post-implantation loss was slightly higher at 300 mg/kg/day and 1000 mg/kg/ day when compared with the control females, the differences were not dose-related and therefore considered to be unrelated to the test item treatment. The total post implantation loss of the dams treated with 300 mg/kg/day was significantly elevated (p<0.01) when compared with the controls, but not dose-dependent and therefore considered to be unrelated to the treatment with the test item. The mean post implantation loss in females at 100 mg/kg/day was nearly identical to the controls.

LITTER SIZE AT FIRST LITTER CHECK
The mean litter sizes of the control group and those at 100 and at 1000 mg/kg/day were similar. The marginally lower mean litter size noted at 300 mg/kg/day coincided with the slightly higher post-implantation loss.

The overall mean numbers of living pups per dam at first litter check compared favorably in the control and test item-treated groups; three pups of litter no. 79 (1000 mg/kg/day), two pups of litter no. 70 (300 mg/kg/day) and four pups of litter no. 49 (control group) were found dead.

The respective birth indices (number of pups borne alive as a percentage of implantations) were similar in test item-treated and control animals.

3. TERMINAL FINDINGS
ORGAN WEIGHTS
No test item-related changes in organ weights were noted at any dose level.

At 300 mg/kg/day, the mean absolute epididymide weight (left side only) were significantly reduced (p<0.05) when compared with the controls. This finding was considered to be incidental as the mean relative organ weight was normal and unilateral organ weight differences are not associated with toxicological relevance.
At 100 mg/kg/day, the mean absolute epididymide weights (bilateral) were significantly reduced (p<0.05) when compared with the controls. These differences were considered to be related to the lower mean terminal body weights; the mean relative organ weights were unaffected.
No further changes of organ weights were noted at any dose level.

MACROSCOPICAL FINDINGS
A small number of typical background changes were noted in test item-treated and control males. Neither type nor incidence were considered to be commensurate with findings of toxicological relevance in males.
Discoloration of the ovaries found in one female at 1000 mg/kg/day and was considered to be within the range of normal background alterations.

HISTOPATHOLOGY FINDINGS
All microscopic findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age. There was no morphological evidence of toxicological properties in the testes, epididymides, prostate and seminal vesicles of male animals and ovaries of female animals examined in this study.
Detailed examination of the testes (including sperm staging) was performed on control males (nos. 1 - 11) and males at 1000 mg/kg/day. The stages were checked for completeness of cell populations, completeness of stages and degenerative changes. No differences on the completeness of stages or cell populations of the testes were recorded between the control males (nos. 1 - 11) and the males at 1000 mg/kg/day (nos. 34 - 44). In male no. 6 of the control group, unilateral Sertoli cell vacuolation (minimal in degree) was noted. Male no. 41 (treated with 1000 mg/kg/day) showed unilateral tubular degeneration/atrophy that was considered to be minimal in degree.

There were no findings or microscopical changes to the reproductive organs that were considered to be related to the infertility noted in male no. 40.

Dose descriptor:

NOAEL

Remarks:

for general toxicity

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed up to 1000 mg/kg bw/day, the highest dose level tested.

Dose descriptor:

NOAEL

Remarks:

for reproduction

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed up to 1000 mg/kg bw/day, the highest dose level tested.

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

POSTNATAL LOSS DAYS 0 - 4 POST PARTUM
From days 2 - 3 post partum, five male and seven female pups of dams treated with 1000 mg/kg/ day were missing or found dead, one female pup of a dam treated with 300 mg/kg/day were missing or found dead, one male and one female pup of dams treated with 100 mg/kg/day were missing or found dead, and three males and five female pups of control dams were missing or found dead. These minor differences were considered to be unrelated to the treatment with the test item. The statistically significant decrease noted in the total postnatal loss noted at 300 mg/kg/day (p<0.05) is not associated with toxicological relevance.

EXTERNAL EXAMINATION AT FIRST LITTER CHECK AND DURING LACTATION
No test item-related observations were noted in pups during the first litter check or during lactation at any dose level. In one litter (no. 49) of the control group, four pups were found to have no milk in the stomach. One litter ( no. 70) at 300 mg/kg/day had two pups without milk in the stomach and one litter (no. 79) at 1000 mg/kg/day had three pups without milk in the stomach. Insofar as these pups were found dead at first litter check, it is assumed they were stillborn or died during parturition.
During the lactation period, five pups of litter no. 49 (control) had missing body parts (most likely due to partial cannibalization) and two pups of litter no. 79 had no milk in the stomach.

SEX RATIOS: The sex ratio of the test item-treated pups did not indicate any effects of toxicological relevance.

BODY WEIGHTS TO DAY 4 POST PARTUM
No effects on pup body weights were noted at any dose level.

MACROSCOPICAL FINDINGS
No test item-related findings were found in pups at any dose level.

Dose descriptor:

NOAEL

Remarks:

for development

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed up to 1000 mg/kg bw/day, the highest dose level tested.

Reproductive effects observed:

not specified

SUMMARY OF PERFORMANCE

 

P Animals Breeding for F1 Litters

 

Group (mg/kg/day)	1 (0)	2 (100)	3 (300)	4 (1000)
Female numbers	45 - 55	56 - 66	67 - 77	78 - 88
Number of females paired	11	11	11	11
Number of females mated	11	11	11	11
Number of females which were not pregnant	1	0	0	2
Number of females which reared their pups until day 4 post partum	10	11	11	9

 

Conclusions:

This study according to OOECD TG 421 is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item to rats. The test item was administered in water as vehicle at dosages of 100, 300, and 1000 mg/kg body weight/day, and controls received the vehicle only. The test item was administered to male rats for 14 days prior to pairing and for 28 days in total, and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.
Test item-related effects were restricted to secondar effects only (discolored feces) and there were no findings of toxicological relevance. Body weight and food consumption of parent animals were unaffected, and no differences in organ weights were noted. There were no macroscopical or microscopical findings related to the treatment with the test item in parental animals nor offspring
No effects on mating performance, fertility, corpora lutea count or duration of gestation were observed at any dose level. Minor differences in the mean post-implantation loss, postnatal loss, and litter size at first litter check and day 4 post partum were either unrelated to dose or within the limits of the historical control data and therefore considered to be incidental.
No test item-related findings were noted at first litter check and during lactation in pups at any dose level. The sex ratios of the pups in test item-treated groups were normal and pup weights recorded until day 4 post partum compared favorably in all groups.
Because fecal discoloration (i.e. a secondary passive finding) was noted in treated rats at all dose levels, a NOEL (No Observed Effect Level) could not be established. The NOAEL (No Observed Adverse Effect Level) for general toxicity in males and females, as well as for reproduction data and F1 offspring was considered to be 1000 mg/kg bw/day, the highest dose level tested.

Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of the test item on male and female reproductive performance such as gonadal function, mating behavior, conception and parturition and to assess major effects on pup development.

Four groups of 11 males and 11 females were treated by gavage with test item once daily (according to OECD 421 and GLP compliant). Males were treated over a 14-day pre-pairing period and during the pairing period up to one day before necropsy. Females were treated throughout the pre-pairing, pairing, gestation and lactation period up to the day 3 post partum.

The following dose levels were used:

Group 1: 0 mg/kg body weight/day (control group)

Group 2: 100 mg/kg body weight/day

Group 3: 300 mg/kg body weight/day

Group 4: 1000 mg/kg body weight/day

 

A standard dose volume of 10 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (bidistilled water).

 

The following results were obtained:  

MORTALITY AND GENERAL TOLERABILITY OF PARENTAL ANIMALS

  All animals survived the scheduled study period.

  No clinical signs of toxicological relevance were noted at any dose level. In both sexes, yellow discoloration of the feces was noted from treatment day 5 of the pre-pairing period and continued throughout treatment. The severity of this finding was generally dose-dependent, but was considered to be a typical secondary passive effect that results from oral administration of a dyestuff and was of no toxicological relevance.

 

FOOD CONSUMPTION OF PARENTAL ANIMALS

  Minor intergroup differences in the mean daily food consumption of males were not considered to be related to the treatment with the test item. The daily food consumption values of the test item-treated females were unaffected.

 

BODY WEIGHTS OF PARENTAL ANIMALS: There were no effects upon body weights of either males or females.

 

REPRODUCTION AND BREEDING DATA

  No effects on mating performance, fertility, corpora lutea count or duration of gestation were observed at any dose level.

  At the dose level of 1000 mg/kg bw/day, higher incidence of post-implantation loss and higher postnatal loss as well as lower litter size at first litter check and on day 4 post partum were noted.

  The mean post implantation loss of the dams at 1000 mg/kg/day was close to the upper limit of the historical control data and was below the mean post implantation loss of the dams treated with 300 mg/kg/day; therefore this was considered to be unrelated to the treatment with the test item.

 

ORGAN WEIGHS OF PARENTAL ANIMALS: No test item-related effects on organ weights were noted at any dose level.

 

MACROSCOPICAL FINDINGS AND HISTOPATHOLOGICAL EXAMINATIONS OF PARENTAL ANIMALS

  No test item-related macroscopical findings were noted at any dose level.

  Under the conditions of this study, the test item produced no morphological evidence of toxicological properties in testes, epididymides, prostate and seminal vesicles of male animals and ovaries of female animals.

 

FINDINGS IN PUPS AT FIRST LITTER CHECK AND DURING LACTATION

  No test item-related findings were noted in pups at any dose level.

  The sex ratio of the test item-treated pups did not indicate any effects of toxicological relevance.

 

PUP WEIGHTS TO DAY 4 POST PARTUM:  No effects on pup body weights were noted at any dose level.

 

MACROSCOPICAL FINDINGS OF PUPS:  No test item-related findings were found in pups at any dose level.

Reference 2

Endpoint:

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

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity

Reference 3

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 05 Jan 2012 to 31 Jul 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 421, GLP-compliant)

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Animals: Rat, RccHanTM: WIST(SPF)
- Rationale: Recognized by international guidelines as a recommended test system.
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Number of Animals: 44 males (11 per group) and 44 females (11 per group)
- Age (at delivery): 10 weeks
- Body Weight Range (at Start of Treatment): 301 to 344 g (males) and 191 to 228 g (females)
- Identification: Cage card and individual animal number (ear tattoo).
- Randomization: Computer-generated random algorithm. In addition body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
- Accommodation: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J.Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland; batch/lot no. 02105111001) with paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey / UK), batch/lot no. 55/T-6060). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet: Pelleted standard Harlan Teklad 2914C (batch no. 73/11) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum. Results of representative analyses for contaminants were included in the study report.
- Water: Community tap-water from Itingen was available ad libitum in water bottles. Results of bacteriological assay, chemical and contaminant analyses of representative samples were included in the study report.
- Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study. At least 5 days.

ENVIRONMENTAL CONDITIONS
Standard laboratory conditions, continuously monitored.
- Temperature (°C): 22 +/- 3
- Humidity (%): 30 to 70
- Air changes (per hr): . Air-conditioned with 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12 (with at least eight hours music during the light period)

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

DOSE FORMULATIONS
The dose formulations were not corrected for purity and were prepared as supplied by the Sponsor. The dose formulations were prepared weekly as indicated by the results of stability analyses in the non-GLP dose range-finding study. The test item was weighed into a glass beaker on a tared Mettler balance and the vehicle was added. The mixtures were stirred using a magnetic stirrer and stored at room temperature (20 °C +/- 5 °C). Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

STORAGE OF DOSE FORMULATIONS
The dose formulations were stored at room temperature (20 +/- 5 °C) in glass beakers. They were stable for at least 8 days, based upon the results of stability analyses performed during the non-GLP dose range-finding study.

TREATMENT
- Method: Oral, by gavage
- Rationale for Method: Administration by gavage is a common and accepted route of exposure for this type of studies.
- Frequency of Administration: Once daily
- Daily Target Dose Level: 0 mg/kg/day (control group), 100 mg/kg/day (group 2), 300 mg/kg/day (group 3), 1000 mg/kg/day (group 4)
- Rationale for Dose Level Selection: The dose levels were selected based on a previous dose range-finding toxicity study in Wistar rats, Harlan Laboratories study (non-GLP).
- Dose Volume: 10 mL/kg body weight
- Dose Concentrations: 0 mg/mL/day (control group), 10 mg/mL/day (group 2), 30 mg/mL/day (group 3), 100 mg/mL/day (group 4)

Details on mating procedure:

During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed. The females were removed and housed individually if the daily vaginal smear was sperm positive, or a copulation plug was observed.

The day on which a positive mating was determined (copulation plug or sperm) was designated day 0 post coitum.

If a female did not mate during the 14-day pairing period, a second pairing of this female with a male in the same group, which had already mated successfully, was considered. If mating was not recorded during this additional pairing period of a maximum of 14 days, the female was sacrificed and, if indicated, the reproductive organs examined histopathologically in order to ascertain the reason for the infertility.

All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

METHOD
After experimental start, a sample from the control group as well as three samples (top, middle and bottom) of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples were also taken to confirm stability (4 hours and 8 days at room temperature). During week 3 of treatment, samples were also taken to confirm homogeneity and concentration. The aliquots for analysis of dose formulations were stored at -20 +/- 5°C until analysis. The test item was used as the analytical standard.

RESULTS
The linearity of the analytical systems used for sample analyses was demonstrated with a good relationship between absorbance measured and working standard concentrations. All calibration points met the acceptance limit of +/- 20% variation from the calibration curve derived by linear regression analysis. The regression coefficients calculated were found to be better than 0.99.

Blank samples showed no significant absorbance and, therefore, it was confirmed that only bidistilled water was applied within the control experiment.

The application formulations investigated during the study were found to comprise test material in the range of 93.8% to 108.3% and, thus, the required content limit of +/-20% with reference to the nominal content was met. The homogeneous distribution of the test item in the preparations was approved because single results found did not deviate more than 3% (acceptance criterion: <15%) from the corresponding mean.

In addition, the test item was found to be stable in application formulations when kept four hours or eight days at room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean.

In conclusion, the results indicate the accurate use of the test item and bidistilled water as vehicle during this study. Application formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.

Duration of treatment / exposure:

Minimum 4 weeks (males) and approximately 7 weeks (females)

Frequency of treatment:

Once daily

Details on study schedule:

MALES
- Acclimatization: 5 days minimum
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum
- Treatment Ends: On day before sacrifice
- Necropsy: After a minimum of 28 days treatment

FEMALES
- Acclimatization: 5 days minimum
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum
- Gestation: Approximately 21 days
- Treatment Ends: On day 3 post partum
- Necropsy: On day 4 post partum

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

11

Control animals:

yes, concurrent vehicle

Details on study design:

The purpose of this study was to generate preliminary information concerning the effects of the test item on male and female reproductive performance such as gonadal function, mating behavior, conception and parturition and to assess major effects on pup development.

Positive control:

No

Parental animals: Observations and examinations:

VIABILITY/MORTALITY: Twice daily

CLINICAL SIGNS: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

FOOD CONSUMPTION
Males: Weekly during pre-pairing and after pairing periods.
Females: Pre-pairing period days 1 - 8 and 8 - 14, gestation period days 0 - 7, 7 - 14 and 14 - 21 post coitum and lactation period days 1 - 4 post partum.
No food consumption was recorded during the pairing period.

BODY WEIGHTS
Recorded daily from treatment start to day of necropsy.

Oestrous cyclicity (parental animals):

Not examined

Sperm parameters (parental animals):

Organ weight:
Testes and epididymides of all parental males were weighed

Histopathology:
Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.

Detailed examination of the testes (including sperm staging) was performed on control males and males at 1000 mg/kg/day. The stages were checked for completeness of cell populations, completeness of stages and degenerative changes.

Litter observations:

The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum.

Postmortem examinations (parental animals):

TERMINATION AND NECROPSY
Males were sacrificed after they had been treated for at least 28 days. Dams were sacrificed on day 4 post partum. If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.
All animals sacrificed or found dead were subjected to a detailed macroscopic examination to establish, if possible, the cause of death. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution. At the scheduled sacrifice, all animals were killed by an injection of sodium pentobarbital. All P generation animals were exsanguinated.
All parent animals were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.
For the parent animals, special attention was directed at the organs of the reproductive system. The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

ORGAN WEIGHTS
At the scheduled sacrifice, the testes and epididymides of all parental males were weighed separately.

TISSUE PRESERVATION
The ovaries from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution. The testes and epididymides from all parental males were preserved in Bouin’s fixative. The prostate and seminal vesicles from all males were fixed in neutral phosphate buffered 4% formaldehyde solution.

HISTOTECHNIQUE
All organ and tissue samples to be examined by the principal investigator for histopathology were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis was stained by PAS-hematoxylin. Special stains were used at the discretion of the principal investigator for histopathology.

HISTOPATHOLGOLGY
Slides of all organs and tissues collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the principal investigator for histopathology. The same applied to all occurring gross lesions and to all animals, which died spontaneously or had to be terminated in extremis.
Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.
Histological examination of ovaries was carried out on any female that did not give birth. In addition, microscopic examination of the reproductive organs of all infertile males was made, if necessary.
A histopathology peer review was performed.

Postmortem examinations (offspring):

Pups were sacrificed on day 4 post partum. At the scheduled sacrifice, all animals were killed by an injection of sodium pentobarbital. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution. Dead pups, except those excessively cannibalized, were examined macroscopically.

Statistics:

The following statistical methods were used to analyze food consumption, body weights and reproduction data:
- Means and standard deviations of various data were calculated.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Reproductive indices:

From the on-line recorded reproduction data, the following parameters were calculated:
fertility indices, mean precoital time, post-implantation losses, mean litter size, pup sex ratios and viability indices.

Offspring viability indices:

From the on-line recorded reproduction data, the following parameters were calculated: dead/live pups at first litter check, pups sex ratio

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Yellow discoloration of feces in males and females of dose groups

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

1. IN-LIFE DATA
VIABILITY / MORTALITY
All animals survived the scheduled study period.

CLINICAL SIGNS OR OBSERVATIONS
There were no findings of toxicological relevance at any dose level.
In males, yellow discoloration of the feces was noted from treatment day 5 of the pre-pairing period and continued throughout the pairing period. The severity of this finding was generally dose-dependent, but was considered to be a typical passive effect that results from oral administration of a dyestuff and was of no toxicological relevance.
In females, a similar finding was noted from day 5 of the pre-pairing period and continued throughout the subsequent pairing, gestation and lactation periods. As in males, the severity of this finding was generally dose-dependent, and was ascribed the same lack of toxicological relevance as in the males. Slight hair loss was noted in a single female during the gestation period, but in view of the late onset and low incidence, was considered to be without toxicological implications.

FOOD CONSUMPTION OF MALES
Pre-pairing Period
In males, although the mean daily food consumption values at 300 mg/kg/day and 1000 mg/kg/ day were significantly lower (p<0.05) than that of the control males during days 8 - 14, these differences were not dose-related and were considered to be a result of an incidentally elevated control value rather than to the treatment with the test item.

FOOD CONSUMPTION OF FEMALES
Pre-pairing, Gestation and Lactation Periods
The mean daily food consumption values of the test item-treated females compared favorably with the control females throughout the pre-pairing, gestation and lactation periods.

BODY WEIGHTS OF MALES
Pre-Pairing and Pairing Periods
There were no differences of toxicological relevance between the mean body weights of test item-treated or control males. The mean body weight gain of all groups compared favorably.

BODY WEIGHTS OF FEMALES
Pre-Pairing, Pairing, Gestation and Lactation Periods
No test item-related effects on body weights and body weight gain of females were observed at any dose level.
In females treated with 1000 mg/kg/day, a significant increase (p<0.05) in mean body weight was noted on day 4 of the gestation period. This isolated finding was considered to be incidental. The body weights recorded during all other periods were similar to those of the control females.
At 300 mg/kg/day, all body weights were similar throughout the various phases.
At 100 mg/kg/day, significantly elevated mean body weights (p<0.05) were noted on days 0 - 5 of the gestation period only. In the absence of a dose-response relationship, these differences were considered to be incidental. The body weights recorded during all other periods were similar to the controls.

2. REPRODUCTION AND BREEDING
MATING PERFORMANCE AND FERTILITY
The mean and median precoital times for all groups were similar.
The respective parameters of the mating performance were unaffected as all dose levels and similar to those of the control values.
Mating of female no. 84 was not detected. Three females were not pregnant: no. 55 (control group) and nos. 81 ad 83 (1000 mg/kg/day).

DURATION OF GESTATION
No effects on duration of gestation were observed at any dose level.

CORPORA LUTEA COUNT
No effects on corpora lutea count were observed at any dose level.

IMPLANTATION RATE AND POST-IMPLANTATION LOSS
The implantation rate was unaffected in all groups.
Although the mean post-implantation loss was slightly higher at 300 mg/kg/day and 1000 mg/kg/ day when compared with the control females, the differences were not dose-related and therefore considered to be unrelated to the test item treatment. The total post implantation loss of the dams treated with 300 mg/kg/day was significantly elevated (p<0.01) when compared with the controls, but not dose-dependent and therefore considered to be unrelated to the treatment with the test item. The mean post implantation loss in females at 100 mg/kg/day was nearly identical to the controls.

LITTER SIZE AT FIRST LITTER CHECK
The mean litter sizes of the control group and those at 100 and at 1000 mg/kg/day were similar. The marginally lower mean litter size noted at 300 mg/kg/day coincided with the slightly higher post-implantation loss.

The overall mean numbers of living pups per dam at first litter check compared favorably in the control and test item-treated groups; three pups of litter no. 79 (1000 mg/kg/day), two pups of litter no. 70 (300 mg/kg/day) and four pups of litter no. 49 (control group) were found dead.

The respective birth indices (number of pups borne alive as a percentage of implantations) were similar in test item-treated and control animals.

3. TERMINAL FINDINGS
ORGAN WEIGHTS
No test item-related changes in organ weights were noted at any dose level.

At 300 mg/kg/day, the mean absolute epididymide weight (left side only) were significantly reduced (p<0.05) when compared with the controls. This finding was considered to be incidental as the mean relative organ weight was normal and unilateral organ weight differences are not associated with toxicological relevance.
At 100 mg/kg/day, the mean absolute epididymide weights (bilateral) were significantly reduced (p<0.05) when compared with the controls. These differences were considered to be related to the lower mean terminal body weights; the mean relative organ weights were unaffected.
No further changes of organ weights were noted at any dose level.

MACROSCOPICAL FINDINGS
A small number of typical background changes were noted in test item-treated and control males. Neither type nor incidence were considered to be commensurate with findings of toxicological relevance in males.
Discoloration of the ovaries found in one female at 1000 mg/kg/day and was considered to be within the range of normal background alterations.

HISTOPATHOLOGY FINDINGS
All microscopic findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age. There was no morphological evidence of toxicological properties in the testes, epididymides, prostate and seminal vesicles of male animals and ovaries of female animals examined in this study.
Detailed examination of the testes (including sperm staging) was performed on control males (nos. 1 - 11) and males at 1000 mg/kg/day. The stages were checked for completeness of cell populations, completeness of stages and degenerative changes. No differences on the completeness of stages or cell populations of the testes were recorded between the control males (nos. 1 - 11) and the males at 1000 mg/kg/day (nos. 34 - 44). In male no. 6 of the control group, unilateral Sertoli cell vacuolation (minimal in degree) was noted. Male no. 41 (treated with 1000 mg/kg/day) showed unilateral tubular degeneration/atrophy that was considered to be minimal in degree.

There were no findings or microscopical changes to the reproductive organs that were considered to be related to the infertility noted in male no. 40.

Dose descriptor:

NOAEL

Remarks:

for general toxicity

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed up to 1000 mg/kg bw/day, the highest dose level tested.

Dose descriptor:

NOAEL

Remarks:

for reproduction

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed up to 1000 mg/kg bw/day, the highest dose level tested.

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

POSTNATAL LOSS DAYS 0 - 4 POST PARTUM
From days 2 - 3 post partum, five male and seven female pups of dams treated with 1000 mg/kg/ day were missing or found dead, one female pup of a dam treated with 300 mg/kg/day were missing or found dead, one male and one female pup of dams treated with 100 mg/kg/day were missing or found dead, and three males and five female pups of control dams were missing or found dead. These minor differences were considered to be unrelated to the treatment with the test item. The statistically significant decrease noted in the total postnatal loss noted at 300 mg/kg/day (p<0.05) is not associated with toxicological relevance.

EXTERNAL EXAMINATION AT FIRST LITTER CHECK AND DURING LACTATION
No test item-related observations were noted in pups during the first litter check or during lactation at any dose level. In one litter (no. 49) of the control group, four pups were found to have no milk in the stomach. One litter ( no. 70) at 300 mg/kg/day had two pups without milk in the stomach and one litter (no. 79) at 1000 mg/kg/day had three pups without milk in the stomach. Insofar as these pups were found dead at first litter check, it is assumed they were stillborn or died during parturition.
During the lactation period, five pups of litter no. 49 (control) had missing body parts (most likely due to partial cannibalization) and two pups of litter no. 79 had no milk in the stomach.

SEX RATIOS: The sex ratio of the test item-treated pups did not indicate any effects of toxicological relevance.

BODY WEIGHTS TO DAY 4 POST PARTUM
No effects on pup body weights were noted at any dose level.

MACROSCOPICAL FINDINGS
No test item-related findings were found in pups at any dose level.

Dose descriptor:

NOAEL

Remarks:

for development

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed up to 1000 mg/kg bw/day, the highest dose level tested.

Reproductive effects observed:

not specified

SUMMARY OF PERFORMANCE

 

P Animals Breeding for F1 Litters

 

Group (mg/kg/day)	1 (0)	2 (100)	3 (300)	4 (1000)
Female numbers	45 - 55	56 - 66	67 - 77	78 - 88
Number of females paired	11	11	11	11
Number of females mated	11	11	11	11
Number of females which were not pregnant	1	0	0	2
Number of females which reared their pups until day 4 post partum	10	11	11	9

 

Conclusions:

This study according to OOECD TG 421 is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item to rats. The test item was administered in water as vehicle at dosages of 100, 300, and 1000 mg/kg body weight/day, and controls received the vehicle only. The test item was administered to male rats for 14 days prior to pairing and for 28 days in total, and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.
Test item-related effects were restricted to secondar effects only (discolored feces) and there were no findings of toxicological relevance. Body weight and food consumption of parent animals were unaffected, and no differences in organ weights were noted. There were no macroscopical or microscopical findings related to the treatment with the test item in parental animals nor offspring
No effects on mating performance, fertility, corpora lutea count or duration of gestation were observed at any dose level. Minor differences in the mean post-implantation loss, postnatal loss, and litter size at first litter check and day 4 post partum were either unrelated to dose or within the limits of the historical control data and therefore considered to be incidental.
No test item-related findings were noted at first litter check and during lactation in pups at any dose level. The sex ratios of the pups in test item-treated groups were normal and pup weights recorded until day 4 post partum compared favorably in all groups.
Because fecal discoloration (i.e. a secondary passive finding) was noted in treated rats at all dose levels, a NOEL (No Observed Effect Level) could not be established. The NOAEL (No Observed Adverse Effect Level) for general toxicity in males and females, as well as for reproduction data and F1 offspring was considered to be 1000 mg/kg bw/day, the highest dose level tested.

Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of the test item on male and female reproductive performance such as gonadal function, mating behavior, conception and parturition and to assess major effects on pup development.

Four groups of 11 males and 11 females were treated by gavage with test item once daily (according to OECD 421 and GLP compliant). Males were treated over a 14-day pre-pairing period and during the pairing period up to one day before necropsy. Females were treated throughout the pre-pairing, pairing, gestation and lactation period up to the day 3 post partum.

The following dose levels were used:

Group 1: 0 mg/kg body weight/day (control group)

Group 2: 100 mg/kg body weight/day

Group 3: 300 mg/kg body weight/day

Group 4: 1000 mg/kg body weight/day

 

A standard dose volume of 10 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (bidistilled water).

 

The following results were obtained:  

MORTALITY AND GENERAL TOLERABILITY OF PARENTAL ANIMALS

  All animals survived the scheduled study period.

  No clinical signs of toxicological relevance were noted at any dose level. In both sexes, yellow discoloration of the feces was noted from treatment day 5 of the pre-pairing period and continued throughout treatment. The severity of this finding was generally dose-dependent, but was considered to be a typical secondary passive effect that results from oral administration of a dyestuff and was of no toxicological relevance.

 

FOOD CONSUMPTION OF PARENTAL ANIMALS

  Minor intergroup differences in the mean daily food consumption of males were not considered to be related to the treatment with the test item. The daily food consumption values of the test item-treated females were unaffected.

 

BODY WEIGHTS OF PARENTAL ANIMALS: There were no effects upon body weights of either males or females.

 

REPRODUCTION AND BREEDING DATA

  No effects on mating performance, fertility, corpora lutea count or duration of gestation were observed at any dose level.

  At the dose level of 1000 mg/kg bw/day, higher incidence of post-implantation loss and higher postnatal loss as well as lower litter size at first litter check and on day 4 post partum were noted.

  The mean post implantation loss of the dams at 1000 mg/kg/day was close to the upper limit of the historical control data and was below the mean post implantation loss of the dams treated with 300 mg/kg/day; therefore this was considered to be unrelated to the treatment with the test item.

 

ORGAN WEIGHS OF PARENTAL ANIMALS: No test item-related effects on organ weights were noted at any dose level.

 

MACROSCOPICAL FINDINGS AND HISTOPATHOLOGICAL EXAMINATIONS OF PARENTAL ANIMALS

  No test item-related macroscopical findings were noted at any dose level.

  Under the conditions of this study, the test item produced no morphological evidence of toxicological properties in testes, epididymides, prostate and seminal vesicles of male animals and ovaries of female animals.

 

FINDINGS IN PUPS AT FIRST LITTER CHECK AND DURING LACTATION

  No test item-related findings were noted in pups at any dose level.

  The sex ratio of the test item-treated pups did not indicate any effects of toxicological relevance.

 

PUP WEIGHTS TO DAY 4 POST PARTUM:  No effects on pup body weights were noted at any dose level.

 

MACROSCOPICAL FINDINGS OF PUPS:  No test item-related findings were found in pups at any dose level.

Reference 4

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From 05 Jan 2012 to 31 Jul 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 421, GLP-compliant)

Justification for type of information:

See read across justification document in chapter 13

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Animals: Rat, RccHanTM: WIST(SPF)
- Rationale: Recognized by international guidelines as a recommended test system.
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Number of Animals: 44 males (11 per group) and 44 females (11 per group)
- Age (at delivery): 10 weeks
- Body Weight Range (at Start of Treatment): 301 to 344 g (males) and 191 to 228 g (females)
- Identification: Cage card and individual animal number (ear tattoo).
- Randomization: Computer-generated random algorithm. In addition body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
- Accommodation: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J.Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland; batch/lot no. 02105111001) with paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey / UK), batch/lot no. 55/T-6060). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet: Pelleted standard Harlan Teklad 2914C (batch no. 73/11) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum. Results of representative analyses for contaminants were included in the study report.
- Water: Community tap-water from Itingen was available ad libitum in water bottles. Results of bacteriological assay, chemical and contaminant analyses of representative samples were included in the study report.
- Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study. At least 5 days.

ENVIRONMENTAL CONDITIONS
Standard laboratory conditions, continuously monitored.
- Temperature (°C): 22 +/- 3
- Humidity (%): 30 to 70
- Air changes (per hr): . Air-conditioned with 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12 (with at least eight hours music during the light period)

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

DOSE FORMULATIONS
The dose formulations were not corrected for purity and were prepared as supplied by the Sponsor. The dose formulations were prepared weekly as indicated by the results of stability analyses in the non-GLP dose range-finding study. The test item was weighed into a glass beaker on a tared Mettler balance and the vehicle was added. The mixtures were stirred using a magnetic stirrer and stored at room temperature (20 °C +/- 5 °C). Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

STORAGE OF DOSE FORMULATIONS
The dose formulations were stored at room temperature (20 +/- 5 °C) in glass beakers. They were stable for at least 8 days, based upon the results of stability analyses performed during the non-GLP dose range-finding study.

TREATMENT
- Method: Oral, by gavage
- Rationale for Method: Administration by gavage is a common and accepted route of exposure for this type of studies.
- Frequency of Administration: Once daily
- Daily Target Dose Level: 0 mg/kg/day (control group), 100 mg/kg/day (group 2), 300 mg/kg/day (group 3), 1000 mg/kg/day (group 4)
- Rationale for Dose Level Selection: The dose levels were selected based on a previous dose range-finding toxicity study in Wistar rats, Harlan Laboratories study (non-GLP).
- Dose Volume: 10 mL/kg body weight
- Dose Concentrations: 0 mg/mL/day (control group), 10 mg/mL/day (group 2), 30 mg/mL/day (group 3), 100 mg/mL/day (group 4)

Details on mating procedure:

During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed. The females were removed and housed individually if the daily vaginal smear was sperm positive, or a copulation plug was observed.

The day on which a positive mating was determined (copulation plug or sperm) was designated day 0 post coitum.

If a female did not mate during the 14-day pairing period, a second pairing of this female with a male in the same group, which had already mated successfully, was considered. If mating was not recorded during this additional pairing period of a maximum of 14 days, the female was sacrificed and, if indicated, the reproductive organs examined histopathologically in order to ascertain the reason for the infertility.

All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

METHOD
After experimental start, a sample from the control group as well as three samples (top, middle and bottom) of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples were also taken to confirm stability (4 hours and 8 days at room temperature). During week 3 of treatment, samples were also taken to confirm homogeneity and concentration. The aliquots for analysis of dose formulations were stored at -20 +/- 5°C until analysis. The test item was used as the analytical standard.

RESULTS
The linearity of the analytical systems used for sample analyses was demonstrated with a good relationship between absorbance measured and working standard concentrations. All calibration points met the acceptance limit of +/- 20% variation from the calibration curve derived by linear regression analysis. The regression coefficients calculated were found to be better than 0.99.

Blank samples showed no significant absorbance and, therefore, it was confirmed that only bidistilled water was applied within the control experiment.

The application formulations investigated during the study were found to comprise test material in the range of 93.8% to 108.3% and, thus, the required content limit of +/-20% with reference to the nominal content was met. The homogeneous distribution of the test item in the preparations was approved because single results found did not deviate more than 3% (acceptance criterion: <15%) from the corresponding mean.

In addition, the test item was found to be stable in application formulations when kept four hours or eight days at room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean.

In conclusion, the results indicate the accurate use of the test item and bidistilled water as vehicle during this study. Application formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.

Duration of treatment / exposure:

Minimum 4 weeks (males) and approximately 7 weeks (females)

Frequency of treatment:

Once daily

Details on study schedule:

MALES
- Acclimatization: 5 days minimum
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum
- Treatment Ends: On day before sacrifice
- Necropsy: After a minimum of 28 days treatment

FEMALES
- Acclimatization: 5 days minimum
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum
- Gestation: Approximately 21 days
- Treatment Ends: On day 3 post partum
- Necropsy: On day 4 post partum

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

11

Control animals:

yes, concurrent vehicle

Details on study design:

The purpose of this study was to generate preliminary information concerning the effects of the test item on male and female reproductive performance such as gonadal function, mating behavior, conception and parturition and to assess major effects on pup development.

Positive control:

No

Parental animals: Observations and examinations:

VIABILITY/MORTALITY: Twice daily

CLINICAL SIGNS: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

FOOD CONSUMPTION
Males: Weekly during pre-pairing and after pairing periods.
Females: Pre-pairing period days 1 - 8 and 8 - 14, gestation period days 0 - 7, 7 - 14 and 14 - 21 post coitum and lactation period days 1 - 4 post partum.
No food consumption was recorded during the pairing period.

BODY WEIGHTS
Recorded daily from treatment start to day of necropsy.

Oestrous cyclicity (parental animals):

Not examined

Sperm parameters (parental animals):

Organ weight:
Testes and epididymides of all parental males were weighed

Histopathology:
Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.

Detailed examination of the testes (including sperm staging) was performed on control males and males at 1000 mg/kg/day. The stages were checked for completeness of cell populations, completeness of stages and degenerative changes.

Litter observations:

The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum.

Postmortem examinations (parental animals):

TERMINATION AND NECROPSY
Males were sacrificed after they had been treated for at least 28 days. Dams were sacrificed on day 4 post partum. If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.
All animals sacrificed or found dead were subjected to a detailed macroscopic examination to establish, if possible, the cause of death. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution. At the scheduled sacrifice, all animals were killed by an injection of sodium pentobarbital. All P generation animals were exsanguinated.
All parent animals were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.
For the parent animals, special attention was directed at the organs of the reproductive system. The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

ORGAN WEIGHTS
At the scheduled sacrifice, the testes and epididymides of all parental males were weighed separately.

TISSUE PRESERVATION
The ovaries from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution. The testes and epididymides from all parental males were preserved in Bouin’s fixative. The prostate and seminal vesicles from all males were fixed in neutral phosphate buffered 4% formaldehyde solution.

HISTOTECHNIQUE
All organ and tissue samples to be examined by the principal investigator for histopathology were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis was stained by PAS-hematoxylin. Special stains were used at the discretion of the principal investigator for histopathology.

HISTOPATHOLGOLGY
Slides of all organs and tissues collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the principal investigator for histopathology. The same applied to all occurring gross lesions and to all animals, which died spontaneously or had to be terminated in extremis.
Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.
Histological examination of ovaries was carried out on any female that did not give birth. In addition, microscopic examination of the reproductive organs of all infertile males was made, if necessary.
A histopathology peer review was performed.

Postmortem examinations (offspring):

Pups were sacrificed on day 4 post partum. At the scheduled sacrifice, all animals were killed by an injection of sodium pentobarbital. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution. Dead pups, except those excessively cannibalized, were examined macroscopically.

Statistics:

The following statistical methods were used to analyze food consumption, body weights and reproduction data:
- Means and standard deviations of various data were calculated.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Reproductive indices:

From the on-line recorded reproduction data, the following parameters were calculated:
fertility indices, mean precoital time, post-implantation losses, mean litter size, pup sex ratios and viability indices.

Offspring viability indices:

From the on-line recorded reproduction data, the following parameters were calculated: dead/live pups at first litter check, pups sex ratio

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Yellow discoloration of feces in males and females of dose groups

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

1. IN-LIFE DATA
VIABILITY / MORTALITY
All animals survived the scheduled study period.

CLINICAL SIGNS OR OBSERVATIONS
There were no findings of toxicological relevance at any dose level.
In males, yellow discoloration of the feces was noted from treatment day 5 of the pre-pairing period and continued throughout the pairing period. The severity of this finding was generally dose-dependent, but was considered to be a typical passive effect that results from oral administration of a dyestuff and was of no toxicological relevance.
In females, a similar finding was noted from day 5 of the pre-pairing period and continued throughout the subsequent pairing, gestation and lactation periods. As in males, the severity of this finding was generally dose-dependent, and was ascribed the same lack of toxicological relevance as in the males. Slight hair loss was noted in a single female during the gestation period, but in view of the late onset and low incidence, was considered to be without toxicological implications.

FOOD CONSUMPTION OF MALES
Pre-pairing Period
In males, although the mean daily food consumption values at 300 mg/kg/day and 1000 mg/kg/ day were significantly lower (p<0.05) than that of the control males during days 8 - 14, these differences were not dose-related and were considered to be a result of an incidentally elevated control value rather than to the treatment with the test item.

FOOD CONSUMPTION OF FEMALES
Pre-pairing, Gestation and Lactation Periods
The mean daily food consumption values of the test item-treated females compared favorably with the control females throughout the pre-pairing, gestation and lactation periods.

BODY WEIGHTS OF MALES
Pre-Pairing and Pairing Periods
There were no differences of toxicological relevance between the mean body weights of test item-treated or control males. The mean body weight gain of all groups compared favorably.

BODY WEIGHTS OF FEMALES
Pre-Pairing, Pairing, Gestation and Lactation Periods
No test item-related effects on body weights and body weight gain of females were observed at any dose level.
In females treated with 1000 mg/kg/day, a significant increase (p<0.05) in mean body weight was noted on day 4 of the gestation period. This isolated finding was considered to be incidental. The body weights recorded during all other periods were similar to those of the control females.
At 300 mg/kg/day, all body weights were similar throughout the various phases.
At 100 mg/kg/day, significantly elevated mean body weights (p<0.05) were noted on days 0 - 5 of the gestation period only. In the absence of a dose-response relationship, these differences were considered to be incidental. The body weights recorded during all other periods were similar to the controls.

2. REPRODUCTION AND BREEDING
MATING PERFORMANCE AND FERTILITY
The mean and median precoital times for all groups were similar.
The respective parameters of the mating performance were unaffected as all dose levels and similar to those of the control values.
Mating of female no. 84 was not detected. Three females were not pregnant: no. 55 (control group) and nos. 81 ad 83 (1000 mg/kg/day).

DURATION OF GESTATION
No effects on duration of gestation were observed at any dose level.

CORPORA LUTEA COUNT
No effects on corpora lutea count were observed at any dose level.

IMPLANTATION RATE AND POST-IMPLANTATION LOSS
The implantation rate was unaffected in all groups.
Although the mean post-implantation loss was slightly higher at 300 mg/kg/day and 1000 mg/kg/ day when compared with the control females, the differences were not dose-related and therefore considered to be unrelated to the test item treatment. The total post implantation loss of the dams treated with 300 mg/kg/day was significantly elevated (p<0.01) when compared with the controls, but not dose-dependent and therefore considered to be unrelated to the treatment with the test item. The mean post implantation loss in females at 100 mg/kg/day was nearly identical to the controls.

LITTER SIZE AT FIRST LITTER CHECK
The mean litter sizes of the control group and those at 100 and at 1000 mg/kg/day were similar. The marginally lower mean litter size noted at 300 mg/kg/day coincided with the slightly higher post-implantation loss.

The overall mean numbers of living pups per dam at first litter check compared favorably in the control and test item-treated groups; three pups of litter no. 79 (1000 mg/kg/day), two pups of litter no. 70 (300 mg/kg/day) and four pups of litter no. 49 (control group) were found dead.

The respective birth indices (number of pups borne alive as a percentage of implantations) were similar in test item-treated and control animals.

3. TERMINAL FINDINGS
ORGAN WEIGHTS
No test item-related changes in organ weights were noted at any dose level.

At 300 mg/kg/day, the mean absolute epididymide weight (left side only) were significantly reduced (p<0.05) when compared with the controls. This finding was considered to be incidental as the mean relative organ weight was normal and unilateral organ weight differences are not associated with toxicological relevance.
At 100 mg/kg/day, the mean absolute epididymide weights (bilateral) were significantly reduced (p<0.05) when compared with the controls. These differences were considered to be related to the lower mean terminal body weights; the mean relative organ weights were unaffected.
No further changes of organ weights were noted at any dose level.

MACROSCOPICAL FINDINGS
A small number of typical background changes were noted in test item-treated and control males. Neither type nor incidence were considered to be commensurate with findings of toxicological relevance in males.
Discoloration of the ovaries found in one female at 1000 mg/kg/day and was considered to be within the range of normal background alterations.

HISTOPATHOLOGY FINDINGS
All microscopic findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age. There was no morphological evidence of toxicological properties in the testes, epididymides, prostate and seminal vesicles of male animals and ovaries of female animals examined in this study.
Detailed examination of the testes (including sperm staging) was performed on control males (nos. 1 - 11) and males at 1000 mg/kg/day. The stages were checked for completeness of cell populations, completeness of stages and degenerative changes. No differences on the completeness of stages or cell populations of the testes were recorded between the control males (nos. 1 - 11) and the males at 1000 mg/kg/day (nos. 34 - 44). In male no. 6 of the control group, unilateral Sertoli cell vacuolation (minimal in degree) was noted. Male no. 41 (treated with 1000 mg/kg/day) showed unilateral tubular degeneration/atrophy that was considered to be minimal in degree.

There were no findings or microscopical changes to the reproductive organs that were considered to be related to the infertility noted in male no. 40.

Dose descriptor:

NOAEL

Remarks:

for general toxicity

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed up to 1000 mg/kg bw/day, the highest dose level tested.

Dose descriptor:

NOAEL

Remarks:

for reproduction

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed up to 1000 mg/kg bw/day, the highest dose level tested.

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

POSTNATAL LOSS DAYS 0 - 4 POST PARTUM
From days 2 - 3 post partum, five male and seven female pups of dams treated with 1000 mg/kg/ day were missing or found dead, one female pup of a dam treated with 300 mg/kg/day were missing or found dead, one male and one female pup of dams treated with 100 mg/kg/day were missing or found dead, and three males and five female pups of control dams were missing or found dead. These minor differences were considered to be unrelated to the treatment with the test item. The statistically significant decrease noted in the total postnatal loss noted at 300 mg/kg/day (p<0.05) is not associated with toxicological relevance.

EXTERNAL EXAMINATION AT FIRST LITTER CHECK AND DURING LACTATION
No test item-related observations were noted in pups during the first litter check or during lactation at any dose level. In one litter (no. 49) of the control group, four pups were found to have no milk in the stomach. One litter ( no. 70) at 300 mg/kg/day had two pups without milk in the stomach and one litter (no. 79) at 1000 mg/kg/day had three pups without milk in the stomach. Insofar as these pups were found dead at first litter check, it is assumed they were stillborn or died during parturition.
During the lactation period, five pups of litter no. 49 (control) had missing body parts (most likely due to partial cannibalization) and two pups of litter no. 79 had no milk in the stomach.

SEX RATIOS: The sex ratio of the test item-treated pups did not indicate any effects of toxicological relevance.

BODY WEIGHTS TO DAY 4 POST PARTUM
No effects on pup body weights were noted at any dose level.

MACROSCOPICAL FINDINGS
No test item-related findings were found in pups at any dose level.

Dose descriptor:

NOAEL

Remarks:

for development

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed up to 1000 mg/kg bw/day, the highest dose level tested.

Reproductive effects observed:

not specified

SUMMARY OF PERFORMANCE

 

P Animals Breeding for F1 Litters

 

Group (mg/kg/day)	1 (0)	2 (100)	3 (300)	4 (1000)
Female numbers	45 - 55	56 - 66	67 - 77	78 - 88
Number of females paired	11	11	11	11
Number of females mated	11	11	11	11
Number of females which were not pregnant	1	0	0	2
Number of females which reared their pups until day 4 post partum	10	11	11	9

 

Conclusions:

This study according to OECD TG 421 is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item to rats. The test item was administered in water as vehicle at dosages of 100, 300, and 1000 mg/kg body weight/day, and controls received the vehicle only. The test item was administered to male rats for 14 days prior to pairing and for 28 days in total, and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.
Test item-related effects were restricted to secondar effects only (discolored feces) and there were no findings of toxicological relevance. Body weight and food consumption of parent animals were unaffected, and no differences in organ weights were noted. There were no macroscopical or microscopical findings related to the treatment with the test item in parental animals nor offspring
No effects on mating performance, fertility, corpora lutea count or duration of gestation were observed at any dose level. Minor differences in the mean post-implantation loss, postnatal loss, and litter size at first litter check and day 4 post partum were either unrelated to dose or within the limits of the historical control data and therefore considered to be incidental.
No test item-related findings were noted at first litter check and during lactation in pups at any dose level. The sex ratios of the pups in test item-treated groups were normal and pup weights recorded until day 4 post partum compared favorably in all groups.
Because fecal discoloration (i.e. a secondary passive finding) was noted in treated rats at all dose levels, a NOEL (No Observed Effect Level) could not be established. The NOAEL (No Observed Adverse Effect Level) for general toxicity in males and females, as well as for reproduction data and F1 offspring was considered to be 1000 mg/kg bw/day, the highest dose level tested.

Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of the test item on male and female reproductive performance such as gonadal function, mating behavior, conception and parturition and to assess major effects on pup development.

Four groups of 11 males and 11 females were treated by gavage with test item once daily (according to OECD 421 and GLP compliant). Males were treated over a 14-day pre-pairing period and during the pairing period up to one day before necropsy. Females were treated throughout the pre-pairing, pairing, gestation and lactation period up to the day 3 post partum.

The following dose levels were used:

Group 1: 0 mg/kg body weight/day (control group)

Group 2: 100 mg/kg body weight/day

Group 3: 300 mg/kg body weight/day

Group 4: 1000 mg/kg body weight/day

 

A standard dose volume of 10 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (bidistilled water).

 

The following results were obtained:  

MORTALITY AND GENERAL TOLERABILITY OF PARENTAL ANIMALS

  All animals survived the scheduled study period.

  No clinical signs of toxicological relevance were noted at any dose level. In both sexes, yellow discoloration of the feces was noted from treatment day 5 of the pre-pairing period and continued throughout treatment. The severity of this finding was generally dose-dependent, but was considered to be a typical secondary passive effect that results from oral administration of a dyestuff and was of no toxicological relevance.

 

FOOD CONSUMPTION OF PARENTAL ANIMALS

  Minor intergroup differences in the mean daily food consumption of males were not considered to be related to the treatment with the test item. The daily food consumption values of the test item-treated females were unaffected.

 

BODY WEIGHTS OF PARENTAL ANIMALS: There were no effects upon body weights of either males or females.

 

REPRODUCTION AND BREEDING DATA

  No effects on mating performance, fertility, corpora lutea count or duration of gestation were observed at any dose level.

  At the dose level of 1000 mg/kg bw/day, higher incidence of post-implantation loss and higher postnatal loss as well as lower litter size at first litter check and on day 4 post partum were noted.

  The mean post implantation loss of the dams at 1000 mg/kg/day was close to the upper limit of the historical control data and was below the mean post implantation loss of the dams treated with 300 mg/kg/day; therefore this was considered to be unrelated to the treatment with the test item.

 

ORGAN WEIGHS OF PARENTAL ANIMALS: No test item-related effects on organ weights were noted at any dose level.

 

MACROSCOPICAL FINDINGS AND HISTOPATHOLOGICAL EXAMINATIONS OF PARENTAL ANIMALS

  No test item-related macroscopical findings were noted at any dose level.

  Under the conditions of this study, the test item produced no morphological evidence of toxicological properties in testes, epididymides, prostate and seminal vesicles of male animals and ovaries of female animals.

 

FINDINGS IN PUPS AT FIRST LITTER CHECK AND DURING LACTATION

  No test item-related findings were noted in pups at any dose level.

  The sex ratio of the test item-treated pups did not indicate any effects of toxicological relevance.

 

PUP WEIGHTS TO DAY 4 POST PARTUM:  No effects on pup body weights were noted at any dose level.

 

MACROSCOPICAL FINDINGS OF PUPS:  No test item-related findings were found in pups at any dose level.

Effect on fertility: via oral route

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

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

Based on the findings in a n OECD 414 study on a close structrual analogue, it is concluded that, No Observed Adverse Effect Level (NOAEL) for:

Maternal toxicity, fetal developmental toxicity and Teratogencity is 1000 mg/kg/day as

- the maternal body weight and weight gain, corrected body weight gain and food consumption was unaffected up to 1000 mg/kg/day.

- fetal resorptions or post implantation loss were comparable to the controls and there were no effects on fetal body weights.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

See read across document in chapter 13

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Source: Hylasco Biotechnology (India) Pvt. Ltd.
Plot 4B, MN Park,
Shameerpet Mandal,
Turkapally Village,
Medchal District, Telangana -500078

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for analysis of homogeneity and active ingredient (a.i.) were collected from the dose formulations intended for first treatment for the first batch of rats and last treatment (-2 days). The prepared formulations were sampled in duplicate sets wherein one set was used for analysis and another as back up set which was stored at ambient condition. For each set, duplicate sample were drawn from top, middle and bottom layers of each preparation and in case of control duplicate samples from the middle layer was drawn. The samples collected were sent to Analytical R&D of Eurofins Advinus Ltd. for formulation analysis to determine the homogeneity and concentration of the dose formulation.

Details on mating procedure:

The female rats were cohabited with males in a 1:1 ratio and vaginal smears and / or vaginal plug were examined in the morning hours of the subsequent day to confirm mating.

Duration of treatment / exposure:

Gestation day 5 to gestation day 19

Frequency of treatment:

Daily from gestation day 5 to gestation day 19

Duration of test:

Upto gestation day 20

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control

Dose / conc.:

111 mg/kg bw/day (nominal)

Remarks:

Low dose

Dose / conc.:

333 mg/kg bw/day (nominal)

Remarks:

Mid dose

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High dose

No. of animals per sex per dose:

24 day 0 pergnant rats per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Group Nos. Groups Dose
(mg/kg/day) Dosage volume (mL/kg) Concent-ration (mg/mL) No. of Day 0 pregnant rats Rat numbers
From To
G1 Vehicle control 0 10 0 24 Rz221 Rz244
G2 Low dose 111 10 11.1 24 Rz245 Rz268
G3 Mid dose 333 10 33.3 24 Rz269 Rz292
G4 High dose 1000 10 100 24 Rz293 Rz316

Maternal examinations:

CAGE SIDE OBSERVATION: Yes
- Time schedule: Twice a day (pre dose and post dose) during treatment period
- Cage side observation checked in table 2 were included

Ovaries and uterine content:

The ovaries and uterine contents were examined after termination GD 20.
• Pregnancy status
• Gravid uterine weight (from all rats subjected to caesarean section)
• Number of corpora lutea
• Number of implantation sites
• Number of early resorptions
• Number of late resorptions
• Gross evaluation of placenta

Fetal examinations:

• Total number of fetuses
• Total number of live fetuses
• Total number of dead fetuses
• Individual fetal body weight
• Fetus sex (during visceral examination)
• External examination of fetus
• Soft tissue evaluation
• Skeletal examination
• Head examination (half the number of fetuses per litter)

Statistics:

The data on maternal body weight, body weight change in interval, gravid uterine weight, body weight change corrected to gravid uterine weight, maternal food consumption, hormone analyses (T4, T3, TSH), weight of thyroid gland was analyzed using Analysis of Variance (ANOVA) after testing for homogeneity for intra group variance using Levene’s test. Where intra group variances were heterogeneous, ANOVA was performed after suitable transformation of data. Dunnett’s pairwise comparison of the treated group means with the control group mean was performed, when the group differences were found significant.

Fetal weight for male and female was analyzed using Analysis of Covariance (ANCOVA) taking litter size as covariate for group. Anogenital distance for male and female was analyzed using Analysis of Covariance (ANCOVA) taking weight as covariate for group.

Number of corpora lutea, number of implantations, early and late resorptions, pre-implantation and post-implantation loss observations were analyzed using Kruskal Wallis test for group comparison. Wilcoxon pairwise comparison of the treated groups with the control group was performed, when the group differences were significant.

The incidence of dams with resorptions were tested for using Chi-square test followed by Fisher’s exact test for group association.

The incidence of fetus and litter (incidence and percent) observations for external, visceral and skeletal observations were tested using Cochran Armitage trend test and pair wise comparison will be tested by Fisher’s exact test for group association.

Statistically significant differences (p<0.05), indicated by the aforementioned tests was designated as * throughout the report.

Indices:

Refer Table 6 of the final report

Historical control data:

Refer Annexure 8 of the final report

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Grossly, orange contents noted in cecum at 333 and 1000 mg/kg/day and in ileum and colon at 1000 mg/kg/day was also related to the physical nature of test item and there were no other gross findings in the intestinal mucosa.

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormone levels (T3, T4 and TSH), thyroid gland weights and histology of thyroid gland were unaffected by treatment with Novoperm-Orange HL (C.I. Pigment Orange 36) up to the highest dose of 1000 mg/kg/day

Number of abortions:

no effects observed

Description (incidence and severity):

No abortions in the study

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

changes in number of pregnant

changes in pregnancy duration

clinical signs

dead fetuses

early or late resorptions

effects on pregnancy duration

food consumption and compound intake

gross pathology

mortality

necropsy findings

organ weights and organ / body weight ratios

pre and post implantation loss

total litter losses by resorption

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

changes in postnatal survival

external malformations

skeletal malformations

visceral malformations

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

Based on the above findings, it is concluded that, No Observed Adverse- Effect Level (NOAEL) for

• Maternal toxicity is 1000 mg/kg/day as the maternal body weight and weight gain, corrected body weight gain and food consumption was unaffected up to 1000 mg/kg/day.

• Fetal developmental toxicity and Teratogencity is 1000 mg/kg/day as fetal resorptions or post implantation loss were comparable to the controls, no effects on fetal body weights and further the fetal external, visceral and skeletal examinations revealed no signs of teratogenicity or developmental toxicity up to 1000 mg/kg/day

Executive summary:

The objective of this study was to evaluate the developmental toxicity (teratogenic) potential of the test item to cause adverse effects on the pregnant female rats and development of the embryo and fetus consequent to exposure of pregnant rats by oral route during gestation days (GD) 5 to 19. This study was intended to provide a rational basis for risk assessment in humans and to establish a No Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity in rats.

 

A total of 96Day 0 pregnant rats[1]were randomly divided into different groups according to the study design as follows:

Group Nos.	Groups	Dose (mg/kg/day)	Dosage volume (mL/kg)	Concentration (mg/mL)	No. of Day 0 pregnant rats
G1	Vehicle control*	0	10	0	24
G2	Low dose	111	10	11.1	24
G3	Mid dose	333	10	33.3	24
G4	High dose	1000	10	100	24

*0.5 % Carboxymethylcellulose Sodium salt (medium viscosity) in Milli-Q^®Water

 

The following parameters and end points were evaluated in this study: Clinical signs, body weights, body weight gains, food consumption, gross pathology, gravid uterine weights, intrauterine growth and survival,number of corpora lutea, and fetal parameters [sex, weight and anogenital distance, and external, visceral and skeletal observations].Approximately half of the fetuses from each litter were examined for visceral malformations and the remaining half were evaluated for skeletal malformations. In addition, from each dam the thyroids were weighed and subjected to microscopic evaluation, and thyroid hormones were estimated from the blood collected at terminal sacrifie (GD20).

 

Results of the study are summarized below:

 

·        Clinical signs and gross necropsy changes: There were no clinical signs, or mortalities in treated rats at any of the doses tested.Expected light to dark orange coloured faeces were observed in the test item treated groups which can be accounted for the physical nature of the test item.

Grossly, orangecontents noted in cecum at 333 and 1000 mg/kg/day and in ileum and colon at 1000 mg/kg/day was also related to the physical nature of test item and there were no other gross findings in the intestinal mucosa.

·        Maternal Parameters: No treatment-related effects on maternal body weights and food consumption up to the highest tested dose of
1000 mg/kg/day. The other maternal parameters comprising of uterine weight, implantations and early and late resorptions, post implantation loss were comparable to vehicle control group up to the high dose of 1000 mg/kg/day. Gross evaluation of placenta revealed no remarkable findings.

·        Litter Parameters: No treatment-related effects on litter parameters comprising of total number of fetuses, fetal weights, anogenital distance in male and female fetuses, were observed.

·        Fetal examination: The fetal external, visceral and skeletal examinations revealed no signs of teratogenicity or developmental toxicity up to
1000 mg/kg/day.

·        Thyroid hormone levels (T3, T4 and TSH), thyroid gland weights and histology of thyroid gland were unaffected by treatment with C.I. Pigment Orange 36 up to the highest dose of
1000 mg/kg/day.

 

Based on the above findings, it is concluded that, No Observed Adverse Effect Level (NOAEL) for

 

·           Maternal toxicity is 1000 mg/kg/day as the maternal body weight and weight gain, corrected body weight gain and food consumption was unaffected up to 1000 mg/kg/day.

 

Fetal developmental toxicity and Teratogencity is 1000 mg/kg/day as fetal resorptions or post implantation loss were comparable to the controls, no effects on fetal body weights and further the fetal external, visceral and skeletal examinations revealed no signs of teratogenicity or developmental toxicity up to 1000 mg/kg/day

[1]The day of confirmed mating (sperm positive vaginal smear or presence of vaginal plug) was designated as GD 0.

 

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Source: Hylasco Biotechnology (India) Pvt. Ltd.
Plot 4B, MN Park,
Shameerpet Mandal,
Turkapally Village,
Medchal District, Telangana -500078

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for analysis of homogeneity and active ingredient (a.i.) were collected from the dose formulations intended for first treatment for the first batch of rats and last treatment (-2 days). The prepared formulations were sampled in duplicate sets wherein one set was used for analysis and another as back up set which was stored at ambient condition. For each set, duplicate sample were drawn from top, middle and bottom layers of each preparation and in case of control duplicate samples from the middle layer was drawn. The samples collected were sent to Analytical R&D of Eurofins Advinus Ltd. for formulation analysis to determine the homogeneity and concentration of the dose formulation.

Details on mating procedure:

The female rats were cohabited with males in a 1:1 ratio and vaginal smears and / or vaginal plug were examined in the morning hours of the subsequent day to confirm mating.

Duration of treatment / exposure:

Gestation day 5 to gestation day 19

Frequency of treatment:

Daily from gestation day 5 to gestation day 19

Duration of test:

Upto gestation day 20

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control

Dose / conc.:

111 mg/kg bw/day (nominal)

Remarks:

Low dose

Dose / conc.:

333 mg/kg bw/day (nominal)

Remarks:

Mid dose

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High dose

No. of animals per sex per dose:

24 day 0 pergnant rats per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Group Nos. Groups Dose
(mg/kg/day) Dosage volume (mL/kg) Concent-ration (mg/mL) No. of Day 0 pregnant rats Rat numbers
From To
G1 Vehicle control 0 10 0 24 Rz221 Rz244
G2 Low dose 111 10 11.1 24 Rz245 Rz268
G3 Mid dose 333 10 33.3 24 Rz269 Rz292
G4 High dose 1000 10 100 24 Rz293 Rz316

Maternal examinations:

CAGE SIDE OBSERVATION: Yes
- Time schedule: Twice a day (pre dose and post dose) during treatment period
- Cage side observation checked in table 2 were included

Ovaries and uterine content:

The ovaries and uterine contents were examined after termination GD 20.
• Pregnancy status
• Gravid uterine weight (from all rats subjected to caesarean section)
• Number of corpora lutea
• Number of implantation sites
• Number of early resorptions
• Number of late resorptions
• Gross evaluation of placenta

Fetal examinations:

• Total number of fetuses
• Total number of live fetuses
• Total number of dead fetuses
• Individual fetal body weight
• Fetus sex (during visceral examination)
• External examination of fetus
• Soft tissue evaluation
• Skeletal examination
• Head examination (half the number of fetuses per litter)

Statistics:

The data on maternal body weight, body weight change in interval, gravid uterine weight, body weight change corrected to gravid uterine weight, maternal food consumption, hormone analyses (T4, T3, TSH), weight of thyroid gland was analyzed using Analysis of Variance (ANOVA) after testing for homogeneity for intra group variance using Levene’s test. Where intra group variances were heterogeneous, ANOVA was performed after suitable transformation of data. Dunnett’s pairwise comparison of the treated group means with the control group mean was performed, when the group differences were found significant.

Fetal weight for male and female was analyzed using Analysis of Covariance (ANCOVA) taking litter size as covariate for group. Anogenital distance for male and female was analyzed using Analysis of Covariance (ANCOVA) taking weight as covariate for group.

Number of corpora lutea, number of implantations, early and late resorptions, pre-implantation and post-implantation loss observations were analyzed using Kruskal Wallis test for group comparison. Wilcoxon pairwise comparison of the treated groups with the control group was performed, when the group differences were significant.

The incidence of dams with resorptions were tested for using Chi-square test followed by Fisher’s exact test for group association.

The incidence of fetus and litter (incidence and percent) observations for external, visceral and skeletal observations were tested using Cochran Armitage trend test and pair wise comparison will be tested by Fisher’s exact test for group association.

Statistically significant differences (p<0.05), indicated by the aforementioned tests was designated as * throughout the report.

Indices:

Refer Table 6 of the final report

Historical control data:

Refer Annexure 8 of the final report

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Grossly, orange contents noted in cecum at 333 and 1000 mg/kg/day and in ileum and colon at 1000 mg/kg/day was also related to the physical nature of test item and there were no other gross findings in the intestinal mucosa.

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormone levels (T3, T4 and TSH), thyroid gland weights and histology of thyroid gland were unaffected by treatment with Novoperm-Orange HL (C.I. Pigment Orange 36) up to the highest dose of 1000 mg/kg/day

Number of abortions:

no effects observed

Description (incidence and severity):

No abortions in the study

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

changes in number of pregnant

changes in pregnancy duration

clinical signs

dead fetuses

early or late resorptions

effects on pregnancy duration

food consumption and compound intake

gross pathology

mortality

necropsy findings

organ weights and organ / body weight ratios

pre and post implantation loss

total litter losses by resorption

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

changes in postnatal survival

external malformations

skeletal malformations

visceral malformations

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

Based on the above findings, it is concluded that, No Observed Adverse- Effect Level (NOAEL) for

• Maternal toxicity is 1000 mg/kg/day as the maternal body weight and weight gain, corrected body weight gain and food consumption was unaffected up to 1000 mg/kg/day.

• Fetal developmental toxicity and Teratogencity is 1000 mg/kg/day as fetal resorptions or post implantation loss were comparable to the controls, no effects on fetal body weights and further the fetal external, visceral and skeletal examinations revealed no signs of teratogenicity or developmental toxicity up to 1000 mg/kg/day

Executive summary:

The objective of this study was to evaluate the developmental toxicity (teratogenic) potential of the test item C.I. Pigment Orange 36 to cause adverse effects on the pregnant female rats and development of the embryo and fetus consequent to exposure of C.I. Pigment Orange 36 to pregnant rats by oral route during gestation days (GD) 5 to 19. This study was intended to provide a rational basis for risk assessment in humans and to establish a No Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity in rats.

 

A total of 96Day 0 pregnant rats[1]were randomly divided into different groups according to the study design as follows:

Group Nos.	Groups	Dose (mg/kg/day)	Dosage volume (mL/kg)	Concentration (mg/mL)	No. of Day 0 pregnant rats
G1	Vehicle control*	0	10	0	24
G2	Low dose	111	10	11.1	24
G3	Mid dose	333	10	33.3	24
G4	High dose	1000	10	100	24

*0.5 % Carboxymethylcellulose Sodium salt (medium viscosity) in Milli-Q^®Water

 

The following parameters and end points were evaluated in this study: Clinical signs, body weights, body weight gains, food consumption, gross pathology, gravid uterine weights, intrauterine growth and survival,number of corpora lutea, and fetal parameters [sex, weight and anogenital distance, and external, visceral and skeletal observations].Approximately half of the fetuses from each litter were examined for visceral malformations and the remaining half were evaluated for skeletal malformations. In addition, from each dam the thyroids were weighed and subjected to microscopic evaluation, and thyroid hormones were estimated from the blood collected at terminal sacrifie (GD20).

 

Results of the study are summarized below:

 

·        Clinical signs and gross necropsy changes: There were no clinical signs, or mortalities in treated rats at any of the doses tested.Expected light to dark orange coloured faeces were observed in the test item treated groups which can be accounted for the physical nature of the test item.

Grossly, orangecontents noted in cecum at 333 and 1000 mg/kg/day and in ileum and colon at 1000 mg/kg/day was also related to the physical nature of test item and there were no other gross findings in the intestinal mucosa.

·        Maternal Parameters: No treatment-related effects on maternal body weights and food consumption up to the highest tested dose of
1000 mg/kg/day. The other maternal parameters comprising of uterine weight, implantations and early and late resorptions, post implantation loss were comparable to vehicle control group up to the high dose of 1000 mg/kg/day. Gross evaluation of placenta revealed no remarkable findings.

·        Litter Parameters: No treatment-related effects on litter parameters comprising of total number of fetuses, fetal weights, anogenital distance in male and female fetuses, were observed.

·        Fetal examination: The fetal external, visceral and skeletal examinations revealed no signs of teratogenicity or developmental toxicity up to
1000 mg/kg/day.

·        Thyroid hormone levels (T3, T4 and TSH), thyroid gland weights and histology of thyroid gland were unaffected by treatment with C.I. Pigment Orange 36 up to the highest dose of
1000 mg/kg/day.

 

Based on the above findings, it is concluded that, No Observed Adverse Effect Level (NOAEL) for

 

·           Maternal toxicity is1000 mg/kg/dayas the maternal body weight and weight gain, corrected body weight gain and food consumption was unaffected up to 1000 mg/kg/day.

 

Fetal developmental toxicity and Teratogencity is1000 mg/kg/dayas fetal resorptions or post implantation loss were comparable to the controls, no effects on fetal body weights and further the fetal external, visceral and skeletal examinations revealed no signs of teratogenicity or developmental toxicity up to 1000 mg/kg/day

[1]The day of confirmed mating (sperm positive vaginal smear or presence of vaginal plug) was designated as GD 0.

 

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 05 Jan 2012 to 31 Jul 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 421, GLP-compliant)

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Animals: Rat, RccHanTM: WIST(SPF)
- Rationale: Recognized by international guidelines as a recommended test system.
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Number of Animals: 44 males (11 per group) and 44 females (11 per group)
- Age (at delivery): 10 weeks
- Body Weight Range (at Start of Treatment): 301 to 344 g (males) and 191 to 228 g (females)
- Identification: Cage card and individual animal number (ear tattoo).
- Randomization: Computer-generated random algorithm. In addition body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
- Accommodation: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J.Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland; batch/lot no. 02105111001) with paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey / UK), batch/lot no. 55/T-6060). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet: Pelleted standard Harlan Teklad 2914C (batch no. 73/11) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum. Results of representative analyses for contaminants were included in the study report.
- Water: Community tap-water from Itingen was available ad libitum in water bottles. Results of bacteriological assay, chemical and contaminant analyses of representative samples were included in the study report.
- Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study. At least 5 days.

ENVIRONMENTAL CONDITIONS
Standard laboratory conditions, continuously monitored.
- Temperature (°C): 22 +/- 3
- Humidity (%): 30 to 70
- Air changes (per hr): . Air-conditioned with 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12 (with at least eight hours music during the light period)

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

DOSE FORMULATIONS
The dose formulations were not corrected for purity and were prepared as supplied by the Sponsor. The dose formulations were prepared weekly as indicated by the results of stability analyses in the non-GLP dose range-finding study. The test item was weighed into a glass beaker on a tared Mettler balance and the vehicle was added. The mixtures were stirred using a magnetic stirrer and stored at room temperature (20 °C +/- 5 °C). Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

STORAGE OF DOSE FORMULATIONS
The dose formulations were stored at room temperature (20 +/- 5 °C) in glass beakers. They were stable for at least 8 days, based upon the results of stability analyses performed during the non-GLP dose range-finding study.

TREATMENT
- Method: Oral, by gavage
- Rationale for Method: Administration by gavage is a common and accepted route of exposure for this type of studies.
- Frequency of Administration: Once daily
- Daily Target Dose Level: 0 mg/kg/day (control group), 100 mg/kg/day (group 2), 300 mg/kg/day (group 3), 1000 mg/kg/day (group 4)
- Rationale for Dose Level Selection: The dose levels were selected based on a previous dose range-finding toxicity study in Wistar rats, Harlan Laboratories study D33665 (non-GLP).
- Dose Volume: 10 mL/kg body weight
- Dose Concentrations: 0 mg/mL/day (control group), 10 mg/mL/day (group 2), 30 mg/mL/day (group 3), 100 mg/mL/day (group 4)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

METHOD
After experimental start, a sample from the control group as well as three samples (top, middle and bottom) of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples were also taken to confirm stability (4 hours and 8 days at room temperature). During week 3 of treatment, samples were also taken to confirm homogeneity and concentration. The aliquots for analysis of dose formulations were stored at -20 +/- 5°C until analysis. The test item was used as the analytical standard.

RESULTS
The linearity of the analytical systems used for sample analyses was demonstrated with a good relationship between absorbance measured and working standard concentrations. All calibration points met the acceptance limit of +/-20% variation from the calibration curve derived by linear regression analysis. The regression coefficients calculated were found to be better than 0.99.
Blank samples showed no significant absorbance and, therefore, it was confirmed that only bidistilled water was applied within the control experiment.
The application formulations investigated during the study were found to comprise test material in the range of 93.8% to 108.3% and, thus, the required content limit of +/-20% with reference to the nominal content was met. The homogeneous distribution of the test item in the preparations was approved because single results found did not deviate more than 3% (acceptance criterion: <15%) from the corresponding mean.

In addition, the test item was found to be stable in application formulations when kept four hours or eight days at room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean.

In conclusion, the results indicate the accurate use of the test item and bidistilled water as vehicle during this study. Application formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.

Details on mating procedure:

During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed. The females were removed and housed individually if the daily vaginal smear was sperm positive, or a copulation plug was observed.

The day on which a positive mating was determined (copulation plug or sperm) was designated day 0 post coitum.

If a female did not mate during the 14-day pairing period, a second pairing of this female with a male in the same group, which had already mated successfully, was considered. If mating was not recorded during this additional pairing period of a maximum of 14 days, the female was sacrificed and, if indicated, the reproductive organs examined histopathologically in order to ascertain the reason for the infertility.

All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.

Duration of treatment / exposure:

Minimum 4 weeks (males) and approximately 7 weeks (females)

Frequency of treatment:

Once daily

Duration of test:

MALES
- Acclimatization: 5 days minimum
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum
- Treatment Ends: On day before sacrifice
- Necropsy: After a minimum of 28 days treatment

FEMALES
- Acclimatization: 5 days minimum
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum
- Gestation: Approximately 21 days
- Treatment Ends: On day 3 post partum
- Necropsy: On day 4 post partum

No. of animals per sex per dose:

11

Control animals:

yes, concurrent vehicle

Details on study design:

The purpose of this study was to generate preliminary information concerning the effects of the test material on male and female reproductive performance such as gonadal function, mating behavior, conception and parturition and to assess major effects on pup development.

Maternal examinations:

VIABILITY/MORTALITY: Twice daily

CLINICAL SIGNS: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

FOOD CONSUMPTION: Pre-pairing period days 1 - 8 and 8 - 14, gestation period days 0 - 7, 7 - 14 and 14 - 21 post coitum and lactation period days 1 - 4 post partum. No food consumption was recorded during the pairing period.

BODY WEIGHTS: Recorded daily from treatment start to day of necropsy.

TERMINATION AND NECROPSY
Dams were sacrificed on day 4 post partum. If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.
All animals sacrificed or found dead were subjected to a detailed macroscopic examination to establish, if possible, the cause of death. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution. At the scheduled sacrifice, all animals were killed by an injection of sodium pentobarbital. All P generation animals were exsanguinated.
All parent animals were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.
For the parent animals, special attention was directed at the organs of the reproductive system. The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

TISSUE PRESERVATION: The ovaries from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution.

HISTOTECHNIQUE
All organ and tissue samples to be examined by the principal investigator for histopathology were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Special stains were used at the discretion of the principal investigator for histopathology.

HISTOPATHOLGOLGY: Slides of all organs and tissues collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the principal investigator for histopathology. The same applied to all occurring gross lesions and to all animals, which died spontaneously or had to be terminated in extremis. Special emphasis was made on the histopathology of interstitial cell structure. Histological examination of ovaries was carried out on any female that did not give birth.

Ovaries and uterine content:

The ovaries and uterus were examined after termination on day 5 post partum. Examinations included the number of corpora lutea and the number of implantation sites.

Fetal examinations:

Not performed

Statistics:

The following statistical methods were used to analyze food consumption, body weights and reproduction data:
- Means and standard deviations of various data were calculated.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Indices:

From the on-line recorded reproduction data, the following parameters were calculated: fertility indices, mean precoital time, post-implantation losses, mean litter size, pup sex ratios and viability indices, dead/live pups at first litter check, pups sex ratio

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
1. IN-LIFE DATA
VIABILITY / MORTALITY: All animals survived the scheduled study period.

CLINICAL SIGNS OR OBSERVATIONS
There were no findings of toxicological relevance at any dose level.
In females, yellow discoloration of the feces was noted from day 5 of the pre-pairing period and continued throughout the subsequent pairing, gestation and lactation periods. The severity of this finding was generally dose-dependent, but was considered to be a typical passive effect that results from oral administration of a dyestuff and was of no toxicological relevance. Slight hair loss was noted in a single female during the gestation period, but in view of the late onset and low incidence, was considered to be without toxicological implications.

FOOD CONSUMPTION OF FEMALES
Pre-pairing, Gestation and Lactation Periods
The mean daily food consumption values of the test item-treated females compared favorably with the control females throughout the pre-pairing, gestation and lactation periods.

BODY WEIGHTS OF FEMALES
Pre-Pairing, Pairing, Gestation and Lactation Periods
No test item-related effects on body weights and body weight gain of females were observed at any dose level.
In females treated with 1000 mg/kg/day, a significant increase (p<0.05) in mean body weight was noted on day 4 of the gestation period. This isolated finding was considered to be incidental. The body weights recorded during all other periods were similar to those of the control females.
At 300 mg/kg/day, all body weights were similar throughout the various phases.
At 100 mg/kg/day, significantly elevated mean body weights (p<0.05) were noted on days 0 - 5 of the gestation period only. In the absence of a dose-response relationship, these differences were considered to be incidental. The body weights recorded during all other periods were similar to the controls.

REPRODUCTION AND BREEDING DATA
 No effects on mating performance, fertility, corpora lutea count or duration of gestation were observed at any dose level.
 At the dose level of 1000 mg/kg bw/day, higher incidence of post-implantation loss and higher postnatal loss as well as lower litter size at first litter check and on day 4 post partum were noted.
 The mean post implantation loss of the dams at 1000 mg/kg/day was close to the upper limit of the historical control data and was below the mean post implantation loss of the dams treated with 300 mg/kg/day; therefore this was considered to be unrelated to the treatment with the test item.

2. TERMINAL FINDINGS
MACROSCOPICAL FINDINGS
Discoloration of the ovaries found in one female at 1000 mg/kg/day and was considered to be within the range of normal background alterations.

HISTOPATHOLOGY FINDINGS
All microscopic findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age. There was no morphological evidence of toxicological properties in ovaries of female animals examined in this study.

Dose descriptor:

NOAEL

Remarks:

for general toxicity (P)

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: effect type not specified

Dose descriptor:

NOAEL

Remarks:

for reproduction (P)

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: effect type not specified

Dose descriptor:

NOAEL

Remarks:

for development (F1)

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: effect type not specified

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:not examined

Abnormalities:

not specified

Developmental effects observed:

not specified

1. REPRODUCTION, BREEDING AND PUP DATA

SUMMARY OF PERFORMANCE

 

P Animals Breeding for F1 Litters 

Group (mg/kg/day)	1 (0)	2 (100)	3 (300)	4 (1000)
Female numbers	45 - 55	56 - 66	67 - 77	78 - 88
Number of females paired	11	11	11	11
Number of females mated	11	11	11	11
Number of females which were not pregnant	1	0	0	2
Number of females which reared their pups until day 4 post partum	10	11	11	9

 

MATING PERFORMANCE AND FERTILITY

  The mean and median precoital times for all groups were similar.

  The respective parameters of the mating performance were unaffected as all dose levels and similar to those of the control values.

  Mating of female no. 84 was not detected. Three females were not pregnant: no. 55 (control group) and nos. 81 ad 83 (1000 mg/kg/day).

 

DURATION OF GESTATION

  No effects on duration of gestation were observed at any dose level.

 

CORPORA LUTEA COUNT

  No effects on corpora lutea count were observed at any dose level.

 

IMPLANTATION RATE AND POST-IMPLANTATION LOSS

  The implantation rate was unaffected in all groups.

 

Although the mean post-implantation loss was slightly higher at 300 mg/kg/day and 1000 mg/kg/ day when compared with the control females, the differences were not dose-related and therefore considered to be unrelated to the test item treatment. The total post implantation loss of the dams treated with 300 mg/kg/day was significantly elevated (p<0.01) when compared with the controls, but not dose-dependent and therefore considered to be unrelated to the treatment with the test item. The mean post implantation loss in females at 100 mg/kg/day was nearly identical to the controls.

 

LITTER SIZE AT FIRST LITTER CHECK

  The mean litter sizes of the control group and those at 1000 mg/kg/day were similar. The marginally lower mean litter size noted at 300 mg/kg/day coincided with the slightly higher post-implantation loss.

  The overall mean numbers of living pups per dam at first litter check compared favorably in the control and test item-treated groups; three pups of litter no. 79 (1000 mg/kg/day), two pups of litter no. 70 (300 mg/kg/day) and four pups of litter no. 49 (control group) were found dead.

  The respective birth indices (number of pups borne alive as a percentage of implantations) were similar in test item-treated and control animals.

 

2. IN-LIFE DATA OF PARENTAL MALES

 

VIABILITY / MORTALITY

  All animals survived the scheduled study period.

 

CLINICAL SIGNS OR OBSERVATIONS

  There were no findings of toxicological relevance at any dose level.

  In males, yellow discoloration of the feces was noted from treatment day 5 of the pre-pairing period and continued throughout the pairing period. The severity of this finding was generally dose-dependent, but was considered to be a typical passive effect that results from oral administration of a dyestuff and was of no toxicological relevance.

 

FOOD CONSUMPTION OF MALES

  Pre-pairing Period

  In males, although the mean daily food consumption values at 300 mg/kg/day and 1000 mg/kg/ day were significantly lower (p<0.05) than that of the control males during days 8 - 14, these differences were not dose-related and were considered to be a result of an incidentally elevated control value rather than to the treatment with the test item.

 

BODY WEIGHTS OF MALES

  Pre-Pairing and Pairing Periods

  There were no differences of toxicological relevance between the mean body weights of test item-treated or control males. The mean body weight gain of all groups compared favorably.

 

3. TERMINAL FINDINGS OF PARENTAL MALES

ORGAN WEIGHTS

 No test item-related changes in organ weights were noted at any dose level.

At 300 mg/kg/day, the mean absolute epididymide weight (left side only) were significantly reduced (p<0.05) when compared with the controls. This finding was considered to be incidental as the mean relative organ weight was normal and unilateral organ weight differences are not associated with toxicological relevance.

At 100 mg/kg/day, the mean absolute epididymide weights (bilateral) were significantly reduced (p<0.05) when compared with the controls. These differences were considered to be related to the lower mean terminal body weights; the mean relative organ weights were unaffected.

No further changes of organ weights were noted at any dose level.

 

MACROSCOPICAL FINDINGS

A small number of typical background changes were noted in test item-treated and control males. Neither type nor incidence were considered to be commensurate with findings of toxicological relevance in males.

 

HISTOPATHOLOGY FINDINGS

All microscopic findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age. There was no morphological evidence of toxicological properties in the testes, epididymides, prostate and seminal vesicles of male animals examined in this study.

 

Detailed examination of the testes (including sperm staging) was performed on control males (nos. 1 - 11) and males at 1000 mg/kg/day. The stages were checked for completeness of cell populations, completeness of stages and degenerative changes. No differences on the completeness of stages or cell populations of the testes were recorded between the control males (nos. 1 - 11) and the males at 1000 mg/kg/day (nos. 34 - 44). In male no. 6 of the control group, unilateral Sertoli cell vacuolation (minimal in degree) was noted. Male no. 41 (treated with 1000 mg/kg/day) showed unilateral tubular degeneration/atrophy that was considered to be minimal in degree.

 

There were no findings or microscopical changes to the reproductive organs that were considered to be related to the infertility noted in male no. 4

Conclusions:

This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item to rats (OECD421, GLP compiant). The test material was administered in water as vehicle at dosages of 100, 300, and 1000 mg/kg body weight/day, and controls received the vehicle only. The test material was administered to male rats for 14 days prior to pairing and for 28 days in total, and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

Test item-related effects were restricted to secondary, passive effects only (discolored feces) and there were no findings of toxicological relevance. Body weight and food consumption of parent animals were unaffected, and no differences in organ weights were noted. There were no macroscopical or microscopical findings related to the treatment with the test item.

No effects on mating performance, fertility, corpora lutea count or duration of gestation were observed at any dose level. Minor differences in the mean post-implantation loss, postnatal loss, and litter size at first litter check and day 4 post partum were either unrelated to dose or within the limits of the historical control data and therefore considered to be incidental.

No test item-related findings were noted at first litter check and during lactation in pups at any dose level. The sex ratios of the pups in test item-treated groups were normal and pup weights recorded until day 4 post partum compared favorably in all groups.

Because fecal discoloration (i.e. a secondary passive finding) was noted in treated rats at all dose levels, a NOEL (No Observed Effect Level) could not be established. The NOAEL (No Observed Adverse Effect Level) for general toxicity in males and females, as well as for reproduction data and F1 offspring was considered to be 1000 mg/kg bw/day, the highest dose level tested.

Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of the test material on male and female reproductive performance such as gonadal function, mating behavior, conception and parturition. 

Four groups of 11 males and 11 females were treated by gavage with test item once daily. Males were treated over a 14-day pre-pairing period and during the pairing period up to one day before necropsy. Females were treated throughout the pre-pairing, pairing, gestation and lactation period up to the day 3 post partum.

 The following dose levels were used:

 Group 1: 0 mg/kg body weight/day (control group)

Group 2: 100 mg/kg body weight/day

Group 3: 300 mg/kg body weight/day

Group 4: 1000 mg/kg body weight/day

 A standard dose volume of 10 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (bidistilled water).

 

The following results were obtained:

 

MORTALITY AND GENERAL TOLERABILITY OF PARENTAL ANIMALS

 All animals survived the scheduled study period.

 No clinical signs of toxicological relevance were noted at any dose level. In both sexes, yellow discoloration of the feces was noted from treatment day 5 of the pre-pairing period and continued throughout treatment. The severity of this finding was generally dose-dependent, but was considered to be a typical secondary passive effect that results from oral administration of a dyestuff and was of no toxicological relevance.

 

FOOD CONSUMPTION OF PARENTAL ANIMALS

 Minor intergroup differences in the mean daily food consumption of males were not considered to be related to the treatment with the test item. The daily food consumption values of the test item-treated females were unaffected.

 

BODY WEIGHTS OF PARENTAL ANIMALS

 There were no effects upon body weights of either males or females.

 

REPRODUCTION AND BREEDING DATA

 No effects on mating performance, fertility, corpora lutea count or duration of gestation were observed at any dose level.

 At the dose level of 1000 mg/kg bw/day, higher incidence of post-implantation loss and higher postnatal loss as well as lower litter size at first litter check and on day 4 post partum were noted.

 The mean post implantation loss of the dams at 1000 mg/kg/day was close to the upper limit of the historical control data and was below the mean post implantation loss of the dams treated with 300 mg/kg/day; therefore this was considered to be unrelated to the treatment with the test item.

 

ORGAN WEIGHS OF PARENTAL ANIMALS

 No test item-related effects on organ weights were noted at any dose level.

 

MACROSCOPICAL FINDINGS AND HISTOPATHOLOGICAL EXAMINATIONS OF PARENTAL ANIMALS

 No test item-related macroscopical findings were noted at any dose level.

 Under the conditions of this study, the test item produced no morphological evidence of toxicological properties in testes, epididymides, prostate and seminal vesicles of male animals and ovaries of female animals.

 

FINDINGS IN PUPS AT FIRST LITTER CHECK AND DURING LACTATION

 No test item-related findings were noted in pups at any dose level.

 The sex ratio of the test item-treated pups did not indicate any effects of toxicological relevance.

 

PUP WEIGHTS TO DAY 4 POST PARTUM

 No effects on pup body weights were noted at any dose level.

 

MACROSCOPICAL FINDINGS OF PUPS

 No test item-related findings were found in pups at any dose level.

Reference 4

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

From 05 Jan 2012 to 31 Jul 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 421, GLP-compliant)

Justification for type of information:

See read across justification document in chapter 13

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Animals: Rat, RccHanTM: WIST(SPF)
- Rationale: Recognized by international guidelines as a recommended test system.
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Number of Animals: 44 males (11 per group) and 44 females (11 per group)
- Age (at delivery): 10 weeks
- Body Weight Range (at Start of Treatment): 301 to 344 g (males) and 191 to 228 g (females)
- Identification: Cage card and individual animal number (ear tattoo).
- Randomization: Computer-generated random algorithm. In addition body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
- Accommodation: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J.Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland; batch/lot no. 02105111001) with paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey / UK), batch/lot no. 55/T-6060). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet: Pelleted standard Harlan Teklad 2914C (batch no. 73/11) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum. Results of representative analyses for contaminants were included in the study report.
- Water: Community tap-water from Itingen was available ad libitum in water bottles. Results of bacteriological assay, chemical and contaminant analyses of representative samples were included in the study report.
- Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study. At least 5 days.

ENVIRONMENTAL CONDITIONS
Standard laboratory conditions, continuously monitored.
- Temperature (°C): 22 +/- 3
- Humidity (%): 30 to 70
- Air changes (per hr): . Air-conditioned with 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12 (with at least eight hours music during the light period)

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

DOSE FORMULATIONS
The dose formulations were not corrected for purity and were prepared as supplied by the Sponsor. The dose formulations were prepared weekly as indicated by the results of stability analyses in the non-GLP dose range-finding study. The test item was weighed into a glass beaker on a tared Mettler balance and the vehicle was added. The mixtures were stirred using a magnetic stirrer and stored at room temperature (20 °C +/- 5 °C). Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

STORAGE OF DOSE FORMULATIONS
The dose formulations were stored at room temperature (20 +/- 5 °C) in glass beakers. They were stable for at least 8 days, based upon the results of stability analyses performed during the non-GLP dose range-finding study.

TREATMENT
- Method: Oral, by gavage
- Rationale for Method: Administration by gavage is a common and accepted route of exposure for this type of studies.
- Frequency of Administration: Once daily
- Daily Target Dose Level: 0 mg/kg/day (control group), 100 mg/kg/day (group 2), 300 mg/kg/day (group 3), 1000 mg/kg/day (group 4)
- Rationale for Dose Level Selection: The dose levels were selected based on a previous dose range-finding toxicity study in Wistar rats, Harlan Laboratories study D33665 (non-GLP).
- Dose Volume: 10 mL/kg body weight
- Dose Concentrations: 0 mg/mL/day (control group), 10 mg/mL/day (group 2), 30 mg/mL/day (group 3), 100 mg/mL/day (group 4)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

METHOD
After experimental start, a sample from the control group as well as three samples (top, middle and bottom) of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples were also taken to confirm stability (4 hours and 8 days at room temperature). During week 3 of treatment, samples were also taken to confirm homogeneity and concentration. The aliquots for analysis of dose formulations were stored at -20 +/- 5°C until analysis. The test item was used as the analytical standard.

RESULTS
The linearity of the analytical systems used for sample analyses was demonstrated with a good relationship between absorbance measured and working standard concentrations. All calibration points met the acceptance limit of +/-20% variation from the calibration curve derived by linear regression analysis. The regression coefficients calculated were found to be better than 0.99.
Blank samples showed no significant absorbance and, therefore, it was confirmed that only bidistilled water was applied within the control experiment.
The application formulations investigated during the study were found to comprise test material in the range of 93.8% to 108.3% and, thus, the required content limit of +/-20% with reference to the nominal content was met. The homogeneous distribution of the test item in the preparations was approved because single results found did not deviate more than 3% (acceptance criterion: <15%) from the corresponding mean.

In addition, the test item was found to be stable in application formulations when kept four hours or eight days at room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean.

In conclusion, the results indicate the accurate use of the test item and bidistilled water as vehicle during this study. Application formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.

Details on mating procedure:

During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed. The females were removed and housed individually if the daily vaginal smear was sperm positive, or a copulation plug was observed.

The day on which a positive mating was determined (copulation plug or sperm) was designated day 0 post coitum.

If a female did not mate during the 14-day pairing period, a second pairing of this female with a male in the same group, which had already mated successfully, was considered. If mating was not recorded during this additional pairing period of a maximum of 14 days, the female was sacrificed and, if indicated, the reproductive organs examined histopathologically in order to ascertain the reason for the infertility.

All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.

Duration of treatment / exposure:

Minimum 4 weeks (males) and approximately 7 weeks (females)

Frequency of treatment:

Once daily

Duration of test:

MALES
- Acclimatization: 5 days minimum
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum
- Treatment Ends: On day before sacrifice
- Necropsy: After a minimum of 28 days treatment

FEMALES
- Acclimatization: 5 days minimum
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum
- Gestation: Approximately 21 days
- Treatment Ends: On day 3 post partum
- Necropsy: On day 4 post partum

No. of animals per sex per dose:

11

Control animals:

yes, concurrent vehicle

Details on study design:

The purpose of this study was to generate preliminary information concerning the effects of the test material on male and female reproductive performance such as gonadal function, mating behavior, conception and parturition and to assess major effects on pup development.

Maternal examinations:

VIABILITY/MORTALITY: Twice daily

CLINICAL SIGNS: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

FOOD CONSUMPTION: Pre-pairing period days 1 - 8 and 8 - 14, gestation period days 0 - 7, 7 - 14 and 14 - 21 post coitum and lactation period days 1 - 4 post partum. No food consumption was recorded during the pairing period.

BODY WEIGHTS: Recorded daily from treatment start to day of necropsy.

TERMINATION AND NECROPSY
Dams were sacrificed on day 4 post partum. If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.
All animals sacrificed or found dead were subjected to a detailed macroscopic examination to establish, if possible, the cause of death. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution. At the scheduled sacrifice, all animals were killed by an injection of sodium pentobarbital. All P generation animals were exsanguinated.
All parent animals were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.
For the parent animals, special attention was directed at the organs of the reproductive system. The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

TISSUE PRESERVATION: The ovaries from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution.

HISTOTECHNIQUE
All organ and tissue samples to be examined by the principal investigator for histopathology were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Special stains were used at the discretion of the principal investigator for histopathology.

HISTOPATHOLGOLGY: Slides of all organs and tissues collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the principal investigator for histopathology. The same applied to all occurring gross lesions and to all animals, which died spontaneously or had to be terminated in extremis. Special emphasis was made on the histopathology of interstitial cell structure. Histological examination of ovaries was carried out on any female that did not give birth.

Ovaries and uterine content:

The ovaries and uterus were examined after termination on day 5 post partum. Examinations included the number of corpora lutea and the number of implantation sites.

Fetal examinations:

Not performed

Statistics:

The following statistical methods were used to analyze food consumption, body weights and reproduction data:
- Means and standard deviations of various data were calculated.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Indices:

From the on-line recorded reproduction data, the following parameters were calculated: fertility indices, mean precoital time, post-implantation losses, mean litter size, pup sex ratios and viability indices, dead/live pups at first litter check, pups sex ratio

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
1. IN-LIFE DATA
VIABILITY / MORTALITY: All animals survived the scheduled study period.

CLINICAL SIGNS OR OBSERVATIONS
There were no findings of toxicological relevance at any dose level.
In females, yellow discoloration of the feces was noted from day 5 of the pre-pairing period and continued throughout the subsequent pairing, gestation and lactation periods. The severity of this finding was generally dose-dependent, but was considered to be a typical passive effect that results from oral administration of a dyestuff and was of no toxicological relevance. Slight hair loss was noted in a single female during the gestation period, but in view of the late onset and low incidence, was considered to be without toxicological implications.

FOOD CONSUMPTION OF FEMALES
Pre-pairing, Gestation and Lactation Periods
The mean daily food consumption values of the test item-treated females compared favorably with the control females throughout the pre-pairing, gestation and lactation periods.

BODY WEIGHTS OF FEMALES
Pre-Pairing, Pairing, Gestation and Lactation Periods
No test item-related effects on body weights and body weight gain of females were observed at any dose level.
In females treated with 1000 mg/kg/day, a significant increase (p<0.05) in mean body weight was noted on day 4 of the gestation period. This isolated finding was considered to be incidental. The body weights recorded during all other periods were similar to those of the control females.
At 300 mg/kg/day, all body weights were similar throughout the various phases.
At 100 mg/kg/day, significantly elevated mean body weights (p<0.05) were noted on days 0 - 5 of the gestation period only. In the absence of a dose-response relationship, these differences were considered to be incidental. The body weights recorded during all other periods were similar to the controls.

REPRODUCTION AND BREEDING DATA
 No effects on mating performance, fertility, corpora lutea count or duration of gestation were observed at any dose level.
 At the dose level of 1000 mg/kg bw/day, higher incidence of post-implantation loss and higher postnatal loss as well as lower litter size at first litter check and on day 4 post partum were noted.
 The mean post implantation loss of the dams at 1000 mg/kg/day was close to the upper limit of the historical control data and was below the mean post implantation loss of the dams treated with 300 mg/kg/day; therefore this was considered to be unrelated to the treatment with the test item.

2. TERMINAL FINDINGS
MACROSCOPICAL FINDINGS
Discoloration of the ovaries found in one female at 1000 mg/kg/day and was considered to be within the range of normal background alterations.

HISTOPATHOLOGY FINDINGS
All microscopic findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age. There was no morphological evidence of toxicological properties in ovaries of female animals examined in this study.

Dose descriptor:

NOAEL

Remarks:

for general toxicity (P)

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: effect type not specified

Dose descriptor:

NOAEL

Remarks:

for reproduction (P)

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: effect type not specified

Dose descriptor:

NOAEL

Remarks:

for development (F1)

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: effect type not specified

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:not examined

Abnormalities:

not specified

Developmental effects observed:

not specified

1. REPRODUCTION, BREEDING AND PUP DATA

SUMMARY OF PERFORMANCE

 

P Animals Breeding for F1 Litters 

Group (mg/kg/day)	1 (0)	2 (100)	3 (300)	4 (1000)
Female numbers	45 - 55	56 - 66	67 - 77	78 - 88
Number of females paired	11	11	11	11
Number of females mated	11	11	11	11
Number of females which were not pregnant	1	0	0	2
Number of females which reared their pups until day 4 post partum	10	11	11	9

 

MATING PERFORMANCE AND FERTILITY

  The mean and median precoital times for all groups were similar.

  The respective parameters of the mating performance were unaffected as all dose levels and similar to those of the control values.

  Mating of female no. 84 was not detected. Three females were not pregnant: no. 55 (control group) and nos. 81 ad 83 (1000 mg/kg/day).

 

DURATION OF GESTATION

  No effects on duration of gestation were observed at any dose level.

 

CORPORA LUTEA COUNT

  No effects on corpora lutea count were observed at any dose level.

 

IMPLANTATION RATE AND POST-IMPLANTATION LOSS

  The implantation rate was unaffected in all groups.

 

Although the mean post-implantation loss was slightly higher at 300 mg/kg/day and 1000 mg/kg/ day when compared with the control females, the differences were not dose-related and therefore considered to be unrelated to the test item treatment. The total post implantation loss of the dams treated with 300 mg/kg/day was significantly elevated (p<0.01) when compared with the controls, but not dose-dependent and therefore considered to be unrelated to the treatment with the test item. The mean post implantation loss in females at 100 mg/kg/day was nearly identical to the controls.

 

LITTER SIZE AT FIRST LITTER CHECK

  The mean litter sizes of the control group and those at 1000 mg/kg/day were similar. The marginally lower mean litter size noted at 300 mg/kg/day coincided with the slightly higher post-implantation loss.

  The overall mean numbers of living pups per dam at first litter check compared favorably in the control and test item-treated groups; three pups of litter no. 79 (1000 mg/kg/day), two pups of litter no. 70 (300 mg/kg/day) and four pups of litter no. 49 (control group) were found dead.

  The respective birth indices (number of pups borne alive as a percentage of implantations) were similar in test item-treated and control animals.

 

2. IN-LIFE DATA OF PARENTAL MALES

 

VIABILITY / MORTALITY

  All animals survived the scheduled study period.

 

CLINICAL SIGNS OR OBSERVATIONS

  There were no findings of toxicological relevance at any dose level.

  In males, yellow discoloration of the feces was noted from treatment day 5 of the pre-pairing period and continued throughout the pairing period. The severity of this finding was generally dose-dependent, but was considered to be a typical passive effect that results from oral administration of a dyestuff and was of no toxicological relevance.

 

FOOD CONSUMPTION OF MALES

  Pre-pairing Period

  In males, although the mean daily food consumption values at 300 mg/kg/day and 1000 mg/kg/ day were significantly lower (p<0.05) than that of the control males during days 8 - 14, these differences were not dose-related and were considered to be a result of an incidentally elevated control value rather than to the treatment with the test item.

 

BODY WEIGHTS OF MALES

  Pre-Pairing and Pairing Periods

  There were no differences of toxicological relevance between the mean body weights of test item-treated or control males. The mean body weight gain of all groups compared favorably.

 

3. TERMINAL FINDINGS OF PARENTAL MALES

ORGAN WEIGHTS

 No test item-related changes in organ weights were noted at any dose level.

At 300 mg/kg/day, the mean absolute epididymide weight (left side only) were significantly reduced (p<0.05) when compared with the controls. This finding was considered to be incidental as the mean relative organ weight was normal and unilateral organ weight differences are not associated with toxicological relevance.

At 100 mg/kg/day, the mean absolute epididymide weights (bilateral) were significantly reduced (p<0.05) when compared with the controls. These differences were considered to be related to the lower mean terminal body weights; the mean relative organ weights were unaffected.

No further changes of organ weights were noted at any dose level.

 

MACROSCOPICAL FINDINGS

A small number of typical background changes were noted in test item-treated and control males. Neither type nor incidence were considered to be commensurate with findings of toxicological relevance in males.

 

HISTOPATHOLOGY FINDINGS

All microscopic findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age. There was no morphological evidence of toxicological properties in the testes, epididymides, prostate and seminal vesicles of male animals examined in this study.

 

Detailed examination of the testes (including sperm staging) was performed on control males (nos. 1 - 11) and males at 1000 mg/kg/day. The stages were checked for completeness of cell populations, completeness of stages and degenerative changes. No differences on the completeness of stages or cell populations of the testes were recorded between the control males (nos. 1 - 11) and the males at 1000 mg/kg/day (nos. 34 - 44). In male no. 6 of the control group, unilateral Sertoli cell vacuolation (minimal in degree) was noted. Male no. 41 (treated with 1000 mg/kg/day) showed unilateral tubular degeneration/atrophy that was considered to be minimal in degree.

 

There were no findings or microscopical changes to the reproductive organs that were considered to be related to the infertility noted in male no. 4

Conclusions:

This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item to rats (OECD421, GLP compiant). The test material was administered in water as vehicle at dosages of 100, 300, and 1000 mg/kg body weight/day, and controls received the vehicle only. The test material was administered to male rats for 14 days prior to pairing and for 28 days in total, and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

Test item-related effects were restricted to secondary, passive effects only (discolored feces) and there were no findings of toxicological relevance. Body weight and food consumption of parent animals were unaffected, and no differences in organ weights were noted. There were no macroscopical or microscopical findings related to the treatment with the test item.

No effects on mating performance, fertility, corpora lutea count or duration of gestation were observed at any dose level. Minor differences in the mean post-implantation loss, postnatal loss, and litter size at first litter check and day 4 post partum were either unrelated to dose or within the limits of the historical control data and therefore considered to be incidental.

No test item-related findings were noted at first litter check and during lactation in pups at any dose level. The sex ratios of the pups in test item-treated groups were normal and pup weights recorded until day 4 post partum compared favorably in all groups.

Because fecal discoloration (i.e. a secondary passive finding) was noted in treated rats at all dose levels, a NOEL (No Observed Effect Level) could not be established. The NOAEL (No Observed Adverse Effect Level) for general toxicity in males and females, as well as for reproduction data and F1 offspring was considered to be 1000 mg/kg bw/day, the highest dose level tested.

Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of the test material on male and female reproductive performance such as gonadal function, mating behavior, conception and parturition. 

Four groups of 11 males and 11 females were treated by gavage with test item once daily. Males were treated over a 14-day pre-pairing period and during the pairing period up to one day before necropsy. Females were treated throughout the pre-pairing, pairing, gestation and lactation period up to the day 3 post partum.

 The following dose levels were used:

 Group 1: 0 mg/kg body weight/day (control group)

Group 2: 100 mg/kg body weight/day

Group 3: 300 mg/kg body weight/day

Group 4: 1000 mg/kg body weight/day

 A standard dose volume of 10 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (bidistilled water).

 

The following results were obtained:

 

MORTALITY AND GENERAL TOLERABILITY OF PARENTAL ANIMALS

 All animals survived the scheduled study period.

 No clinical signs of toxicological relevance were noted at any dose level. In both sexes, yellow discoloration of the feces was noted from treatment day 5 of the pre-pairing period and continued throughout treatment. The severity of this finding was generally dose-dependent, but was considered to be a typical secondary passive effect that results from oral administration of a dyestuff and was of no toxicological relevance.

 

FOOD CONSUMPTION OF PARENTAL ANIMALS

 Minor intergroup differences in the mean daily food consumption of males were not considered to be related to the treatment with the test item. The daily food consumption values of the test item-treated females were unaffected.

 

BODY WEIGHTS OF PARENTAL ANIMALS

 There were no effects upon body weights of either males or females.

 

REPRODUCTION AND BREEDING DATA

 No effects on mating performance, fertility, corpora lutea count or duration of gestation were observed at any dose level.

 At the dose level of 1000 mg/kg bw/day, higher incidence of post-implantation loss and higher postnatal loss as well as lower litter size at first litter check and on day 4 post partum were noted.

 The mean post implantation loss of the dams at 1000 mg/kg/day was close to the upper limit of the historical control data and was below the mean post implantation loss of the dams treated with 300 mg/kg/day; therefore this was considered to be unrelated to the treatment with the test item.

 

ORGAN WEIGHS OF PARENTAL ANIMALS

 No test item-related effects on organ weights were noted at any dose level.

 

MACROSCOPICAL FINDINGS AND HISTOPATHOLOGICAL EXAMINATIONS OF PARENTAL ANIMALS

 No test item-related macroscopical findings were noted at any dose level.

 Under the conditions of this study, the test item produced no morphological evidence of toxicological properties in testes, epididymides, prostate and seminal vesicles of male animals and ovaries of female animals.

 

FINDINGS IN PUPS AT FIRST LITTER CHECK AND DURING LACTATION

 No test item-related findings were noted in pups at any dose level.

 The sex ratio of the test item-treated pups did not indicate any effects of toxicological relevance.

 

PUP WEIGHTS TO DAY 4 POST PARTUM

 No effects on pup body weights were noted at any dose level.

 

MACROSCOPICAL FINDINGS OF PUPS

 No test item-related findings were found in pups at any dose level.

Reference 5

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Source: Hylasco Biotechnology (India) Pvt. Ltd.
Plot 4B, MN Park, Shameerpet Mandal,
Turkapally Village,
Medchal District, Telangana -500078

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for analysis of homogeneity and active ingredient (a.i.) were collected from the dose formulations intended for first treatment and last treatment (-2 days). The prepared formulations were sampled in duplicate sets wherein one set was used for analysis and another as back up set which was stored at ambient condition. For each set, duplicate sample were drawn from top, middle and bottom layers of each preparation and in case of control duplicate samples from the middle layer was drawn. The samples collected were sent to Analytical R&D of Eurofins Advinus Ltd. for formulation analysis to determine the homogeneity and concentration of the dose formulation.

Details on mating procedure:

The female rats were cohabited with males in a 1:1 ratio and vaginal smears and / or vaginal plug we re examined in the morning hours of the subsequent day to confirm mating

Duration of treatment / exposure:

Gestation day 5 to gestation day 19

Frequency of treatment:

Daily from gestation day 5 to gestation day 19

Duration of test:

Upto gestation day 20

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control

Dose / conc.:

111 mg/kg bw/day (nominal)

Remarks:

Low dose

Dose / conc.:

333 mg/kg bw/day (nominal)

Remarks:

Mid dose

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High dose

No. of animals per sex per dose:

24 day 0 pergnant rats per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Group Nos. Groups Dose
(mg/kg/day) Dosage volume (mL/kg) Concent-ration (mg/mL) No. of Day 0 pregnant rats Rat numbers
From To
G1 Vehicle control 0 10 0 24 Rz121 Rz144
G2 Low dose 111 10 11.1 24 Rz145 Rz168
G3 Mid dose 333 10 33.3 24 Rz169 Rz192
G4 High dose 1000 10 100 24 Rz193 Rz216

Maternal examinations:

CAGE SIDE OBSERVATION: Yes
- Time schedule: Twice a day (pre dose and post dose) during treatment period
- Cage side observation checked in table 2 were included

Ovaries and uterine content:

The ovaries and uterine contents were examined after termination GD 20.
• Pregnancy status
• Gravid uterine weight (from all rats subjected to caesarean section)
• Number of corpora lutea
• Number of implantation sites
• Number of early resorptions
• Number of late resorptions
• Gross evaluation of placenta

Fetal examinations:

• Total number of fetuses
• Total number of live fetuses
• Total number of dead fetuses
• Individual fetal body weight
• Fetus sex (during visceral examination)
• External examination of fetus
• Soft tissue evaluation
• Skeletal examination
• Head examination (half the number of fetuses per litter)

Statistics:

The data on maternal body weight, body weight change in interval, gravid uterine weight, body weight change corrected to gravid uterine weight, maternal food consumption, hormone analyses (T4, T3, TSH), weight of thyroid gland was analyzed using Analysis of Variance (ANOVA) after testing for homogeneity for intra group variance using Levene’s test. Where intra group variances were heterogeneous, ANOVA was performed after suitable transformation of data. Dunnett’s pairwise comparison of the treated group means with the control group mean was performed, when the group differences were found significant.

Fetal weight for male and female was analyzed using Analysis of Covariance (ANCOVA) taking litter size as covariate for group. Anogenital distance for male and female was analyzed using Analysis of Covariance (ANCOVA) taking weight as covariate for group. Number of corpora lutea, number of implantations, early and late resorptions, pre-implantation and post-implantation loss observations were analyzed using Kruskal Wallis test for group comparison. Wilcoxon pairwise comparison of the treated groups with the control group was performed, when the
group differences were significant. The incidence of dams with resorptions were tested for using Chi-square test followed by Fisher’s
exact test for group association. The incidence of fetus and litter (incidence and percent) observations for external, visceral and skeletal observations were tested using Cochran Armitage trend test and pair wise comparison will be tested by Fisher’s exact test for group association.
Statistically significant differences (p<0.05), indicated by the aforementioned tests was designated as * throughout the report.

Indices:

Refer Table 6 of the final report

Historical control data:

Refer Annexure 8 of the final report

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Grossly observed yellow intestinal contents in cecum and colon at ≥333 mg/kg/day was attributed to the physical nature of the test item

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormone levels (T3, T4 and TSH), thyroid gland weights and histology of thyroid gland were unaffected by treatment with Novoperm-Gelb F2G (C.I. Pigment Yellow 194) up to the highest dose of 1000 mg/kg/day.

Number of abortions:

no effects observed

Description (incidence and severity):

No abortions in the study

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: There were no adverse effects at all

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no effects at all

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

Based on the above findings, it is concluded that, No Observed Adverse Effect Level (NOAEL) for

Maternal toxicity, fetal developmental toxicity and Teratogencity is 1000 mg/kg/day as
- the maternal body weight and weight gain, corrected body weight gain and food consumption was unaffected up to 1000 mg/kg/day.
- fetal resorptions or post implantation loss were comparable to the controls and there were no effects on fetal body weights.
- the fetal external, visceral and skeletal examinations revealed no signs of teratogenicity or developmental toxicity up to 1000 mg/kg/day

Executive summary:

The objective of this study was to evaluate the developmental toxicity (teratogenic) potential of the test item C.I. Pigment Yellow 194 to cause adverse effects on the pregnant rats and development of the embryo and fetus consequent to exposure of pregnant rats by oral route during gestation days (GD) 5 to 19. This study was intended to provide a rational basis for  a No Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity in rats.

A total of 96 Day 0 pregnant rats[1]were randomly divided into different groups according to the study design as follows:

Group Nos.	Groups	Dose (mg/kg/day)	Dosage volume (mL/kg)	Concentration (mg/mL)	No. of Day 0 pregnant rats
G1	Vehicle control*	0	10	0	24
G2	Low dose	111	10	11.1	24
G3	Mid dose	333	10	33.3	24
G4	High dose	1000	10	100	24

*0.5 % Carboxymethylcellulose Sodium salt (medium viscosity) in Milli-Q^®Water

 

The following parameters and end points were evaluated in this study: Clinical signs, body weights, body weight gains, food consumption, gross pathology, gravid uterine weights, intrauterine growth and survival,number of corpora lutea, and fetal parameters [sex, weight and anogenital distance, and external, visceral and skeletal observations].Approximately half the number of fetuses from each litter were examined for visceral malformations and the remaining half were evaluated for skeletal malformations. In addition, from each dam the thyroid glands were weighed and subjected to microscopic evaluation, and thyroid hormones were estimated from the blood collected at terminal sacrifice (GD20).

 

Results of the study are summarized below:

 

·        Clinical signs and gross necropsy changes: There were no clinical signs, or mortalities in treated rats at any of the doses tested.Expected yellow coloured faeces was observed in the test item treated groups which was attributed to the physical nature of the test item.

·        Grossly observed yellow intestinal contents in cecum and colon at ≥333 mg/kg/day was attributed to the physical nature of the test item. 

 ·       Maternal Parameters: There were no treatment-related effects on maternal body weights and food consumption up to the highest tested dose of 1000 mg/kg/day. The other maternal parameters comprising of uterine weight, implantations and early and late resorptions, post implantation loss were comparable to vehicle control group up to the high dose of 1000 mg/kg/day. Gross evaluation of placenta revealed no findings.

·        Litter Parameters: There were no treatment-related effects on litter parameters comprising of total number of fetuses, fetal weights and anogenital distance in male and female foetuses.

·        Fetal examination: The fetal external, visceral and skeletal examinations revealed no signs of teratogenicity or developmental toxicity up to 1000 mg/kg/day.

·        Thyroid hormone levels (T3, T4 and TSH), thyroid gland weights and histology of thyroid gland were unaffected by treatment with C.I. Pigment Yellow 194 up to the highest dose of
1000 mg/kg/day.

 

Based on the above findings, it is concluded that, No Observed Adverse Effect Level (NOAEL) for  Maternal toxicity, fetal developmental toxicity and Teratogencity is 1000 mg/kg/day as

-            the maternal body weight and weight gain, corrected body weight gain and food consumption was unaffected up to 1000 mg/kg/day.

-            fetal resorptions or post implantation loss were comparable to the controls and there were no effects on fetal body weights. 

The fetal external, visceral and skeletal examinations revealed no signs of teratogenicity or developmental toxicity up to 1000 mg/kg/day

 

Reference 6

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

See read across justification document in chapter13

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Source: Hylasco Biotechnology (India) Pvt. Ltd.
Plot 4B, MN Park, Shameerpet Mandal,
Turkapally Village,
Medchal District, Telangana -500078

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for analysis of homogeneity and active ingredient (a.i.) were collected from the dose formulations intended for first treatment and last treatment (-2 days). The prepared formulations were sampled in duplicate sets wherein one set was used for analysis and another as back up set which was stored at ambient condition. For each set, duplicate sample were drawn from top, middle and bottom layers of each preparation and in case of control duplicate samples from the middle layer was drawn. The samples collected were sent to Analytical R&D of Eurofins Advinus Ltd. for formulation analysis to determine the homogeneity and concentration of the dose formulation.

Details on mating procedure:

The female rats were cohabited with males in a 1:1 ratio and vaginal smears and / or vaginal plug we re examined in the morning hours of the subsequent day to confirm mating

Duration of treatment / exposure:

Gestation day 5 to gestation day 19

Frequency of treatment:

Daily from gestation day 5 to gestation day 19

Duration of test:

Upto gestation day 20

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control

Dose / conc.:

111 mg/kg bw/day (nominal)

Remarks:

Low dose

Dose / conc.:

333 mg/kg bw/day (nominal)

Remarks:

Mid dose

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High dose

No. of animals per sex per dose:

24 day 0 pergnant rats per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Group Nos. Groups Dose
(mg/kg/day) Dosage volume (mL/kg) Concent-ration (mg/mL) No. of Day 0 pregnant rats Rat numbers
From To
G1 Vehicle control 0 10 0 24 Rz121 Rz144
G2 Low dose 111 10 11.1 24 Rz145 Rz168
G3 Mid dose 333 10 33.3 24 Rz169 Rz192
G4 High dose 1000 10 100 24 Rz193 Rz216

Maternal examinations:

CAGE SIDE OBSERVATION: Yes
- Time schedule: Twice a day (pre dose and post dose) during treatment period
- Cage side observation checked in table 2 were included

Ovaries and uterine content:

The ovaries and uterine contents were examined after termination GD 20.
• Pregnancy status
• Gravid uterine weight (from all rats subjected to caesarean section)
• Number of corpora lutea
• Number of implantation sites
• Number of early resorptions
• Number of late resorptions
• Gross evaluation of placenta

Fetal examinations:

• Total number of fetuses
• Total number of live fetuses
• Total number of dead fetuses
• Individual fetal body weight
• Fetus sex (during visceral examination)
• External examination of fetus
• Soft tissue evaluation
• Skeletal examination
• Head examination (half the number of fetuses per litter)

Statistics:

The data on maternal body weight, body weight change in interval, gravid uterine weight, body weight change corrected to gravid uterine weight, maternal food consumption, hormone analyses (T4, T3, TSH), weight of thyroid gland was analyzed using Analysis of Variance (ANOVA) after testing for homogeneity for intra group variance using Levene’s test. Where intra group variances were heterogeneous, ANOVA was performed after suitable transformation of data. Dunnett’s pairwise comparison of the treated group means with the control group mean was performed, when the group differences were found significant.

Fetal weight for male and female was analyzed using Analysis of Covariance (ANCOVA) taking litter size as covariate for group. Anogenital distance for male and female was analyzed using Analysis of Covariance (ANCOVA) taking weight as covariate for group. Number of corpora lutea, number of implantations, early and late resorptions, pre-implantation and post-implantation loss observations were analyzed using Kruskal Wallis test for group comparison. Wilcoxon pairwise comparison of the treated groups with the control group was performed, when the
group differences were significant. The incidence of dams with resorptions were tested for using Chi-square test followed by Fisher’s
exact test for group association. The incidence of fetus and litter (incidence and percent) observations for external, visceral and skeletal observations were tested using Cochran Armitage trend test and pair wise comparison will be tested by Fisher’s exact test for group association.
Statistically significant differences (p<0.05), indicated by the aforementioned tests was designated as * throughout the report.

Indices:

Refer Table 6 of the final report

Historical control data:

Refer Annexure 8 of the final report

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Grossly observed yellow intestinal contents in cecum and colon at ≥333 mg/kg/day was attributed to the physical nature of the test item

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormone levels (T3, T4 and TSH), thyroid gland weights and histology of thyroid gland were unaffected by treatment with Novoperm-Gelb F2G (C.I. Pigment Yellow 194) up to the highest dose of 1000 mg/kg/day.

Number of abortions:

no effects observed

Description (incidence and severity):

No abortions in the study

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: There were no adverse effects at all

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no effects at all

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

Based on the above findings, it is concluded that, No Observed Adverse Effect Level (NOAEL) for

Maternal toxicity, fetal developmental toxicity and Teratogencity is 1000 mg/kg/day as
- the maternal body weight and weight gain, corrected body weight gain and food consumption was unaffected up to 1000 mg/kg/day.
- fetal resorptions or post implantation loss were comparable to the controls and there were no effects on fetal body weights.
- the fetal external, visceral and skeletal examinations revealed no signs of teratogenicity or developmental toxicity up to 1000 mg/kg/day

Executive summary:

The objective of this study was to evaluate the developmental toxicity (teratogenic) potential of the test item to cause adverse effects on the pregnant rats and development of the embryo and fetus consequent to exposure of pregnant rats by oral route during gestation days (GD) 5 to 19. This study was intended to provide a rational basis for  a No Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity in rats.

A total of 96 Day 0 pregnant rats[1]were randomly divided into different groups according to the study design as follows:

Group Nos.	Groups	Dose (mg/kg/day)	Dosage volume (mL/kg)	Concentration (mg/mL)	No. of Day 0 pregnant rats
G1	Vehicle control*	0	10	0	24
G2	Low dose	111	10	11.1	24
G3	Mid dose	333	10	33.3	24
G4	High dose	1000	10	100	24

*0.5 % Carboxymethylcellulose Sodium salt (medium viscosity) in Milli-Q^®Water

 

The following parameters and end points were evaluated in this study: Clinical signs, body weights, body weight gains, food consumption, gross pathology, gravid uterine weights, intrauterine growth and survival,number of corpora lutea, and fetal parameters [sex, weight and anogenital distance, and external, visceral and skeletal observations].Approximately half the number of fetuses from each litter were examined for visceral malformations and the remaining half were evaluated for skeletal malformations. In addition, from each dam the thyroid glands were weighed and subjected to microscopic evaluation, and thyroid hormones were estimated from the blood collected at terminal sacrifice (GD20).

 

Results of the study are summarized below:

 

·        Clinical signs and gross necropsy changes: There were no clinical signs, or mortalities in treated rats at any of the doses tested.Expected yellow coloured faeces was observed in the test item treated groups which was attributed to the physical nature of the test item.

·        Grossly observed yellow intestinal contents in cecum and colon at ≥333 mg/kg/day was attributed to the physical nature of the test item. 

 ·       Maternal Parameters: There were no treatment-related effects on maternal body weights and food consumption up to the highest tested dose of 1000 mg/kg/day. The other maternal parameters comprising of uterine weight, implantations and early and late resorptions, post implantation loss were comparable to vehicle control group up to the high dose of 1000 mg/kg/day. Gross evaluation of placenta revealed no findings.

·        Litter Parameters: There were no treatment-related effects on litter parameters comprising of total number of fetuses, fetal weights and anogenital distance in male and female foetuses.

·        Fetal examination: The fetal external, visceral and skeletal examinations revealed no signs of teratogenicity or developmental toxicity up to 1000 mg/kg/day.

·        Thyroid hormone levels (T3, T4 and TSH), thyroid gland weights and histology of thyroid gland were unaffected by treatment with the test item up to the highest dose of
1000 mg/kg/day.

 

Based on the above findings, it is concluded that, No Observed Adverse Effect Level (NOAEL) for  Maternal toxicity, fetal developmental toxicity and Teratogencity is 1000 mg/kg/day as

-            the maternal body weight and weight gain, corrected body weight gain and food consumption was unaffected up to 1000 mg/kg/day.

-            fetal resorptions or post implantation loss were comparable to the controls and there were no effects on fetal body weights. 

The fetal external, visceral and skeletal examinations revealed no signs of teratogenicity or developmental toxicity up to 1000 mg/kg/day

 

Reference 7

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

See read across document in chapter 13

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Source: Hylasco Biotechnology (India) Pvt. Ltd.
Plot 4B, MN Park, Shameerpet Mandal,
Turkapally Village,
Medchal District, Telangana -500078

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for analysis of homogeneity and active ingredient (a.i.) were collected from the dose formulations intended for first treatment and last treatment (-2 days). The prepared formulations were sampled in duplicate sets wherein one set was used for analysis and another as back up set which was stored at ambient condition. For each set, duplicate sample were drawn from top, middle and bottom layers of each preparation and in case of control duplicate samples from the middle layer was drawn. The samples collected were sent to Analytical R&D of Eurofins Advinus Ltd. for formulation analysis to determine the homogeneity and concentration of the dose formulation.

Details on mating procedure:

The female rats were cohabited with males in a 1:1 ratio and vaginal smears and / or vaginal plug we re examined in the morning hours of the subsequent day to confirm mating

Duration of treatment / exposure:

Gestation day 5 to gestation day 19

Frequency of treatment:

Daily from gestation day 5 to gestation day 19

Duration of test:

Upto gestation day 20

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control

Dose / conc.:

111 mg/kg bw/day (nominal)

Remarks:

Low dose

Dose / conc.:

333 mg/kg bw/day (nominal)

Remarks:

Mid dose

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High dose

No. of animals per sex per dose:

24 day 0 pergnant rats per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Group Nos. Groups Dose
(mg/kg/day) Dosage volume (mL/kg) Concent-ration (mg/mL) No. of Day 0 pregnant rats Rat numbers
From To
G1 Vehicle control 0 10 0 24 Rz121 Rz144
G2 Low dose 111 10 11.1 24 Rz145 Rz168
G3 Mid dose 333 10 33.3 24 Rz169 Rz192
G4 High dose 1000 10 100 24 Rz193 Rz216

Maternal examinations:

CAGE SIDE OBSERVATION: Yes
- Time schedule: Twice a day (pre dose and post dose) during treatment period
- Cage side observation checked in table 2 were included

Ovaries and uterine content:

The ovaries and uterine contents were examined after termination GD 20.
• Pregnancy status
• Gravid uterine weight (from all rats subjected to caesarean section)
• Number of corpora lutea
• Number of implantation sites
• Number of early resorptions
• Number of late resorptions
• Gross evaluation of placenta

Fetal examinations:

• Total number of fetuses
• Total number of live fetuses
• Total number of dead fetuses
• Individual fetal body weight
• Fetus sex (during visceral examination)
• External examination of fetus
• Soft tissue evaluation
• Skeletal examination
• Head examination (half the number of fetuses per litter)

Statistics:

The data on maternal body weight, body weight change in interval, gravid uterine weight, body weight change corrected to gravid uterine weight, maternal food consumption, hormone analyses (T4, T3, TSH), weight of thyroid gland was analyzed using Analysis of Variance (ANOVA) after testing for homogeneity for intra group variance using Levene’s test. Where intra group variances were heterogeneous, ANOVA was performed after suitable transformation of data. Dunnett’s pairwise comparison of the treated group means with the control group mean was performed, when the group differences were found significant.

Fetal weight for male and female was analyzed using Analysis of Covariance (ANCOVA) taking litter size as covariate for group. Anogenital distance for male and female was analyzed using Analysis of Covariance (ANCOVA) taking weight as covariate for group. Number of corpora lutea, number of implantations, early and late resorptions, pre-implantation and post-implantation loss observations were analyzed using Kruskal Wallis test for group comparison. Wilcoxon pairwise comparison of the treated groups with the control group was performed, when the
group differences were significant. The incidence of dams with resorptions were tested for using Chi-square test followed by Fisher’s
exact test for group association. The incidence of fetus and litter (incidence and percent) observations for external, visceral and skeletal observations were tested using Cochran Armitage trend test and pair wise comparison will be tested by Fisher’s exact test for group association.
Statistically significant differences (p<0.05), indicated by the aforementioned tests was designated as * throughout the report.

Indices:

Refer Table 6 of the final report

Historical control data:

Refer Annexure 8 of the final report

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Grossly observed yellow intestinal contents in cecum and colon at ≥333 mg/kg/day was attributed to the physical nature of the test item

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormone levels (T3, T4 and TSH), thyroid gland weights and histology of thyroid gland were unaffected by treatment with Novoperm-Gelb F2G (C.I. Pigment Yellow 194) up to the highest dose of 1000 mg/kg/day.

Number of abortions:

no effects observed

Description (incidence and severity):

No abortions in the study

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: There were no adverse effects at all

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no effects at all

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

Based on the above findings, it is concluded that, No Observed Adverse Effect Level (NOAEL) for

Maternal toxicity, fetal developmental toxicity and Teratogencity is 1000 mg/kg/day as
- the maternal body weight and weight gain, corrected body weight gain and food consumption was unaffected up to 1000 mg/kg/day.
- fetal resorptions or post implantation loss were comparable to the controls and there were no effects on fetal body weights.
- the fetal external, visceral and skeletal examinations revealed no signs of teratogenicity or developmental toxicity up to 1000 mg/kg/day

Executive summary:

The objective of this study was to evaluate the developmental toxicity (teratogenic) potential of the test item to cause adverse effects on the pregnant rats and development of the embryo and fetus consequent to exposure of pregnant rats by oral route during gestation days (GD) 5 to 19. This study was intended to provide a rational basis for  a No Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity in rats.

A total of 96 Day 0 pregnant rats[1]were randomly divided into different groups according to the study design as follows:

Group Nos.	Groups	Dose (mg/kg/day)	Dosage volume (mL/kg)	Concentration (mg/mL)	No. of Day 0 pregnant rats
G1	Vehicle control*	0	10	0	24
G2	Low dose	111	10	11.1	24
G3	Mid dose	333	10	33.3	24
G4	High dose	1000	10	100	24

*0.5 % Carboxymethylcellulose Sodium salt (medium viscosity) in Milli-Q^®Water

 

The following parameters and end points were evaluated in this study: Clinical signs, body weights, body weight gains, food consumption, gross pathology, gravid uterine weights, intrauterine growth and survival,number of corpora lutea, and fetal parameters [sex, weight and anogenital distance, and external, visceral and skeletal observations].Approximately half the number of fetuses from each litter were examined for visceral malformations and the remaining half were evaluated for skeletal malformations. In addition, from each dam the thyroid glands were weighed and subjected to microscopic evaluation, and thyroid hormones were estimated from the blood collected at terminal sacrifice (GD20).

 

Results of the study are summarized below:

 

·        Clinical signs and gross necropsy changes: There were no clinical signs, or mortalities in treated rats at any of the doses tested.Expected yellow coloured faeces was observed in the test item treated groups which was attributed to the physical nature of the test item.

·        Grossly observed yellow intestinal contents in cecum and colon at ≥333 mg/kg/day was attributed to the physical nature of the test item. 

 ·       Maternal Parameters: There were no treatment-related effects on maternal body weights and food consumption up to the highest tested dose of 1000 mg/kg/day. The other maternal parameters comprising of uterine weight, implantations and early and late resorptions, post implantation loss were comparable to vehicle control group up to the high dose of 1000 mg/kg/day. Gross evaluation of placenta revealed no findings.

·        Litter Parameters: There were no treatment-related effects on litter parameters comprising of total number of fetuses, fetal weights and anogenital distance in male and female foetuses.

·        Fetal examination: The fetal external, visceral and skeletal examinations revealed no signs of teratogenicity or developmental toxicity up to 1000 mg/kg/day.

·        Thyroid hormone levels (T3, T4 and TSH), thyroid gland weights and histology of thyroid gland were unaffected by treatment with the test item up to the highest dose of
1000 mg/kg/day.

 

Based on the above findings, it is concluded that, No Observed Adverse Effect Level (NOAEL) for  Maternal toxicity, fetal developmental toxicity and Teratogencity is 1000 mg/kg/day as

-            the maternal body weight and weight gain, corrected body weight gain and food consumption was unaffected up to 1000 mg/kg/day.

-            fetal resorptions or post implantation loss were comparable to the controls and there were no effects on fetal body weights. 

The fetal external, visceral and skeletal examinations revealed no signs of teratogenicity or developmental toxicity up to 1000 mg/kg/day

 

Effect on developmental toxicity: via oral route

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

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

No classification

There were no adverse effects to reproduction in any of the relevant studies (OECD 421, OECD 414)