2-[[1-[[(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)amino]carbonyl]-2-oxopropyl]azo]benzoic acid

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Repeated oral-gavage administration of pigment (bulk- and nanoforms) to rats (according to OECD 422, GLP compliant) did not result in any adverse effects in rats. The test item was administered in vehicle at dosages of 100, 300, and 1000 mg/kg body weight/day, animals in control groups received the vehicle only. Test item was administered to male rats for 28 days 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. The NOAEL was 1000 mg/kg bw.

Daily oral (gavage) administration of the test item to Wistar rats at the dose levels of 111, 333 and 1000 mg/kg bwt/day for 2 weeks prior to mating, during mating, and post mating (males) or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery (females) had no effects on general health, food consumption, pre-coital time, gestation length, mating and fertility parameters. Male and female reproductive organs did not reveal any changes. There were no treatment-related effects on the uterine/implantation data and mean litter size. There were no external abnormalities in live or pups in any of the groups. No treatment-related changes in the ano-genital distance and ano-genital ratio were observed at any of the doses. The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested.

Link to relevant study records

Reference

Reference 1

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 2

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 422, GLP-compliant)

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Tocixity 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, Inc., Maasheseweg 87c, 5800 AN Vernay / Netherlands
- Number of Animals: 44 males (11 per group) and 44 females (11 per group)
- Age (at Start of Treatment): 11 weeks
- Body Weight Range (at Start of Treatment): 301 to 362 g (males), 216 to 247 g (females)
- Identification: Parent animals had cage card and individual animal number (ear tattoo), pups were individually tattooed with Indian ink on day 1 post partum
- Randomization: Performed after at least three days of acclimatization using a computer-generated random algorithm. Body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
- Accommodation: In groups of five in Makrolon type-4 cages with wire mesh tops up to the day of randomization and afterwards 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) with paper enrichment (ISO-BLOX from Harlan Laboratories B.V., Netherlands), batch/lot nos. 02105111001, 02105111201, 02105120301 and 6960C.CS-100099). 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 2018C (batch no. 80/11) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum.
- Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles.
- Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

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)

IN-LIFE DATES From 26 January 2012 to 28 March 2012.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

DOSE FORMULATIONS
The dose formulations were prepared weekly using the test item as supplied by the Sponsor.
The test item was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using a magnetic stirrer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration.
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

STORAGE OF DOSE FORMULATIONS
Dose formulations were stored at room temperature (20 +/- 5 °C) in glass beakers.
Based upon the results of stability analyses performed within the non-GLP Harlan Laboratories study no. D33711 (Dose Range-Finding Study for a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test in the Han Wistar Rat), dose formulations were stable for at least 8 days if stored at room temperature.

TREATMENT
- Method: Oral, by gavage
- Rationale for Method: Administration by gavage is a common and accepted route of exposure for this type studies.
- Frequency of Administration: Once daily
- Target Dose Levels: 0 mg/kg/day (control group), 100 mg/kg/day (group 2), 300 mg/kg/day (group 3) and 1000 mg/kg/day (group 4)
- Rationale for Dose Level Selection: The dose levels were selected based on a previous non-GLP dose range-finding toxicity study in Han Wistar rats, Harlan Laboratories Study D33711, using dose levels of 0, 100, 300 and 1000 mg/kg/ day, where no adverse effects were observed up to and including the highest dose level.
- 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) and 100 mg/mL/day (group 4).
- Duration of Acclimatization Period: 7 days.

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.
For a female which 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 performed.
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
On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 0.5 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 0.5 g of each concentration were taken from the middle only to confirm stability (8 days). During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 +/- 5 °C) and delivered on dry ice to the responsible for formulation analysis (Harlan Laboratories Ltd., Itingen / Switzerland) and stored there at -20 +/- 5 °C until analysis.
The samples were analyzed by UV-VIS spectroscopy following an analytical procedure developed at Harlan Laboratories. The test item was used as the analytical standard.

RESULTS
Blank samples showed no significant absorbance and, therefore, it was confirmed that only highly purified water was applied within the control experiment.
The application formulations investigated during the study were found to comprise test material in the range of 93.1% to 105.6% and, thus, the required content limit of +/-20% with reference to the nominal content was met. The homogeneous distribution of test item in the preparations was approved because single results found did not deviate more than 5.5% (<15%) from the corresponding mean.
The test item was found to be stable in application formulations when kept eight days at 20 +/- 5 °C 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 highly purified water as vehicle during this study. Application formulations were found to be homogeneously prepared and stable over a storage period of eight days (20 +/- 5 °C).

Duration of treatment / exposure:

MALES: 40 days
FEMALES: approximately 7 weeks

Frequency of treatment:

once daily

Details on study schedule:

MALES

- Acclimatization: 7 days
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum
- Treatment Ends: On day before sacrifice
- Blood Sampling: After 28 days of Treatment
- Necropsy: After treatment for 39 days, when no longer needed for assessment of reproductive effects

FEMALES

- Acclimatization: 7 days
- 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 4 post partum
- Blood Sampling: Day 5 post partum
- Necropsy: On day 5 post partum (pups on day 4 post partum)

Remarks:

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

No. of animals per sex per dose:

11

Control animals:

yes, concurrent vehicle

Positive control:

Not required

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: on days 1 - 4, 4 - 8, 8 - 11 and 11 - 14 during pre-pairing period and weekly during after pairing period.
Females: on days 1 - 4, 4 - 8, 8 - 11 and 11 - 14 during pre-pairing period; on days 0 - 7, 7 14 and 14 - 21 during gestation period and on days 1 - 4 of during lactation period.
No food consumption was recorded during the pairing period.

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

DETAILED CLINICAL OBSERVATIONS
Detailed clinical observations were performed outside the home cage in all animals. In males, it was performed once prior to the first administration of the test item and weekly thereafter. In females, it was performed once prior to the first administration of the test item, weekly during the pre-pairing and pairing periods and on days 0, 6, 13 and 20 of the gestation period.
Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior were also reported.

FUNCTIONAL OBSERVATIONAL BATTERY
At one time during the study (males shortly before the scheduled sacrifice and females on day 3 or 4 post partum) relevant parameters were performed with five P generation males and five P generation females from each group. This FOB assessment was conducted following the daily dose administration. Animals were observed for the following:
- Cage-side observations: faeces-balls, urine and posture as well as resistance to removal.
- Hand-held observations: muscle tone, constituation, skin, pupile size, palpebral closure, lacrimation, salivation, reaction to handling and general abnormalities.
- Open field observations: level of ambulatory activity including rearing (one minute evaluation), unusual body movements (e.g. spasms, convulsions), gait evaluation, behavior, hair coat, respiration, quantity of faeces-balls and urine.
- Reflexes: blinking, palpebral closure, pinna reflex, extensor thrust response, paw pinch, responsiveness to sharp noise, righting reflex and hearing ability (Preyer’s reflex).
- Measurements / Counts: hind limb / fore limb grip strength, landing foot splay, rectal temperature.

Any abnormal findings were recorded and, where appropriate, graded in severity.

Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with an Activity Monitor AMS-0151 (FMI, Germany). Activity of the animals (based on beam count) was recorded for 6-minute intervals over a period of 30 minutes. These data and the total activity over 30 minutes were reported.

CLINICAL LABORATORY INVESTIGATIONS
Blood samples were obtained on the day of the scheduled necropsy from 5 males from each group. Blood samples from 5 lactating females from each group were obtained on day 5 post partum. Blood samples were drawn sublingually from all animals under light isoflurane anesthesia. The animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.

The following hematology parameters were determined:
- Erythrocyte count
- Hemoglobin
- Hematocrit
- Mean corpuscular volume
- Red cell volume distribution width
- Mean corpuscular hemoglobin
- Mean corpuscular hemoglobin concentration
- Hemoglobin concentration distribution width
- Leukocyte count, total
- Differential leukocyte count
- Platelet count
- Prothrombin time (= Thromboplastin time)
- Activated partial Thromboplastin time

The following clinical biochemistry parameters were determined:
- Glucose
- Urea
- Creatinine
- Bilirubin, total
- Cholesterol, total
- Triglycerides
- Aspartate aminotransferase
- Alanine aminotransferase
- Alkaline phosphatase
- Gamma-glutamyl-transferase
- Bile acids
- Sodium
- Potassium
- Chloride
- Calcium
- Phosphorus
- Protein, total
- Albumin
- Globulin
- Albumin/Globulin ratio

URINALYSIS
The following urinalysis parameters were determined in five males of each group, which are allocated to the blood analysis, during the last week of the study using timed urine volume collection:
- Volume (18 hours)
- Specific gravity (relative density)
- Color
- Appearance
- pH
- Nitrite
- Osmolality
- Protein
- Glucose
- Ketones
- Urobilinogen
- Bilirubin
- Blood/Blood cells

Oestrous cyclicity (parental animals):

Not examined

Sperm parameters (parental animals):

Sperm analysis was performed on the first 5 males per group.

MOTILITY
At necropsy of adult males an epididymal sperm sample was obtained from the left cauda epididymidis of each male. The sample was diluted with a pre-warmed (about 35 °C) physiological medium, and shortly after being obtained, one hundred sperm were counted microscopically for determination of percentage of not motile, stationary motile and progressively motile sperm.

MORPHOLOGY
A second sperm sample from the left cauda epididymidis was used for morphological assessment after fixation and Eosin staining. 500 sperm per sample were evaluated microscopically and classified into the following categories:
A: Normal, complete sperm
B: Normal head only (tail detached)
C: Complete sperm, misshapen hook
D: Complete sperm, abnormally curved hook
E: Complete sperm, reversed head
F: Abnormal head only (tail detached)

Morphological sperm evaluation was performed only for group 1 and 4 males. In the absence of a treatment-related effect the slides for the group 2 and 3 males were not evaluated.

SPERM, SPERMATID COUNT
The left caudal epididymis and left testis were taken for determination of homogenization-resistant spermatids and caudal epididymal sperm reserve. These tissues were frozen at -20 +/- 5 °C pending evaluation. For evaluation the weighed tissues were placed in Triton-X-100 solution and homogenized with a blender (Ultra Turrax) and an ultrasonic water bath. Sperm or spermatid heads were counted microscopically using a modified Neubauer chamber. These evaluations were performed in the first instance only for group 1 and 4 males. In the absence of a treatment-related effect the remaining frozen tissues were not evaluated.

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 treatment for 39 days, when no longer needed for the assessment of reproductive effects. Dams were sacrificed on day 5 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 were subjected to a detailed macroscopic examination. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution. At the scheduled sacrifice, all animals were sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated. All parent animals were examined macroscopically for any structural changes. 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, testes and epididymides from all parental males were weighed separately. In addition, from 5 males and 5 females sacrificed at the end of the study which were selected from each group, the following organs were trimmed from any adherent tissue, as appropriate, and their wet weight taken.
- Adrenal glands (weighed as pairs)
- Brain
- Heart
- Kidneys (weighed as pairs)
- Liver
- Thymus
- Spleen

TISSUE PRESERVATION
The following tissues from all parental males were preserved in neutral phosphate buffered 4% formaldehyde solution:
- Prostate
- Seminal vesicles with coagulating gland
- Testes (in Bouin’s fixative)*
- Epididymides (in Bouin’s fixative)*
*From the first five males in each group which were used for sperm analysis, only the right testis and right epididymis were preserved for histopathological examination.

The following tissues from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution:
- Ovaries
In addition, from 5 males and 5 females per group selected for organ weights, the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution:
- Gross lesions
- Brain (representative regions including cerebrum, cerebellum and pons)
- Spinal chord
- Small and large intestines (incl. Peyer’s patches)
- Stomach
- Liver
- Kidneys
- Adrenals
- Spleen
- Heart
- Thymus
- Thyroids, and parathyroids if possible
- Trachea and lungs (preserved by inflation with fixative and then immersion)
- Uterus (with vagina)
- Urinary bladder
- Lymph nodes (mesenterial, mandibular)
- Peripheral nerve (sciatic)
- Bone marrow

HISTOTECHNIQUE
All organ and tissue samples to be examined by the study pathologist 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 study pathologist.

HISTOPATHOLOGY
Slides of all organs and tissues listed collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the study pathologist. The same applied to all occurring gross lesions.
Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.
Histological examination of ovaries was carried out on any females that did not give birth. In addition, microscopic examination of the reproductive organs of all infertile males was made, where necessary.
A histopathology peer review was performed. A histopathology phase report was provided by the principal investigator which is included in the report.

Postmortem examinations (offspring):

Pups were sacrificed on day 4 post partum. All animals were sacrificedby by an injection of sodium pentobarbital and subjected to a detailed macroscopic examination. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution. Pups found dead during the study, 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 when the variables could be dichotomized without loss of information.

Reproductive indices:

From the on-line recorded reproduction data, the following parameters were calculated: mean precoital time, percentage mating, fertility index, conception rate, post-implantation loss, gestation index, birth index and viability index.

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 and viability index.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Red stained faeces was noted in all males and females in dose groups; this was due to the staining properties of the test item.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In males at the dose level of 1000 mg/kg bw/day, lower body weight gain during the pre-pairing period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In males at the dose level of 1000 mg/kg bw/day, lower body weight gain during the pre-pairing period.

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:

effects observed, treatment-related

Description (incidence and severity):

Sperm motility was reduced in all dose groups.

Reproductive performance:

no effects observed

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

DAILY CLINICAL SIGNS OR OBSERVATIONS
Red stained feces was noted in all males and females in all dose groups starting from day 2 of the treatment until completion of the study with dose-related intensity of discoloration. This observation was due to staining properties of the test item.
No further test item-related clinical signs or observations were noted in males or females at any dose level.
Incidentally, in one male (no. 16) at the dose level of 100 mg/kg bw/day chromodacryorrhea was noted during the study (starting on day 1 of the pre-pairing period) and eye reduced in size was noted in the same animal from day 13 of the pre-pairing period.
No further test item-related findings were noted at any dose level.

FINDINGS AT DETAILED WEEKLY CLINICAL OBSERVATIONS
No test item-related findings were noted during detailed weekly clinical observations.
The only findings noted were chromodacryorrhea and eye reduced in size in male no. 16 at the dose level of 100 mg/kg bw/day recorded already during the daily clinical observations.

FUNCTIONAL OBSERVATIONAL BATTERY
No test item-related findings were noted during the functional observational battery tests in males or females at any dose level.
Statistically significantly lower body temperature was noted in both sexes. In males mean body temperature was 37.9 °C and 37.8 °C at the high- and mid-dose levels, respectively, compared to 38.4 °C in the control group. In females, 38.5 °C was noted at the high-dose level, compared to 38.9 °C in the control group. The differences noted in males and females were only minor, not clearly dose dependent and all values remained in the historical control range. For these reasons, changes in body temperature were considered not to be test item-related.
No further findings were noted during functional observational battery in males or females at any dose level except for the eye findings in male no. 16 at 100 mg/kg bw/day.

LOCOMOTOR ACTIVITY
No effects on locomotor activity were noted in males or females at any dose level.
Mean beam counts during the 30 minutes of measurement at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were respectively: 1255, 1137, 1219 and 1209 in males and 924, 882, 1002 and 1050 in females.

FOOD CONSUMPTION OF MALES
No effects on food consumption were noted in males at any dose level.
Mean differences in food consumption at the dose levels 100, 300 and 1000 mg/kg bw/day were respectively: +1.1%, -3.4% and -3.4% during the pre-pairing period and -1.5%, -3.1% and -3.8% during the after pairing period ( percentages refer to the respective values in the control group).

FOOD CONSUMPTION OF FEMALES
No test item-related effects on food consumption were noted in females at any dose level.
Incidentally, statistically significantly higher food consumption was noted at the dose level of 100 mg/kg bw/day during lactation period. In the absence of an effect in females at the dose levels of 300 and 1000 mg/kg bw/day, this difference was considered not to be related to the treatment.
Mean differences in food consumption at the dose levels 100, 300 and 1000 mg/kg bw/day were respectively: +5.9%, +2.7%, and +1.1% during the pre-pairing period, +7.0%, +2.5% and +2.9% during the gestation period and +23.2%, -5.9% and +11.0% during the lactation period (percentages refer to the respective values in the control group).

BODY WEIGHTS OF MALES
At the dose level of 1000 mg/kg bw/day, a slightly lower body weight gain if compared to the controls was noted during the pre-pairing period. Mean body weight gain within this period was +10%, compared to +13% in the control group. The difference in body weight gain was statistically significant during the most days starting from day 3 until the end of the pre-pairing period. This effect was considered to be test item-related. During the pairing and after pairing periods, body weight gain was similar at all dose levels.
No significant changes in body weights were noted in males at any time during the study.
Because the lower body weight gain at the high-dose level was reversible despite treatment continued and did not result in any significant changes in body weights, this finding was considered not to be adverse.
No significant changes in body weight gain or body weights were noted in males at the dose levels of 100 and 300 mg/kg bw/day.
Mean differences in body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were respectively: +13%, +13%, +11% and +10% during the pre-pairing period, +10%, +10%, +9% and +9% during the pairing period and +7%, +6%, +7% and +6% during the after pairing period (percentages refer to the body weight change within the respective period).

BODY WEIGHTS OF FEMALES
Body weights and body weight gain of females were not affected by the treatment with the test item at any dose level.
On individual days some statistically significantly changed values of body weight gain were noted at the low-, mid- and high-dose levels. The changes did not follow a dose dependency and were therefore not related to the treatment.
Mean differences in body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were respectively: 5%, 7%, 6% and 7% during the pre-pairing period, 48%, 56%, 47% and 54% during the gestation period and 3%, 8%, 4% and 5% during the lactation period (percentages refer to the body weight change within the respective period).

2. CLINICAL LABORATORY INVESTIGATIONS
HEMATOLOGY
No test item-related effects on hematology parameters were noted in males or females at any dose level.
In males, statistically significant changes of several parameters: higher distribution width of red cell volume (RDW) at the low-dose level and higher distribution width of hemoglobin concentration (HDW) at the low- and mid-dose levels occurred in the absence of an effect at the high dose and therefore were considered not to be test item-related.
In females, at the low-dose level, statistically significantly higher platelets count was noted in the absence of any increase of this value at the mid- and high-dose levels and therefore it was not test item-related.
No further changes of hematology parameters were noted in males or females at any dose level.

CLINICAL BIOCHEMISTRY
No test item-related effects on biochemistry parameters were noted in males or females at any dose level.
In males, at the mid-dose level, statistically significantly lower concentration of triglycerides was noted. In the absence of dose dependency, this finding was not test item-related.
In females at the low dose level, following statistically significant changes were noted: higher concentration of cholesterol, higher concentration of globulin, and lower globulin to albumin ratio. These changes were not dose-dependent and therefore they were considered not to be test item-related.
No further changes of biochemistry parameters were noted in either males or females at any dose level.

URINALYSIS
No changes in urine parameters were noted in males at any dose level.

3. TERMINAL FINDINGS
SEMINOLOGY AND SPERMATID COUNT
In all dose groups, statistically significant changes in motility of sperms were noted. Following values were assessed in sperm samples at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day respectively: 81.1%, 64.6%, 57.3% and 47.5% of progressive sperms (changes were statistically significant in all dose groups), 3.7%, 11.3%, 6.0% and 11.9% of stationary sperms (changes were statistically significant at the dose levels of 1000 and 100 mg/kg bw/day) and 15.2%, 24.1, 36.7 and 40.6% of not motile sperms (changes were statistically significant at the dose levels of 1000 and 300 mg/kg bw/day). These changes might be test item-related. However no significant dose dependent trend indicated by probability values of <0.05 was determined for any of these changes when performing a linear regression analysis (least squares).
No further changes were noted during sperm analysis. At the high-dose level, all morphological categories of sperms were represented with similar frequency to that in the control group whereas sperm count was similar to the respective control values in samples from both testis and epididymidis.

ORGAN WEIGHTS
No changes in absolute organ weights or organ weights to body weights and to brain weights ratios were noted in males or females at any dose level.

MACROSCOPICAL FINDINGS
Type and distribution of findings noted during macroscopical examination of males or females did not indicate any test item-related effect.

HISTOPATHOLOGY FINDINGS
Under the conditions of this experiment, treatment with the test item did not cause pathological findings. All findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.

4. REPRODUCTION AND BREEDING
MATING PERFORMANCE AND FERTILITY
Mating performance and fertility were not affected by the treatment at any dose level.
All females in groups 2, 3 and 4 mated within the first pairing period. In group 1, one female (no. 54) was mated during the second pairing period.
Mean (median) precoital times were 4.5 (3), 2.5 (3), 4.0 (2) and 2.6 (3) days at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.
Seven females were not pregnant: three in the control group and in the mid-dose level and one in the low-dose level. Consequently, fertility indexes (number of females pregnant as percentages of females paired) and conception rate (number of females pregnant as percentages of females mated) were 72.7%, 90.9%, 72.7% and 100.0% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.
One female at the high dose level had one implantation site but delivered no pups. Consequently, gestation index (number of females with living pups as percentages of females pregnant) was 100% in the control group and at low- and mid-dose levels and 90.9% at the high-dose level.

CORPORA LUTEA COUNT
No test item-related effects on corpora lutea count were observed at any dose level.
Mean number of corpora lutea per dam was 16.0, 17.2, 16.3 and 18.4 in order of ascending dose levels.

DURATION OF GESTATION
No effects on duration of gestation were observed at any dose level.
Mean duration of gestation was 21.6, 21.6, 21.5 and 21.7 days, in order of ascending dose level.

IMPLANTATION RATE AND POST-IMPLANTATION LOSS
No effects on implantation rate and post-implantation loss were observed at any dose level.
In order of ascending dose levels, mean number of implantations per dam was 12.6, 14.9, 12.6 and 14.0 whereas mean incidence of post-implantation loss per dam was 1.5, 0.8, 0.6 and 0.5 per dam.

LITTER SIZE AT FIRST LITTER CHECK
No effects on litter size were noted at any dose level.
During the first litter check, one dead pup was found in a litter at the dose level of 1000 mg/kg bw/day. Because of isolated occurrence, this finding was considered to be incidental.
Mean number of living pups per dam at first litter check was 11.1, 14.3, 12.0 and 13.5 in order of ascending dose levels.
Birth index (number of pups born alive as a percentage of implantations) was 88.1%, 94.8%, 95.0% and 96.4% at the dose level of 0, 100, 300 and 1000 mg/kg bw/day.
Birth index at the dose level of 1000 mg kg bw/day was statistically significantly higher than the respective control value. This was considered to be a result of biological variability.

Dose descriptor:

NOAEL

Remarks:

for general toxicity

Effect level:

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

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 (actual dose received)

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
No test item-related effects on postnatal loss were noted at any dose level.
In the control group one pup was missing on day 4, at the low-dose level one pup was missing on day 2, at the mid dose level three pups (from two litters) were missing on day 2 and at the high dose level no postnatal loss was noted in any litter.
Mean postnatal loss per dam during four days of lactation was 0.1%, 0.1%, 0.4% and 0.0% at the dose level of 0, 100, 300 and 1000 mg/kg bw/day, respectively. Consequently, viability index (number of pups alive at termination on day 4 p.p. as a percentage of pups born alive) was 98.9%, 99.3%, 96.9% and 100% in order of ascending dose levels.

EXTERNAL EXAMINATION AT FIRST LITTER CHECK AND DURING LACTATION
No test item-related findings were noted in pups during first litter check and during lactation at any dose level.

Incidentally, one pup in the control group was found with a wound and missing tail tip, two further pups, each one at the low- and mid-dose levels, had a wound at first litter check. These findings were also noted during the remaining lactation period.

SEX RATIOS
Pups sex ratio was not affected by exposure to the test item at any dose level.
At first litter check, percentages of male pups were 56%, 48%, 51% and 56% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day.

BODY WEIGHTS TO DAY 4 POST PARTUM
Body weights and body weight gain of pups were not affected by the treatment with the test item at any dose level.
Mean body weights of pups on day 1 post partum were: 6.4 g, 6.1 g, 6.4 and 6.2 g and mean differences in body weights during lactation were +49.9%, +43.6%, +47.8% and +42.6%, at the dose levels of 0, 100, 300 and 1000 mg/kg/day, respectively.
At the dose levels of 100 and 1000 mg/kg bw/day, slightly not statistically significantly lower body weight gain of pups was noted. This effect was considered to be due to a higher number of pups at these dose levels which was supported by observation that reduction of body weight gain was more pronounced in litters of higher size. Therefore this effect was considered not to be test item-related.

MACROSCOPICAL FINDINGS
No test item-related findings were noted at macroscopic examination of pups at any dose level.
Incidentally, in the control group one pup had a sore in the thoraco-dorsal region, one further pup in this group had a missing tail tip. These findings were already recorded during the in life phase. At the high-dose level, one pup had a watery cyst in the left kidney.
No further findings were noted in pups at any dose level.

Dose descriptor:

NOAEL

Remarks:

for development

Generation:

F1

Effect level:

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

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 (100)
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 non pregnant females (A)	3	1	3	0
Numbers of pregnant females, which did not deliver any pups (B)	0	0	0	1
Number of females which reared their pups until day 4 post partum	8	10	8	10

(A) Female Nos. 45, 46, 55, 62, 74, 75 and 77

(B) Female No. 85 had implantations only

 

Conclusions:

Repeated oral-gavage administration of the test item to rats (according to OECD 422, GLP compliant) did not result in any adverse deveolpmental effects in rats. The test item was administered in vehicle (highly purified water) at dosages of 100, 300, and 1000 mg/kg body weight/day, animals in control groups received the vehicle only. Test item was administered to male rats for 28 days 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. The NOAEL was 1000 mg/kg bw.

Under the conditions of this study, no adverse effects were found in males or females up to the highest dose level of 1000 mg/kg bw/day.

All animals survived the scheduled study period.
During the treatment, faeces stained red with dose-dependent intensity of discoloration were noted in all males and females receiving test material. This observation was due to staining properties of the test item.

No effects on food consumption were noted in males at any dose level. Body weight gain was slightly but statistically significantly reduced in males at the dose level of 1000 mg/kg bw/day during the pre-pairing period. No differences in body weight gain were noted at any dose level during the remaining study period. Body weights of males in all dose groups were similar to the respective control values during the entire study period. Because lower body weight gain at the high-dose levels was reversible and did not cause significant changes in body weights, this effect was considered not to be adverse.

Food consumption, body weights and body weight gain of females were not affected by the treatment at any dose level.

No further test item-related observations were noted in males or females at any dose level during the live part of the study.

Terminal examinations revealed changes in motility of sperms in all dose groups. Statistically significant decrease in mean count of progressive sperms was noted at the dose levels of 100, 300 and 1000 mg/kg bw/day, statistically significant increase in mean count of stationary sperms was noted at the dose levels of 100 and 1000 mg/kg bw/day and statistically significant increase in mean count in not motile sperms was noted at the dose levels of 300 and 1000 mg/kg bw/day. However a significant dose dependent trend indicated by probability values of <0.05 was not established for any of these changes when performing a linear regression analysis (least squares).
No further effects on male reproductive system were noted during the study. Sperm morphology and sperm count at the high-dose level was similar to the control values. Weights of male reproductive organs, macroscopical and histopathological examination of testes and epididymides gave no indication of any treatment-related effect. Further, no indication of effects on reproduction was noted within this study up to and including the highest dose level. For this reason, changes in motility of sperms were considered not to be adverse in this study.


Reproduction and development were not affected by the treatment. Mating performance, fertility, duration of gestation, corpora lutea count, implantation rate, post implantation and postnatal loss or litter size were similar in the control and all dose groups. There were no test item-related findings in pups noted during the first litter check, the first 4 days post partum or during the necropsy, pups body weights and body weight gain were not affected by the treatment at any dose level.

Based on these results, the NOAEL (No Observed Adverse Effect Level) for general toxicity in males and females and for reproduction/developmental toxicity in this study was considered to be 1000 mg/kg bw/day, the highest dose level used.

Executive summary:

The purpose of this study was to generatepreliminaryinformation concerning the effects of the test material on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. In addition it provides information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

 

The test item was administered to male rats for 39 days 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.

 

The following dose levels were applied:

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 (highly purified water).

 

The following results were obtained:

 MORTALITY AND GENERAL TOLERABILITY OF PARENTAL ANIMALS

  All animals survived the scheduled study period.

Feces stained red with dose-dependent intensity of discoloration were noted in all males and females in all dose groups starting from day 2 of the treatment until completion of the study. This observation was due to staining properties of the test item.

  No further test item-related clinical signs or observations were noted in males or females at any dose level.

 

FUNCTIONAL OBSERVATIONAL BATTERY IN PARENTAL ANIMALS

  No test item-related findings were noted during the functional observational battery tests in males or females at any dose level.

 

FOOD CONSUMPTION OF PARENTAL ANIMALS

  No effects on food consumption were noted in males or females at any dose level.

 

BODY WEIGHTS OF PARENTAL ANIMALS

  In males at the dose level of 1000 mg/kg bw/day, a slight but statistically significant lower body weight gain was noted during the pre-pairing period. No differences in body weight gain were noted at any dose level during the remaining study period. Body weights of males in all dose groups were similar to the respective control values during the entire study period. Because reduction in body weight gain at the high-dose levels was reversible and did not cause significant changes in body weights, this effect was considered not to be adverse.

  Body weights and body weight gain of females were not affected by the treatment at any dose level.

 

CLINICAL LABORATORY INVESTIGATIONS IN PARENTAL ANIMALS

  No test item-related effects on hematology and clinical biochemistry parameters were noted in males or females at any dose level.

  No changes in urine parameters were noted in males at any dose level.

 

REPRODUCTION AND BREEDING DATA

  Mating performance, fertility, corpora lutea count, duration of gestation, implantation rate and post-implantation loss, litter size or postnatal loss were not affected by the treatment with the test item.

 

SEMINOLOGY AND SPERMATID COUNT IN PARENTAL MALES

Effects on sperm motility which might be test item-related were noted in all dose groups. Mean count of progressive sperms was statistically significantly reduced at the dose levels of 1000, 300 and 100 mg/kg bw/day, mean count of stationary sperms was statistically significantly increased at the dose levels of 1000 and 100 mg/kg bw/day and mean count of not motile sperms was statistically significantly increased at the dose level of 1000 and 300 mg/kg bw/day. But a significant dose dependent trend couldn’t be established.

In the absence of any findings during necropsy or histopathological examination of male reproductive organs as well as in the absence of any effects on reproduction, the differences in sperm motility were considered not to be adverse.

 

ORGAN WEIGHTS OF PARENTAL ANIMALS

  No changes in absolute organ weights or organ weights to body weights and to brain weights ratios were noted in males or females at any dose level.

 

MACROSCOPICAL FINDINGS AND HISTOPATHOLOGICAL EXAMINATIONS OF PARENTAL ANIMALS

  Type and distribution of findings noted during macroscopical examination did not indicate any test item-related effect.

  Treatment with test item did not cause pathological findings. All findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.

 

FINDINGS IN PUPS AT FIRST LITTER CHECK AND DURING LACTATION

  No test item-related findings were noted in pups during first litter check and during lactation at any dose level.

  Pups sex ratio was not affected by the exposure to the test item at any dose level.

 

PUP WEIGHTS TO DAY 4 POST PARTUM

  Body Weights and body weight gain of pups were not affected by the treatment with the test item at any dose level.

 

MACROSCOPICAL FINDINGS OF PUPS

  No test item-related findings were noted at macroscopic examination of pups at any dose level.

 

CONCLUSION

  Based on these results, the NOAEL (No Observed Adverse Effect Level) for general toxicity in males and females and for reproduction/developmental toxicity in this study was considered to be 1000 mg/kg bw/day, the highest dose level used.

Reference 3

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 November 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Justification for study design:

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar Rats was to determine the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. Further this study also provides initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition. This study also provides information on reversibility, persistence or delayed occurrence of systemic toxic effects, for 14 days post treatment

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Source: Hylasco Biotechnology (India) Pvt. Ltd., 4B MN Park, Turkapally Village, Shameerpet Mandal, Medchal Dist, Telangana 500078
Justification for selection of species: Rat is the standard laboratory rodent species used for toxicity assessment and recommended by various regulatory authorities. The Wistar rat was selected due to the large amount of background data available for this strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Rats were housed in an environment controlled room. The temperature maintained during the experiment was between 19 to 24°C and relative humidity between 49 and 68 %. The photoperiod was a 12 hours light and 12 hours dark cycle. Adequate fresh air supply of 12.5 – 12.9 air changes/hour was maintained in the experimental room. The maximum and minimum temperature in the experimental room was recorded once daily. The relative humidity in the experimental room was calculated daily from dry and wet bulb temperature recordings.

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

The test item was administered by oral gavage in graduated doses to three groups of male and female rats. The males were dosed for 57 days, up to and including the day before scheduled sacrifice (this includes two weeks prior to mating, during mating period and approximately, two weeks post mating period).
Females were dosed throughout the treatment period. This includes two weeks prior to mating (with the objective of covering at least two complete oestrous cycles), the variable time to conception, the duration of pregnancy and up to and including the day before scheduled sacrifice (i.e., up to LD13).
Animals in the recovery groups were kept only for observations of reversibility, persistence or delayed occurrence of systemic toxic effects for 14 days of recovery period and these animals were not mated and consequently were not used for assessment of reproduction/developmental toxicity. The recovery period of the study started from the first scheduled kill of dams.

Details on mating procedure:

One female was placed with one male from the same group in a 1:1 ratio. Cohabitation was continued until there was evidence of sperms in the vaginal smear. All the females copulated successfully within seven days from the day of cohabitation. Subsequently, pregnant females were housed individually until LD 14. Not-littered females were sacrificed after 25 days from the day they were found sperm positive (by vaginal smear examination).
The day of confirmed mating was designated as GD 0. The pre-coital time (days) was calculated for each female.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For homogeneity and test item concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 and during Week 4 (Day 23) of treatment period and was analysed in-house. For each set, duplicate samples were drawn from the top, middle and bottom layers of each preparation and in case of the control duplicate samples from the middle layer were drawn.
The analysis was done as per the method validated under Eurofins Advinus Study No.: G19468. One set of samples (first set) were analysed for test item concentration analysis and other set (second set) of samples were stored at ambient condition for reanalysis purpose as a backup.
Both set I and set II analysis results of G3 and G4 groups prepared on Day 1(16 June 2020) were out of acceptance limits. Hence, the samples from subsequent preparations were analysed on 17 June 2020. The results of G3 group from the subsequent preparation (17 June 2020) was within the acceptance limits, however G4 group results were out of acceptance limits. Hence the backup samples (formulations prepared on 17 June) of G4 group samples were analysed on 18 June 2020.
Formulations were considered acceptable when the mean results (calculated using all the replicate values) of all the layers and mean of each layer was within ±15.0 % of the claimed concentration and the relative standard deviation (% RSD) was equal to or less than 10.0 %.

Duration of treatment / exposure:

Males: The dose formulation was administered orally by gavage to the rats of the specific groups once daily at approximately the same time each day (varying by ± 3 hours) for 57 days which includes 2 weeks prior to mating, during mating and post-mating, up to and including the day before scheduled sacrifice.
Females: The dose formulations were administered orally by gavage to the specific group of rats once daily at approximately the same time each day (varying by ± 3 hours) for total of 51-58 days which includes 2 weeks prior to the mating, during mating, pregnancy and up to LD 13.
The dose formulations were administered to the high dose recovery group of rats once daily at approximately the same time each day (varying by ± 3 hours).
The dose volume administered to each rat was at an equivolume of 10 mL/kg body weight throughout the study. The dose volume was adjusted based on the most recent body weight of individual rat.
Similarly, vehicle was administered to rats in the vehicle control and vehicle control recovery groups at an equivolume of 10 mL/kg bwt.
The vehicle and the test item were not administered for vehicle control recovery and high dose recovery groups, respectively for 14 days from the first scheduled kill of dams.

Frequency of treatment:

Daily

Details on study schedule:

Study initiation date: 27 May 2020
Experimental starting date: 28 May 2020
Acclimatization: Start: 28 May 2020, End: 01 June 2020
Pre-treatment period: Start : 02 June 2020, End: 15 June 2020
Treatment start: Start: 16 June 2020, End: 12 August 2020
Experiment completion Date: 31 August 2020
Submission of Draft report: 31 August 2020
Study completion: 13 November 2020

Dose / conc.:

111 mg/kg bw/day (nominal)

Dose / conc.:

333 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Main groups : 10 males and 10 females
Recovery groups : 5 males and 5 females

Control animals:

yes

Details on study design:

Group
No. Group Colour
of
cage card Dose
(mg/kg bwt/day) Concen-tration (mg/mL) Dose volume (mL/ kg bwt/day) No. of rats Sex Rat Numbers
From To
Main Groups
G1 Vehicle Control White 0 0 10 10 M Rx7481 Rx7490
10 F Rx7491 Rx7500
G2 Low dose Yellow 111 11.1 10 10 M Rx7501 Rx7510
10 F Rx7511 Rx7520
G3 Mid dose Green 333 33.3 10 10 M Rx7521 Rx7530
10 F Rx7531 Rx7540
G4 High dose Pink 1000 100 10 10 M Rx7541 Rx7550
10 F Rx7551 Rx7560
Recovery Groups
G1R Vehicle Control recovery White 0 0 10 5 M Rx7561 Rx7565
5 F Rx7566 Rx7570
G4R High dose recovery Pink 1000 100 10 5 M Rx7571 Rx7575
5 F Rx7576 Rx7580

Oestrous cyclicity (parental animals):

Vaginal smear was examined and the stage of oestrous cycle was recorded daily for two weeks before start of the treatment to select females with regular
4-5 days cyclicity for the study. The vaginal smear was also examined daily from the beginning of the treatment period until evidence of mating to determine the Day 0 of pregnancy/treatment-related effects on mating or pre-coital time. The time interval (in days) from the diestrus of an oestrous cycle to the next diestrus was considered as the oestrous cycle length of an animal.
Vaginal smears were also examined on the day of necropsy to determine the stage of the oestrous cycle.

Litter observations:

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

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

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

d. On LD 4, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four pups per sex per litter. Partial adjustment was done when the number of male or female pups prevents having four of each sex per litter. Pups were not eliminated when the litter size drop below the culling target (8 pups/litter). Blood samples were collected from the available surplus pups of either sex, pooled, and used for determination of serum Thyroxine (T4) and Thyroid stimulating hormone (TSH) levels.

e. After standardization, the individual pup body weight was measured on LD13.

f. The number of nipples/areolae in male pups was counted on LD 13.

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

h. The litters were observed daily to note the number of alive, dead and cannibalized pups.

i. In addition to daily clinical observations, all pups were observed for any abnormal behaviour.

j. Fertility index for dams, sires as well as the pup survival index until LD 4 was calculated.

Postmortem examinations (parental animals):

All adult animals and pups were subjected for detailed necropsy and findings were recorded. The adult animals sacrificed at term were fasted overnight (water allowed), weighed and exsanguinated under isoflurane anaesthesia. The dams were sacrificed on LD 14. All the surviving pups were necropsied on LD 13 and findings were recorded. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development. Dead pups were examined for defects and/or cause of death.
For apparently non-pregnant rats, the uteri was stained with 10% aqueous ammonium sulphide (Salewski staining method) to identify the peri-implantation loss of the embryos (by staining the implantation sites) thereby, for confirmation of pregnancy.
The number of implantation sites were recorded for all the dams.

Statistics:

Parameters such as body weight, body weight change, food consumption, oestrous cycle, gestation length (days), mean litter size, mean viable litter size, ano-genital distance, sex ratio, survival index and pup body weight, laboratory Investigations – haematology, coagulation, clinical chemistry, urinalysis & thyroid hormone profile, organ weights, organ weight ratios (organ to body weight and organ to brain weight), no. of implantations, post implantation loss (%) were evaluated using the Levene’s Test for homogeneity of variances and the Shapiro-Wilks Test for normality of distributions. When the data was found to be homogeneous and of normal distribution, the data was analysed by analysis of variance (ANOVA). When the data was found to be nonhomogeneous or of non-normal, the data was subjected for transformation and ANOVA was performed on transformed data. When ANOVA was found to be significant, pairwise comparisons of treated groups to the control group was made using a parametric test, Dunnett, to identify statistical differences.
Data like pre-coital interval, mating and fertility indices were analysed using Chi-square test. When Chi-square was found to be significant, pairwise comparisons of treated groups to the control group was made using a Fisher Exact test, to identify statistical difference in ProvantisTM built-in statistical tests.
Data captured outside of ProvantisTM: The statistical analysis of the experimental data was carried out using licensed copies of SYSTAT Statistical package Ver.12.0. All quantitative data was tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene’s test) within the group before performing a one-factor ANOVA modelling by treatment groups. Non-optimal (non-normal or heteroscedastic) data was transformed, before ANOVA was performed. Comparison of means between treatment groups and control group was done using Dunnett’s test when the overall treatment, ‘F’ test was found significant.

Reproductive indices:

11.1 Reproductive Performance Data of Parents
a. Male mating index (%)

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

b. Male fertility index (%)

Number of males siring a litter/impregnated a female
= ----------------------------------------------------------------- x 100
Number of males with evidence of mating

c. Female mating index (%)

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

d. Female fertility index (%)

Number of pregnant females
= ------------------------------------------- x 100
Number of females with evidence of mating

e. Mean number of implantations/group

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


f. Post implantation loss (%)

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

Offspring viability indices:

11.2 Litter Data
a. Mean litter size per group

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

b. Mean viable litter size

No. of viable pups
= -----------------------------------------
Total Number of littered animals

c. Live birth index (%)

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


d. Day 4 survival index (%)

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

e. Sex Ratio/ Percentage of male offspring (%)

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

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

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

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical signs and mortality observed throughout the treatment period in either sex at all the doses tested, except for an incidental observation of dehydration, piloerection and weakness in a female rat (Rx7557) of high dose group on days 42 and 43. This dam was sacrificed on Day 43 due to total litter loss. The yellowish colour faeces was observed at all the tested doses in both the sexes which recovered by Day 2 of recovery period. This yellowish coloured faecal matter is related to physical nature of the test item.
There were no abnormalities observed in pups

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The mean body weights and body weight gains were unaffected throughout treatment and recovery period in males and two weeks pre-mating period in females at all the tested doses when compared to vehicle control group.
The significantly lower weekly absolute weight change during Days 52-59 in recovery group females was considered incidental as the mean body weights were not altered by the treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The food consumption was not altered throughout treatment and recovery period when compared to the concurrent vehicle control.
Incidences of significantly decreased food consumption during Days 1-8 in 1000 mg/kg bwt/day recovery dose group males and during Days 8-14 in both 111 and 333 mg/kg bwt/day dosed females and significantly increased food consumption during Days 43-50 in 1000 mg/kg bwt/day recovery dose group males was observed. These isolated statistically significant differences observed in food consumption were toxicologically not significant as the mean body weights were not altered by the treatment.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related changes in the hematology parameters including PT and APTT values in both males and females. A few variations in coagulation parameters that reached statistical significance were considered incidental and were likely due to random biological variation as the percent change was minimal and/or there was no dose correlation.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The clinical chemistry parameters were not affected by test item administration. Increased creatinine kinase activity in 1000 mg/kg bwt/day dose males was considered as incidental change as it was not associated with any microscopic changes in muscle tissues examined. Increased creatinine concentration in 1000 mg/kg bwt/day dose females was considered as incidental change as it was not associated microscopic changes in kidneys examined. A few other variations in clinical chemistry parameters that reached statistical significance were considered incidental and were likely due to random biological variation as the percent change was minimal and/or there was no dose correlation.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Motor Activity: No treatment-related abnormalities were observed in any of the doses tested in both sexes except for an incidence of lower distance travelled at interval 1 and lower total distance travelled in recovery males treated at 1000 mg/kg bwt/day.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related microscopic changes at any of the dose levels tested.
All other single or few incidences of microscopic findings observed were considered incidental and not related to test item as they were randomly distributed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Litter Date: Test item had no treatment-related effects on the mean litter size, mean viable litter size and number of dead pups at first observation. There were no external abnormalities in live or dead pups in any of the groups. No treatment-related changes were observed in the survival data of pups up to LD 4 at all the tested doses.
Body Weight of Pups: The mean body weight of male and female pups per litter and litter mean pup body weight were not affected by the treatment at all the doses.
Anogental Distance (AGD): No changes attributable to the test item were detected in the Ano-genital distance and Ano-genital ratio in either sex. An incidence of higher ano-genital distance in female pups at 111 mg/kg bwt/day was considered as not related to treatment as this difference was not dose related and was minimal (̴ 3%).
Uterine/Implantation Data: No test item-related changes were observed in the number of implantations and percentage of post implantation loss.
Areolae/Nipple Retention in Pups: The male pups did not exhibit areola/nipple retention on PND 13.
Oestrous Cycle Prior to Sacrifice: The vaginal smear was examined for all the animals prior to necropsy and following is the details of various stages observed;

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects on the mean pre-coital time, gestation length (average days to litter), number of pregnancies and number of dams littered. No treatment-related changes were observed in the mating and fertility indices of sires and dams at all the doses tested. The lower gestation length at high dose was considered not treatment related as the difference was minimal (̴ 3%).

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:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

urinalysis

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

reproductive performance

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

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

not examined

Sexual maturation:

no effects observed

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Gross pathological findings:

no effects observed

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

sexual maturation

clinical signs

mortality

body weight and weight gain

gross pathology

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

To summarize, oral (gavage) administration of the test item to Wistar rats at the dose levels of 111, 333 and 1000 mg/kg bwt/day for 2 weeks prior to mating, during mating, and post mating (males) or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery (females) had no effects on general health, neurological parameters, body weights, food consumption, pre-coital time, gestation length, mating and fertility parameters. There were no treatment-related effects on the uterine/implantation data and mean litter size. There were no external abnormalities in live or dead pups in any of the groups. No treatment-related changes in the ano-genital distance and ano-genital ratio were observed at any of the doses. The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested.

Treatment did not induce any test item-related adverse changes with respect to terminal fasting body weights, organ weights/ratios (including reproductive organs), clinical pathology parameters and thyroid hormone profile. Grossly, yellowish gastro-intestinal contents noted in parental rats at 333 mg/kg and 1000 mg/kg was attributed to the physical appearance of the test item. Histopathology did not reveal any changes in adult animals and pups at all dose levels tested.

No Observed Adverse Effect Level
As there were no treatment related effects on systemic, reproduction and fertility parameters up to and including the highest dose tested 1000 mg/kg bwt/day, the No Observed Adverse Effect Level (NOAEL) for Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test by Oral Gavage in Wistar Rats for the test item is determined to be 1000 mg/kg bwt/day under the test conditions and doses employed.

Executive summary:

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

The test item was suspended in vehicle [0.5 % (w/v) of carboxymethyl cellulose sodium salt (medium viscosity) in Milli-Q^®water] and administered at the graduated dose levels of 111, 333 and 1000 mg/kg bwt/day for low dose (G2), mid dose (G3) and high dose (G4)/high dose recovery (G4R) group rats, respectively. The rats in the vehicle control (G1)/vehicle control recovery (G1R) groups received vehicle alone. The dose volume administered was 10 mL/kg body weight/day. Each main group in the experiment comprised of 10 male and 10 female rats and each recovery group comprised of 5 male and 5 female rats.

The dose formulations were administered once daily to a specific group of rats for two weeks prior to mating, during mating and post-mating periods (for males), during pregnancy and up to Lactation Day (LD) 13 for females.

The identity of the test item was provided by the Sponsor by a Certificate of Analysis (CoA). The authenticity of the test item was not determined at the test facility. The stability of the test item in the vehicle was established separately under Eurofins Advinus Study No. G19468 at 1 and 100 mg/mL. Based on the results, the test item was found to be stable and homogeneous in the vehicle for up to 48 hours when stored at room temperature.

During the conduct of this study, the prepared dose formulations were analysed for test item concentration prior to dosing on Day 1 and during Week 4 (Day 23) of the treatment period. The results indicated that the analysed concentrations were within ± 15 % variation from the claimed concentrations.

All rats were observed for clinical signs once daily. Body weight was recorded prior to the start of treatment on Day 1 and at weekly intervals thereafter. The body weights were also recorded at termination. Food consumption was recorded at weekly intervals except during the cohabitation period.

After confirmation of mating by vaginal smear, the dams were weighed on presumedGestation Days (GDs) 0, 7, 14 and 20 and the food consumption was recorded on GD 7, 14 and 20.

The littered dams were weighed on LDs 0, 4 and 13 and the food consumption was recorded on LD 4 and 13.

The number, survival and mortality of pups were observed during the lactation period. The body weight and ano-genital distance of each live pup was measured on LD 0. The size of each litter was adjusted by eliminating extra pups by random selection on LD 4 after recording the body weight of each live pup. After standardization, the individual pup body weight was recorded on LD 13. All the surviving male pups were examined for the appearance of nipples/areolae on LD 13.

Neurological examinations were conducted for randomly selected 5 main group females on LD 13 and randomly selected 5 main group males on treatment day 56 and towards the end of recovery period for the recovery group animals.

Laboratory investigations such as hematology, coagulation, clinical chemistry and urinalysis were performed on randomly selected 5 parental males and 5 parental females from each group at the end of the pre-mating period after overnight fasting. Thyroxine 4 (T4) and Thyroid Stimulating Hormone (TSH) analysis were performed in all main group males at termination, all dams on LD 13 and from available pups on LD 4 and 13.

At sacrifice, the parental males (Day 58), parental females (LD14) and the recovery animals (Day 65) were subjected to detailed necropsy after overnight fasting (water allowed) and the study plan specified tissues were collected. The pups were sacrificed on LD 13 after examining the external reproductive genitals for signs of altered development.

Tissues/organs collected from randomly selected 5 males and females in the control and high dose groups (including reproductive organs) were examined microscopically for histopathological changes. Histopathological examination of the testes also included a qualitative assessment of stages of spermatogenesis and interstitial testicular cell structure. All gross lesions were examined in all the groups.In the absence of test item related histopathological changes in any of the suspected tissues in the high dose group (G4), tissues from the lower dose groups were not evaluated. The available thyroid gland from a male and a female pup per litter (randomly selected) were also evaluated from all the groups.

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

Clinical signs and Mortality:There were no treatment related clinical signs or mortality observed at any of the doses tested. The yellowish faeces were observed in the test item administered rats from treatment day 2 to end of treatment. It was not observed in the high dose recovery group rats from day 2 of recovery period. This clearly indicates that the yellowish coloured faecal matter was due to physical nature of the test item.

There were no abnormalities observed in pups.

Functional Observation Battery:No treatment-related neurological abnormalities were observed at any of the doses tested.

 

Body weights:The mean body weights and body weight gainswere unaffected by the treatment at all the tested doses in both sexes.

 

Food consumption:Treatment did not affect the food consumption at any of the tested doses in either sex.

 

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

 

Fertility parameters:Treatment had no effect on the pre-coital interval, gestation length, oestrous cycle length. The mating and fertility parameters in both sexes were unaffected by the treatment.

 

Litter parameters:There were no treatment-related effects on the uterine/implantation data, mean litter size and mean viable litter size. There were no external abnormalities in live or dead pups in any of the groups. No treatment-related changes in the ano-genital distance, ano-genital ratio, pup body weights were observed at any of the doses tested when compared to the control. The male pups did not exhibit areola/nipple retention on LD 13 at any of the doses tested.

 

Haematology, Coagulation, Clinical chemistry and Urine Parameters:No test item-related changes were observed in the haematology, coagulation, clinical chemistry, and urine parameters at all the doses tested in both sexes.

 

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

 

Terminal fasting body weights, organ weights and its ratios:There were no test item-related changes in terminal fasting body weights, organ weight and their ratios in adult male and female rats of all groups compared to the control group.There were no significant intergroup differences observed in the terminal body weights and thyroid gland weights in male/female pups.

 

Gross and histopathology:

Grossly noted yellow contents in intestinal segments ≥333 mg/kg bwt/day were attributed to the test item colour.

There were no test item-related adverse histopathological changes observed either in parents or the offspring. The staging of spermatogenesis did not reveal any stage specific changes in testes and the spermatogenic cycles observed in the different seminiferous tubules were complete. The qualitative assessment of stages of spermatogenesis and evaluation of interstitial testicular structures did not reveal any test item associated findings in parental rats.

In view of the results observed:

As there were no treatment related effects on systemic, reproduction and fertility parameters up to and including the highest dose tested 1000 mg/kg bwt/day, the No Observed Adverse Effect Level (NOAEL) for Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test by Oral Gavage in Wistar ratsfor the test itemHostaperm-Gelb H6G (C. I. Pigment Yellow 175) is determined to be 1000 mg/kg bwt/dayunder the test conditions and doses employed.

Reference 4

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

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

Adequacy of study:

key study

Study period:

13 November 2020

Reliability:

1 (reliable without restriction)

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

GLP compliance:

yes

Justification for study design:

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Wistar Rats was to determine the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. Further this study also provides initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition. This study also provides information on reversibility, persistence or delayed occurrence of systemic toxic effects, for 14 days post treatment

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Source: Hylasco Biotechnology (India) Pvt. Ltd., 4B MN Park, Turkapally Village, Shameerpet Mandal, Medchal Dist, Telangana 500078
Justification for selection of species: Rat is the standard laboratory rodent species used for toxicity assessment and recommended by various regulatory authorities. The Wistar rat was selected due to the large amount of background data available for this strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Rats were housed in an environment controlled room. The temperature maintained during the experiment was between 19 to 24°C and relative humidity between 49 and 68 %. The photoperiod was a 12 hours light and 12 hours dark cycle. Adequate fresh air supply of 12.5 – 12.9 air changes/hour was maintained in the experimental room. The maximum and minimum temperature in the experimental room was recorded once daily. The relative humidity in the experimental room was calculated daily from dry and wet bulb temperature recordings.

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

The test item was administered by oral gavage in graduated doses to three groups of male and female rats. The males were dosed for 57 days, up to and including the day before scheduled sacrifice (this includes two weeks prior to mating, during mating period and approximately, two weeks post mating period).
Females were dosed throughout the treatment period. This includes two weeks prior to mating (with the objective of covering at least two complete oestrous cycles), the variable time to conception, the duration of pregnancy and up to and including the day before scheduled sacrifice (i.e., up to LD13).
Animals in the recovery groups were kept only for observations of reversibility, persistence or delayed occurrence of systemic toxic effects for 14 days of recovery period and these animals were not mated and consequently were not used for assessment of reproduction/developmental toxicity. The recovery period of the study started from the first scheduled kill of dams.

Details on mating procedure:

One female was placed with one male from the same group in a 1:1 ratio. Cohabitation was continued until there was evidence of sperms in the vaginal smear. All the females copulated successfully within seven days from the day of cohabitation. Subsequently, pregnant females were housed individually until LD 14. Not-littered females were sacrificed after 25 days from the day they were found sperm positive (by vaginal smear examination).
The day of confirmed mating was designated as GD 0. The pre-coital time (days) was calculated for each female.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For homogeneity and test item concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 and during Week 4 (Day 23) of treatment period and was analysed in-house. For each set, duplicate samples were drawn from the top, middle and bottom layers of each preparation and in case of the control duplicate samples from the middle layer were drawn.
The analysis was done as per the method validated under Eurofins Advinus Study No.: G19468. One set of samples (first set) were analysed for test item concentration analysis and other set (second set) of samples were stored at ambient condition for reanalysis purpose as a backup.
Both set I and set II analysis results of G3 and G4 groups prepared on Day 1(16 June 2020) were out of acceptance limits. Hence, the samples from subsequent preparations were analysed on 17 June 2020. The results of G3 group from the subsequent preparation (17 June 2020) was within the acceptance limits, however G4 group results were out of acceptance limits. Hence the backup samples (formulations prepared on 17 June) of G4 group samples were analysed on 18 June 2020.
Formulations were considered acceptable when the mean results (calculated using all the replicate values) of all the layers and mean of each layer was within ±15.0 % of the claimed concentration and the relative standard deviation (% RSD) was equal to or less than 10.0 %.

Duration of treatment / exposure:

Males: The dose formulation was administered orally by gavage to the rats of the specific groups once daily at approximately the same time each day (varying by ± 3 hours) for 57 days which includes 2 weeks prior to mating, during mating and post-mating, up to and including the day before scheduled sacrifice.
Females: The dose formulations were administered orally by gavage to the specific group of rats once daily at approximately the same time each day (varying by ± 3 hours) for total of 51-58 days which includes 2 weeks prior to the mating, during mating, pregnancy and up to LD 13.
The dose formulations were administered to the high dose recovery group of rats once daily at approximately the same time each day (varying by ± 3 hours).
The dose volume administered to each rat was at an equivolume of 10 mL/kg body weight throughout the study. The dose volume was adjusted based on the most recent body weight of individual rat.
Similarly, vehicle was administered to rats in the vehicle control and vehicle control recovery groups at an equivolume of 10 mL/kg bwt.
The vehicle and the test item were not administered for vehicle control recovery and high dose recovery groups, respectively for 14 days from the first scheduled kill of dams.

Frequency of treatment:

Daily

Details on study schedule:

Study initiation date: 27 May 2020
Experimental starting date: 28 May 2020
Acclimatization: Start: 28 May 2020, End: 01 June 2020
Pre-treatment period: Start : 02 June 2020, End: 15 June 2020
Treatment start: Start: 16 June 2020, End: 12 August 2020
Experiment completion Date: 31 August 2020
Submission of Draft report: 31 August 2020
Study completion: 13 November 2020

Dose / conc.:

111 mg/kg bw/day (nominal)

Dose / conc.:

333 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Main groups : 10 males and 10 females
Recovery groups : 5 males and 5 females

Control animals:

yes

Details on study design:

Group
No. Group Colour
of
cage card Dose
(mg/kg bwt/day) Concen-tration (mg/mL) Dose volume (mL/ kg bwt/day) No. of rats Sex Rat Numbers
From To
Main Groups
G1 Vehicle Control White 0 0 10 10 M Rx7481 Rx7490
10 F Rx7491 Rx7500
G2 Low dose Yellow 111 11.1 10 10 M Rx7501 Rx7510
10 F Rx7511 Rx7520
G3 Mid dose Green 333 33.3 10 10 M Rx7521 Rx7530
10 F Rx7531 Rx7540
G4 High dose Pink 1000 100 10 10 M Rx7541 Rx7550
10 F Rx7551 Rx7560
Recovery Groups
G1R Vehicle Control recovery White 0 0 10 5 M Rx7561 Rx7565
5 F Rx7566 Rx7570
G4R High dose recovery Pink 1000 100 10 5 M Rx7571 Rx7575
5 F Rx7576 Rx7580

Oestrous cyclicity (parental animals):

Vaginal smear was examined and the stage of oestrous cycle was recorded daily for two weeks before start of the treatment to select females with regular
4-5 days cyclicity for the study. The vaginal smear was also examined daily from the beginning of the treatment period until evidence of mating to determine the Day 0 of pregnancy/treatment-related effects on mating or pre-coital time. The time interval (in days) from the diestrus of an oestrous cycle to the next diestrus was considered as the oestrous cycle length of an animal.
Vaginal smears were also examined on the day of necropsy to determine the stage of the oestrous cycle.

Litter observations:

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

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

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

d. On LD 4, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four pups per sex per litter. Partial adjustment was done when the number of male or female pups prevents having four of each sex per litter. Pups were not eliminated when the litter size drop below the culling target (8 pups/litter). Blood samples were collected from the available surplus pups of either sex, pooled, and used for determination of serum Thyroxine (T4) and Thyroid stimulating hormone (TSH) levels.

e. After standardization, the individual pup body weight was measured on LD13.

f. The number of nipples/areolae in male pups was counted on LD 13.

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

h. The litters were observed daily to note the number of alive, dead and cannibalized pups.

i. In addition to daily clinical observations, all pups were observed for any abnormal behaviour.

j. Fertility index for dams, sires as well as the pup survival index until LD 4 was calculated.

Postmortem examinations (parental animals):

All adult animals and pups were subjected for detailed necropsy and findings were recorded. The adult animals sacrificed at term were fasted overnight (water allowed), weighed and exsanguinated under isoflurane anaesthesia. The dams were sacrificed on LD 14. All the surviving pups were necropsied on LD 13 and findings were recorded. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development. Dead pups were examined for defects and/or cause of death.
For apparently non-pregnant rats, the uteri was stained with 10% aqueous ammonium sulphide (Salewski staining method) to identify the peri-implantation loss of the embryos (by staining the implantation sites) thereby, for confirmation of pregnancy.
The number of implantation sites were recorded for all the dams.

Statistics:

Parameters such as body weight, body weight change, food consumption, oestrous cycle, gestation length (days), mean litter size, mean viable litter size, ano-genital distance, sex ratio, survival index and pup body weight, laboratory Investigations – haematology, coagulation, clinical chemistry, urinalysis & thyroid hormone profile, organ weights, organ weight ratios (organ to body weight and organ to brain weight), no. of implantations, post implantation loss (%) were evaluated using the Levene’s Test for homogeneity of variances and the Shapiro-Wilks Test for normality of distributions. When the data was found to be homogeneous and of normal distribution, the data was analysed by analysis of variance (ANOVA). When the data was found to be nonhomogeneous or of non-normal, the data was subjected for transformation and ANOVA was performed on transformed data. When ANOVA was found to be significant, pairwise comparisons of treated groups to the control group was made using a parametric test, Dunnett, to identify statistical differences.
Data like pre-coital interval, mating and fertility indices were analysed using Chi-square test. When Chi-square was found to be significant, pairwise comparisons of treated groups to the control group was made using a Fisher Exact test, to identify statistical difference in ProvantisTM built-in statistical tests.
Data captured outside of ProvantisTM: The statistical analysis of the experimental data was carried out using licensed copies of SYSTAT Statistical package Ver.12.0. All quantitative data was tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene’s test) within the group before performing a one-factor ANOVA modelling by treatment groups. Non-optimal (non-normal or heteroscedastic) data was transformed, before ANOVA was performed. Comparison of means between treatment groups and control group was done using Dunnett’s test when the overall treatment, ‘F’ test was found significant.

Reproductive indices:

11.1 Reproductive Performance Data of Parents
a. Male mating index (%)

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

b. Male fertility index (%)

Number of males siring a litter/impregnated a female
= ----------------------------------------------------------------- x 100
Number of males with evidence of mating

c. Female mating index (%)

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

d. Female fertility index (%)

Number of pregnant females
= ------------------------------------------- x 100
Number of females with evidence of mating

e. Mean number of implantations/group

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


f. Post implantation loss (%)

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

Offspring viability indices:

11.2 Litter Data
a. Mean litter size per group

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

b. Mean viable litter size

No. of viable pups
= -----------------------------------------
Total Number of littered animals

c. Live birth index (%)

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


d. Day 4 survival index (%)

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

e. Sex Ratio/ Percentage of male offspring (%)

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

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

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

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical signs and mortality observed throughout the treatment period in either sex at all the doses tested, except for an incidental observation of dehydration, piloerection and weakness in a female rat (Rx7557) of high dose group on days 42 and 43. This dam was sacrificed on Day 43 due to total litter loss. The yellowish colour faeces was observed at all the tested doses in both the sexes which recovered by Day 2 of recovery period. This yellowish coloured faecal matter is related to physical nature of the test item.
There were no abnormalities observed in pups

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The mean body weights and body weight gains were unaffected throughout treatment and recovery period in males and two weeks pre-mating period in females at all the tested doses when compared to vehicle control group.
The significantly lower weekly absolute weight change during Days 52-59 in recovery group females was considered incidental as the mean body weights were not altered by the treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The food consumption was not altered throughout treatment and recovery period when compared to the concurrent vehicle control.
Incidences of significantly decreased food consumption during Days 1-8 in 1000 mg/kg bwt/day recovery dose group males and during Days 8-14 in both 111 and 333 mg/kg bwt/day dosed females and significantly increased food consumption during Days 43-50 in 1000 mg/kg bwt/day recovery dose group males was observed. These isolated statistically significant differences observed in food consumption were toxicologically not significant as the mean body weights were not altered by the treatment.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item related changes in the hematology parameters including PT and APTT values in both males and females. A few variations in coagulation parameters that reached statistical significance were considered incidental and were likely due to random biological variation as the percent change was minimal and/or there was no dose correlation.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The clinical chemistry parameters were not affected by test item administration. Increased creatinine kinase activity in 1000 mg/kg bwt/day dose males was considered as incidental change as it was not associated with any microscopic changes in muscle tissues examined. Increased creatinine concentration in 1000 mg/kg bwt/day dose females was considered as incidental change as it was not associated microscopic changes in kidneys examined. A few other variations in clinical chemistry parameters that reached statistical significance were considered incidental and were likely due to random biological variation as the percent change was minimal and/or there was no dose correlation.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Motor Activity: No treatment-related abnormalities were observed in any of the doses tested in both sexes except for an incidence of lower distance travelled at interval 1 and lower total distance travelled in recovery males treated at 1000 mg/kg bwt/day.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related microscopic changes at any of the dose levels tested.
All other single or few incidences of microscopic findings observed were considered incidental and not related to test item as they were randomly distributed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Litter Date: Test item had no treatment-related effects on the mean litter size, mean viable litter size and number of dead pups at first observation. There were no external abnormalities in live or dead pups in any of the groups. No treatment-related changes were observed in the survival data of pups up to LD 4 at all the tested doses.
Body Weight of Pups: The mean body weight of male and female pups per litter and litter mean pup body weight were not affected by the treatment at all the doses.
Anogental Distance (AGD): No changes attributable to the test item were detected in the Ano-genital distance and Ano-genital ratio in either sex. An incidence of higher ano-genital distance in female pups at 111 mg/kg bwt/day was considered as not related to treatment as this difference was not dose related and was minimal (̴ 3%).
Uterine/Implantation Data: No test item-related changes were observed in the number of implantations and percentage of post implantation loss.
Areolae/Nipple Retention in Pups: The male pups did not exhibit areola/nipple retention on PND 13.
Oestrous Cycle Prior to Sacrifice: The vaginal smear was examined for all the animals prior to necropsy and following is the details of various stages observed;

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related effects on the mean pre-coital time, gestation length (average days to litter), number of pregnancies and number of dams littered. No treatment-related changes were observed in the mating and fertility indices of sires and dams at all the doses tested. The lower gestation length at high dose was considered not treatment related as the difference was minimal (̴ 3%).

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:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

urinalysis

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

reproductive performance

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

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

not examined

Sexual maturation:

no effects observed

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Gross pathological findings:

no effects observed

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

sexual maturation

clinical signs

mortality

body weight and weight gain

gross pathology

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

To summarize, oral (gavage) administration of the test item to Wistar rats at the dose levels of 111, 333 and 1000 mg/kg bwt/day for 2 weeks prior to mating, during mating, and post mating (males) or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery (females) had no effects on general health, neurological parameters, body weights, food consumption, pre-coital time, gestation length, mating and fertility parameters. There were no treatment-related effects on the uterine/implantation data and mean litter size. There were no external abnormalities in live or dead pups in any of the groups. No treatment-related changes in the ano-genital distance and ano-genital ratio were observed at any of the doses. The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested.

Treatment did not induce any test item-related adverse changes with respect to terminal fasting body weights, organ weights/ratios (including reproductive organs), clinical pathology parameters and thyroid hormone profile. Grossly, yellowish gastro-intestinal contents noted in parental rats at 333 mg/kg and 1000 mg/kg was attributed to the physical appearance of the test item. Histopathology did not reveal any changes in adult animals and pups at all dose levels tested.

No Observed Adverse Effect Level
As there were no treatment related effects on systemic, reproduction and fertility parameters up to and including the highest dose tested 1000 mg/kg bwt/day, the No Observed Adverse Effect Level (NOAEL) for Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test by Oral Gavage in Wistar Rats for the test item is determined to be 1000 mg/kg bwt/day under the test conditions and doses employed.

Executive summary:

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

The test item was suspended in vehicle [0.5 % (w/v) of carboxymethyl cellulose sodium salt (medium viscosity) in Milli-Q^®water] and administered at the graduated dose levels of 111, 333 and 1000 mg/kg bwt/day for low dose (G2), mid dose (G3) and high dose (G4)/high dose recovery (G4R) group rats, respectively. The rats in the vehicle control (G1)/vehicle control recovery (G1R) groups received vehicle alone. The dose volume administered was 10 mL/kg body weight/day. Each main group in the experiment comprised of 10 male and 10 female rats and each recovery group comprised of 5 male and 5 female rats.

The dose formulations were administered once daily to a specific group of rats for two weeks prior to mating, during mating and post-mating periods (for males), during pregnancy and up to Lactation Day (LD) 13 for females.

The identity of the test item was provided by the Sponsor by a Certificate of Analysis (CoA). The authenticity of the test item was not determined at the test facility. The stability of the test item in the vehicle was established separately under Eurofins Advinus Study No. G19468 at 1 and 100 mg/mL. Based on the results, the test item was found to be stable and homogeneous in the vehicle for up to 48 hours when stored at room temperature.

During the conduct of this study, the prepared dose formulations were analysed for test item concentration prior to dosing on Day 1 and during Week 4 (Day 23) of the treatment period. The results indicated that the analysed concentrations were within ± 15 % variation from the claimed concentrations.

All rats were observed for clinical signs once daily. Body weight was recorded prior to the start of treatment on Day 1 and at weekly intervals thereafter. The body weights were also recorded at termination. Food consumption was recorded at weekly intervals except during the cohabitation period.

After confirmation of mating by vaginal smear, the dams were weighed on presumedGestation Days (GDs) 0, 7, 14 and 20 and the food consumption was recorded on GD 7, 14 and 20.

The littered dams were weighed on LDs 0, 4 and 13 and the food consumption was recorded on LD 4 and 13.

The number, survival and mortality of pups were observed during the lactation period. The body weight and ano-genital distance of each live pup was measured on LD 0. The size of each litter was adjusted by eliminating extra pups by random selection on LD 4 after recording the body weight of each live pup. After standardization, the individual pup body weight was recorded on LD 13. All the surviving male pups were examined for the appearance of nipples/areolae on LD 13.

Neurological examinations were conducted for randomly selected 5 main group females on LD 13 and randomly selected 5 main group males on treatment day 56 and towards the end of recovery period for the recovery group animals.

Laboratory investigations such as hematology, coagulation, clinical chemistry and urinalysis were performed on randomly selected 5 parental males and 5 parental females from each group at the end of the pre-mating period after overnight fasting. Thyroxine 4 (T4) and Thyroid Stimulating Hormone (TSH) analysis were performed in all main group males at termination, all dams on LD 13 and from available pups on LD 4 and 13.

At sacrifice, the parental males (Day 58), parental females (LD14) and the recovery animals (Day 65) were subjected to detailed necropsy after overnight fasting (water allowed) and the study plan specified tissues were collected. The pups were sacrificed on LD 13 after examining the external reproductive genitals for signs of altered development.

Tissues/organs collected from randomly selected 5 males and females in the control and high dose groups (including reproductive organs) were examined microscopically for histopathological changes. Histopathological examination of the testes also included a qualitative assessment of stages of spermatogenesis and interstitial testicular cell structure. All gross lesions were examined in all the groups.In the absence of test item related histopathological changes in any of the suspected tissues in the high dose group (G4), tissues from the lower dose groups were not evaluated. The available thyroid gland from a male and a female pup per litter (randomly selected) were also evaluated from all the groups.

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

Clinical signs and Mortality:There were no treatment related clinical signs or mortality observed at any of the doses tested. The yellowish faeces were observed in the test item administered rats from treatment day 2 to end of treatment. It was not observed in the high dose recovery group rats from day 2 of recovery period. This clearly indicates that the yellowish coloured faecal matter was due to physical nature of the test item.

There were no abnormalities observed in pups.

Functional Observation Battery:No treatment-related neurological abnormalities were observed at any of the doses tested.

 

Body weights:The mean body weights and body weight gainswere unaffected by the treatment at all the tested doses in both sexes.

 

Food consumption:Treatment did not affect the food consumption at any of the tested doses in either sex.

 

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

 

Fertility parameters:Treatment had no effect on the pre-coital interval, gestation length, oestrous cycle length. The mating and fertility parameters in both sexes were unaffected by the treatment.

 

Litter parameters:There were no treatment-related effects on the uterine/implantation data, mean litter size and mean viable litter size. There were no external abnormalities in live or dead pups in any of the groups. No treatment-related changes in the ano-genital distance, ano-genital ratio, pup body weights were observed at any of the doses tested when compared to the control. The male pups did not exhibit areola/nipple retention on LD 13 at any of the doses tested.

 

Haematology, Coagulation, Clinical chemistry and Urine Parameters:No test item-related changes were observed in the haematology, coagulation, clinical chemistry, and urine parameters at all the doses tested in both sexes.

 

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

 

Terminal fasting body weights, organ weights and its ratios:There were no test item-related changes in terminal fasting body weights, organ weight and their ratios in adult male and female rats of all groups compared to the control group.There were no significant intergroup differences observed in the terminal body weights and thyroid gland weights in male/female pups.

 

Gross and histopathology:

Grossly noted yellow contents in intestinal segments ≥333 mg/kg bwt/day were attributed to the test item colour.

There were no test item-related adverse histopathological changes observed either in parents or the offspring. The staging of spermatogenesis did not reveal any stage specific changes in testes and the spermatogenic cycles observed in the different seminiferous tubules were complete. The qualitative assessment of stages of spermatogenesis and evaluation of interstitial testicular structures did not reveal any test item associated findings in parental rats.

In view of the results observed:

As there were no treatment related effects on systemic, reproduction and fertility parameters up to and including the highest dose tested 1000 mg/kg bwt/day, the No Observed Adverse Effect Level (NOAEL) for Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test by Oral Gavage in Wistar ratsfor the test itemHostaperm-Gelb H6G (C. I. Pigment Yellow 175) is determined to be 1000 mg/kg bwt/dayunder the test conditions and doses employed.

Reference 5

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 422, GLP-compliant)

Justification for type of information:

See read across justification document in chapter 13

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Tocixity 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, Inc., Maasheseweg 87c, 5800 AN Vernay / Netherlands
- Number of Animals: 44 males (11 per group) and 44 females (11 per group)
- Age (at Start of Treatment): 11 weeks
- Body Weight Range (at Start of Treatment): 301 to 362 g (males), 216 to 247 g (females)
- Identification: Parent animals had cage card and individual animal number (ear tattoo), pups were individually tattooed with Indian ink on day 1 post partum
- Randomization: Performed after at least three days of acclimatization using a computer-generated random algorithm. Body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
- Accommodation: In groups of five in Makrolon type-4 cages with wire mesh tops up to the day of randomization and afterwards 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) with paper enrichment (ISO-BLOX from Harlan Laboratories B.V., Netherlands), batch/lot nos. 02105111001, 02105111201, 02105120301 and 6960C.CS-100099). 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 2018C (batch no. 80/11) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum.
- Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles.
- Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

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)

IN-LIFE DATES From 26 January 2012 to 28 March 2012.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

DOSE FORMULATIONS
The dose formulations were prepared weekly using the test item as supplied by the Sponsor.
The test item was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using a magnetic stirrer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration.
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

STORAGE OF DOSE FORMULATIONS
Dose formulations were stored at room temperature (20 +/- 5 °C) in glass beakers.
Based upon the results of stability analyses performed within the non-GLP Harlan Laboratories study no. D33711 (Dose Range-Finding Study for a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test in the Han Wistar Rat), dose formulations were stable for at least 8 days if stored at room temperature.

TREATMENT
- Method: Oral, by gavage
- Rationale for Method: Administration by gavage is a common and accepted route of exposure for this type studies.
- Frequency of Administration: Once daily
- Target Dose Levels: 0 mg/kg/day (control group), 100 mg/kg/day (group 2), 300 mg/kg/day (group 3) and 1000 mg/kg/day (group 4)
- Rationale for Dose Level Selection: The dose levels were selected based on a previous non-GLP dose range-finding toxicity study in Han Wistar rats, Harlan Laboratories Study D33711, using dose levels of 0, 100, 300 and 1000 mg/kg/ day, where no adverse effects were observed up to and including the highest dose level.
- 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) and 100 mg/mL/day (group 4).
- Duration of Acclimatization Period: 7 days.

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.
For a female which 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 performed.
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
On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 0.5 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 0.5 g of each concentration were taken from the middle only to confirm stability (8 days). During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 +/- 5 °C) and delivered on dry ice to the responsible for formulation analysis (Harlan Laboratories Ltd., Itingen / Switzerland) and stored there at -20 +/- 5 °C until analysis.
The samples were analyzed by UV-VIS spectroscopy following an analytical procedure developed at Harlan Laboratories. The test item was used as the analytical standard.

RESULTS
Blank samples showed no significant absorbance and, therefore, it was confirmed that only highly purified water was applied within the control experiment.
The application formulations investigated during the study were found to comprise test material in the range of 93.1% to 105.6% and, thus, the required content limit of +/-20% with reference to the nominal content was met. The homogeneous distribution of test item in the preparations was approved because single results found did not deviate more than 5.5% (<15%) from the corresponding mean.
The test item was found to be stable in application formulations when kept eight days at 20 +/- 5 °C 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 highly purified water as vehicle during this study. Application formulations were found to be homogeneously prepared and stable over a storage period of eight days (20 +/- 5 °C).

Duration of treatment / exposure:

MALES: 40 days
FEMALES: approximately 7 weeks

Frequency of treatment:

once daily

Details on study schedule:

MALES

- Acclimatization: 7 days
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum
- Treatment Ends: On day before sacrifice
- Blood Sampling: After 28 days of Treatment
- Necropsy: After treatment for 39 days, when no longer needed for assessment of reproductive effects

FEMALES

- Acclimatization: 7 days
- 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 4 post partum
- Blood Sampling: Day 5 post partum
- Necropsy: On day 5 post partum (pups on day 4 post partum)

Remarks:

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

No. of animals per sex per dose:

11

Control animals:

yes, concurrent vehicle

Positive control:

Not required

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: on days 1 - 4, 4 - 8, 8 - 11 and 11 - 14 during pre-pairing period and weekly during after pairing period.
Females: on days 1 - 4, 4 - 8, 8 - 11 and 11 - 14 during pre-pairing period; on days 0 - 7, 7 14 and 14 - 21 during gestation period and on days 1 - 4 of during lactation period.
No food consumption was recorded during the pairing period.

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

DETAILED CLINICAL OBSERVATIONS
Detailed clinical observations were performed outside the home cage in all animals. In males, it was performed once prior to the first administration of the test item and weekly thereafter. In females, it was performed once prior to the first administration of the test item, weekly during the pre-pairing and pairing periods and on days 0, 6, 13 and 20 of the gestation period.
Animals were observed for the following: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior were also reported.

FUNCTIONAL OBSERVATIONAL BATTERY
At one time during the study (males shortly before the scheduled sacrifice and females on day 3 or 4 post partum) relevant parameters were performed with five P generation males and five P generation females from each group. This FOB assessment was conducted following the daily dose administration. Animals were observed for the following:
- Cage-side observations: faeces-balls, urine and posture as well as resistance to removal.
- Hand-held observations: muscle tone, constituation, skin, pupile size, palpebral closure, lacrimation, salivation, reaction to handling and general abnormalities.
- Open field observations: level of ambulatory activity including rearing (one minute evaluation), unusual body movements (e.g. spasms, convulsions), gait evaluation, behavior, hair coat, respiration, quantity of faeces-balls and urine.
- Reflexes: blinking, palpebral closure, pinna reflex, extensor thrust response, paw pinch, responsiveness to sharp noise, righting reflex and hearing ability (Preyer’s reflex).
- Measurements / Counts: hind limb / fore limb grip strength, landing foot splay, rectal temperature.

Any abnormal findings were recorded and, where appropriate, graded in severity.

Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with an Activity Monitor AMS-0151 (FMI, Germany). Activity of the animals (based on beam count) was recorded for 6-minute intervals over a period of 30 minutes. These data and the total activity over 30 minutes were reported.

CLINICAL LABORATORY INVESTIGATIONS
Blood samples were obtained on the day of the scheduled necropsy from 5 males from each group. Blood samples from 5 lactating females from each group were obtained on day 5 post partum. Blood samples were drawn sublingually from all animals under light isoflurane anesthesia. The animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.

The following hematology parameters were determined:
- Erythrocyte count
- Hemoglobin
- Hematocrit
- Mean corpuscular volume
- Red cell volume distribution width
- Mean corpuscular hemoglobin
- Mean corpuscular hemoglobin concentration
- Hemoglobin concentration distribution width
- Leukocyte count, total
- Differential leukocyte count
- Platelet count
- Prothrombin time (= Thromboplastin time)
- Activated partial Thromboplastin time

The following clinical biochemistry parameters were determined:
- Glucose
- Urea
- Creatinine
- Bilirubin, total
- Cholesterol, total
- Triglycerides
- Aspartate aminotransferase
- Alanine aminotransferase
- Alkaline phosphatase
- Gamma-glutamyl-transferase
- Bile acids
- Sodium
- Potassium
- Chloride
- Calcium
- Phosphorus
- Protein, total
- Albumin
- Globulin
- Albumin/Globulin ratio

URINALYSIS
The following urinalysis parameters were determined in five males of each group, which are allocated to the blood analysis, during the last week of the study using timed urine volume collection:
- Volume (18 hours)
- Specific gravity (relative density)
- Color
- Appearance
- pH
- Nitrite
- Osmolality
- Protein
- Glucose
- Ketones
- Urobilinogen
- Bilirubin
- Blood/Blood cells

Oestrous cyclicity (parental animals):

Not examined

Sperm parameters (parental animals):

Sperm analysis was performed on the first 5 males per group.

MOTILITY
At necropsy of adult males an epididymal sperm sample was obtained from the left cauda epididymidis of each male. The sample was diluted with a pre-warmed (about 35 °C) physiological medium, and shortly after being obtained, one hundred sperm were counted microscopically for determination of percentage of not motile, stationary motile and progressively motile sperm.

MORPHOLOGY
A second sperm sample from the left cauda epididymidis was used for morphological assessment after fixation and Eosin staining. 500 sperm per sample were evaluated microscopically and classified into the following categories:
A: Normal, complete sperm
B: Normal head only (tail detached)
C: Complete sperm, misshapen hook
D: Complete sperm, abnormally curved hook
E: Complete sperm, reversed head
F: Abnormal head only (tail detached)

Morphological sperm evaluation was performed only for group 1 and 4 males. In the absence of a treatment-related effect the slides for the group 2 and 3 males were not evaluated.

SPERM, SPERMATID COUNT
The left caudal epididymis and left testis were taken for determination of homogenization-resistant spermatids and caudal epididymal sperm reserve. These tissues were frozen at -20 +/- 5 °C pending evaluation. For evaluation the weighed tissues were placed in Triton-X-100 solution and homogenized with a blender (Ultra Turrax) and an ultrasonic water bath. Sperm or spermatid heads were counted microscopically using a modified Neubauer chamber. These evaluations were performed in the first instance only for group 1 and 4 males. In the absence of a treatment-related effect the remaining frozen tissues were not evaluated.

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 treatment for 39 days, when no longer needed for the assessment of reproductive effects. Dams were sacrificed on day 5 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 were subjected to a detailed macroscopic examination. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution. At the scheduled sacrifice, all animals were sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated. All parent animals were examined macroscopically for any structural changes. 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, testes and epididymides from all parental males were weighed separately. In addition, from 5 males and 5 females sacrificed at the end of the study which were selected from each group, the following organs were trimmed from any adherent tissue, as appropriate, and their wet weight taken.
- Adrenal glands (weighed as pairs)
- Brain
- Heart
- Kidneys (weighed as pairs)
- Liver
- Thymus
- Spleen

TISSUE PRESERVATION
The following tissues from all parental males were preserved in neutral phosphate buffered 4% formaldehyde solution:
- Prostate
- Seminal vesicles with coagulating gland
- Testes (in Bouin’s fixative)*
- Epididymides (in Bouin’s fixative)*
*From the first five males in each group which were used for sperm analysis, only the right testis and right epididymis were preserved for histopathological examination.

The following tissues from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution:
- Ovaries
In addition, from 5 males and 5 females per group selected for organ weights, the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution:
- Gross lesions
- Brain (representative regions including cerebrum, cerebellum and pons)
- Spinal chord
- Small and large intestines (incl. Peyer’s patches)
- Stomach
- Liver
- Kidneys
- Adrenals
- Spleen
- Heart
- Thymus
- Thyroids, and parathyroids if possible
- Trachea and lungs (preserved by inflation with fixative and then immersion)
- Uterus (with vagina)
- Urinary bladder
- Lymph nodes (mesenterial, mandibular)
- Peripheral nerve (sciatic)
- Bone marrow

HISTOTECHNIQUE
All organ and tissue samples to be examined by the study pathologist 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 study pathologist.

HISTOPATHOLOGY
Slides of all organs and tissues listed collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the study pathologist. The same applied to all occurring gross lesions.
Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.
Histological examination of ovaries was carried out on any females that did not give birth. In addition, microscopic examination of the reproductive organs of all infertile males was made, where necessary.
A histopathology peer review was performed. A histopathology phase report was provided by the principal investigator which is included in the report.

Postmortem examinations (offspring):

Pups were sacrificed on day 4 post partum. All animals were sacrificedby by an injection of sodium pentobarbital and subjected to a detailed macroscopic examination. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution. Pups found dead during the study, 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 when the variables could be dichotomized without loss of information.

Reproductive indices:

From the on-line recorded reproduction data, the following parameters were calculated: mean precoital time, percentage mating, fertility index, conception rate, post-implantation loss, gestation index, birth index and viability index.

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 and viability index.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Red stained faeces was noted in all males and females in dose groups; this was due to the staining properties of the test item.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In males at the dose level of 1000 mg/kg bw/day, lower body weight gain during the pre-pairing period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In males at the dose level of 1000 mg/kg bw/day, lower body weight gain during the pre-pairing period.

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:

effects observed, treatment-related

Description (incidence and severity):

Sperm motility was reduced in all dose groups.

Reproductive performance:

no effects observed

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

DAILY CLINICAL SIGNS OR OBSERVATIONS
Red stained feces was noted in all males and females in all dose groups starting from day 2 of the treatment until completion of the study with dose-related intensity of discoloration. This observation was due to staining properties of the test item.
No further test item-related clinical signs or observations were noted in males or females at any dose level.
Incidentally, in one male (no. 16) at the dose level of 100 mg/kg bw/day chromodacryorrhea was noted during the study (starting on day 1 of the pre-pairing period) and eye reduced in size was noted in the same animal from day 13 of the pre-pairing period.
No further test item-related findings were noted at any dose level.

FINDINGS AT DETAILED WEEKLY CLINICAL OBSERVATIONS
No test item-related findings were noted during detailed weekly clinical observations.
The only findings noted were chromodacryorrhea and eye reduced in size in male no. 16 at the dose level of 100 mg/kg bw/day recorded already during the daily clinical observations.

FUNCTIONAL OBSERVATIONAL BATTERY
No test item-related findings were noted during the functional observational battery tests in males or females at any dose level.
Statistically significantly lower body temperature was noted in both sexes. In males mean body temperature was 37.9 °C and 37.8 °C at the high- and mid-dose levels, respectively, compared to 38.4 °C in the control group. In females, 38.5 °C was noted at the high-dose level, compared to 38.9 °C in the control group. The differences noted in males and females were only minor, not clearly dose dependent and all values remained in the historical control range. For these reasons, changes in body temperature were considered not to be test item-related.
No further findings were noted during functional observational battery in males or females at any dose level except for the eye findings in male no. 16 at 100 mg/kg bw/day.

LOCOMOTOR ACTIVITY
No effects on locomotor activity were noted in males or females at any dose level.
Mean beam counts during the 30 minutes of measurement at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were respectively: 1255, 1137, 1219 and 1209 in males and 924, 882, 1002 and 1050 in females.

FOOD CONSUMPTION OF MALES
No effects on food consumption were noted in males at any dose level.
Mean differences in food consumption at the dose levels 100, 300 and 1000 mg/kg bw/day were respectively: +1.1%, -3.4% and -3.4% during the pre-pairing period and -1.5%, -3.1% and -3.8% during the after pairing period ( percentages refer to the respective values in the control group).

FOOD CONSUMPTION OF FEMALES
No test item-related effects on food consumption were noted in females at any dose level.
Incidentally, statistically significantly higher food consumption was noted at the dose level of 100 mg/kg bw/day during lactation period. In the absence of an effect in females at the dose levels of 300 and 1000 mg/kg bw/day, this difference was considered not to be related to the treatment.
Mean differences in food consumption at the dose levels 100, 300 and 1000 mg/kg bw/day were respectively: +5.9%, +2.7%, and +1.1% during the pre-pairing period, +7.0%, +2.5% and +2.9% during the gestation period and +23.2%, -5.9% and +11.0% during the lactation period (percentages refer to the respective values in the control group).

BODY WEIGHTS OF MALES
At the dose level of 1000 mg/kg bw/day, a slightly lower body weight gain if compared to the controls was noted during the pre-pairing period. Mean body weight gain within this period was +10%, compared to +13% in the control group. The difference in body weight gain was statistically significant during the most days starting from day 3 until the end of the pre-pairing period. This effect was considered to be test item-related. During the pairing and after pairing periods, body weight gain was similar at all dose levels.
No significant changes in body weights were noted in males at any time during the study.
Because the lower body weight gain at the high-dose level was reversible despite treatment continued and did not result in any significant changes in body weights, this finding was considered not to be adverse.
No significant changes in body weight gain or body weights were noted in males at the dose levels of 100 and 300 mg/kg bw/day.
Mean differences in body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were respectively: +13%, +13%, +11% and +10% during the pre-pairing period, +10%, +10%, +9% and +9% during the pairing period and +7%, +6%, +7% and +6% during the after pairing period (percentages refer to the body weight change within the respective period).

BODY WEIGHTS OF FEMALES
Body weights and body weight gain of females were not affected by the treatment with the test item at any dose level.
On individual days some statistically significantly changed values of body weight gain were noted at the low-, mid- and high-dose levels. The changes did not follow a dose dependency and were therefore not related to the treatment.
Mean differences in body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were respectively: 5%, 7%, 6% and 7% during the pre-pairing period, 48%, 56%, 47% and 54% during the gestation period and 3%, 8%, 4% and 5% during the lactation period (percentages refer to the body weight change within the respective period).

2. CLINICAL LABORATORY INVESTIGATIONS
HEMATOLOGY
No test item-related effects on hematology parameters were noted in males or females at any dose level.
In males, statistically significant changes of several parameters: higher distribution width of red cell volume (RDW) at the low-dose level and higher distribution width of hemoglobin concentration (HDW) at the low- and mid-dose levels occurred in the absence of an effect at the high dose and therefore were considered not to be test item-related.
In females, at the low-dose level, statistically significantly higher platelets count was noted in the absence of any increase of this value at the mid- and high-dose levels and therefore it was not test item-related.
No further changes of hematology parameters were noted in males or females at any dose level.

CLINICAL BIOCHEMISTRY
No test item-related effects on biochemistry parameters were noted in males or females at any dose level.
In males, at the mid-dose level, statistically significantly lower concentration of triglycerides was noted. In the absence of dose dependency, this finding was not test item-related.
In females at the low dose level, following statistically significant changes were noted: higher concentration of cholesterol, higher concentration of globulin, and lower globulin to albumin ratio. These changes were not dose-dependent and therefore they were considered not to be test item-related.
No further changes of biochemistry parameters were noted in either males or females at any dose level.

URINALYSIS
No changes in urine parameters were noted in males at any dose level.

3. TERMINAL FINDINGS
SEMINOLOGY AND SPERMATID COUNT
In all dose groups, statistically significant changes in motility of sperms were noted. Following values were assessed in sperm samples at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day respectively: 81.1%, 64.6%, 57.3% and 47.5% of progressive sperms (changes were statistically significant in all dose groups), 3.7%, 11.3%, 6.0% and 11.9% of stationary sperms (changes were statistically significant at the dose levels of 1000 and 100 mg/kg bw/day) and 15.2%, 24.1, 36.7 and 40.6% of not motile sperms (changes were statistically significant at the dose levels of 1000 and 300 mg/kg bw/day). These changes might be test item-related. However no significant dose dependent trend indicated by probability values of <0.05 was determined for any of these changes when performing a linear regression analysis (least squares).
No further changes were noted during sperm analysis. At the high-dose level, all morphological categories of sperms were represented with similar frequency to that in the control group whereas sperm count was similar to the respective control values in samples from both testis and epididymidis.

ORGAN WEIGHTS
No changes in absolute organ weights or organ weights to body weights and to brain weights ratios were noted in males or females at any dose level.

MACROSCOPICAL FINDINGS
Type and distribution of findings noted during macroscopical examination of males or females did not indicate any test item-related effect.

HISTOPATHOLOGY FINDINGS
Under the conditions of this experiment, treatment with the test item did not cause pathological findings. All findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.

4. REPRODUCTION AND BREEDING
MATING PERFORMANCE AND FERTILITY
Mating performance and fertility were not affected by the treatment at any dose level.
All females in groups 2, 3 and 4 mated within the first pairing period. In group 1, one female (no. 54) was mated during the second pairing period.
Mean (median) precoital times were 4.5 (3), 2.5 (3), 4.0 (2) and 2.6 (3) days at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.
Seven females were not pregnant: three in the control group and in the mid-dose level and one in the low-dose level. Consequently, fertility indexes (number of females pregnant as percentages of females paired) and conception rate (number of females pregnant as percentages of females mated) were 72.7%, 90.9%, 72.7% and 100.0% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.
One female at the high dose level had one implantation site but delivered no pups. Consequently, gestation index (number of females with living pups as percentages of females pregnant) was 100% in the control group and at low- and mid-dose levels and 90.9% at the high-dose level.

CORPORA LUTEA COUNT
No test item-related effects on corpora lutea count were observed at any dose level.
Mean number of corpora lutea per dam was 16.0, 17.2, 16.3 and 18.4 in order of ascending dose levels.

DURATION OF GESTATION
No effects on duration of gestation were observed at any dose level.
Mean duration of gestation was 21.6, 21.6, 21.5 and 21.7 days, in order of ascending dose level.

IMPLANTATION RATE AND POST-IMPLANTATION LOSS
No effects on implantation rate and post-implantation loss were observed at any dose level.
In order of ascending dose levels, mean number of implantations per dam was 12.6, 14.9, 12.6 and 14.0 whereas mean incidence of post-implantation loss per dam was 1.5, 0.8, 0.6 and 0.5 per dam.

LITTER SIZE AT FIRST LITTER CHECK
No effects on litter size were noted at any dose level.
During the first litter check, one dead pup was found in a litter at the dose level of 1000 mg/kg bw/day. Because of isolated occurrence, this finding was considered to be incidental.
Mean number of living pups per dam at first litter check was 11.1, 14.3, 12.0 and 13.5 in order of ascending dose levels.
Birth index (number of pups born alive as a percentage of implantations) was 88.1%, 94.8%, 95.0% and 96.4% at the dose level of 0, 100, 300 and 1000 mg/kg bw/day.
Birth index at the dose level of 1000 mg kg bw/day was statistically significantly higher than the respective control value. This was considered to be a result of biological variability.

Dose descriptor:

NOAEL

Remarks:

for general toxicity

Effect level:

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

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 (actual dose received)

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
No test item-related effects on postnatal loss were noted at any dose level.
In the control group one pup was missing on day 4, at the low-dose level one pup was missing on day 2, at the mid dose level three pups (from two litters) were missing on day 2 and at the high dose level no postnatal loss was noted in any litter.
Mean postnatal loss per dam during four days of lactation was 0.1%, 0.1%, 0.4% and 0.0% at the dose level of 0, 100, 300 and 1000 mg/kg bw/day, respectively. Consequently, viability index (number of pups alive at termination on day 4 p.p. as a percentage of pups born alive) was 98.9%, 99.3%, 96.9% and 100% in order of ascending dose levels.

EXTERNAL EXAMINATION AT FIRST LITTER CHECK AND DURING LACTATION
No test item-related findings were noted in pups during first litter check and during lactation at any dose level.

Incidentally, one pup in the control group was found with a wound and missing tail tip, two further pups, each one at the low- and mid-dose levels, had a wound at first litter check. These findings were also noted during the remaining lactation period.

SEX RATIOS
Pups sex ratio was not affected by exposure to the test item at any dose level.
At first litter check, percentages of male pups were 56%, 48%, 51% and 56% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day.

BODY WEIGHTS TO DAY 4 POST PARTUM
Body weights and body weight gain of pups were not affected by the treatment with the test item at any dose level.
Mean body weights of pups on day 1 post partum were: 6.4 g, 6.1 g, 6.4 and 6.2 g and mean differences in body weights during lactation were +49.9%, +43.6%, +47.8% and +42.6%, at the dose levels of 0, 100, 300 and 1000 mg/kg/day, respectively.
At the dose levels of 100 and 1000 mg/kg bw/day, slightly not statistically significantly lower body weight gain of pups was noted. This effect was considered to be due to a higher number of pups at these dose levels which was supported by observation that reduction of body weight gain was more pronounced in litters of higher size. Therefore this effect was considered not to be test item-related.

MACROSCOPICAL FINDINGS
No test item-related findings were noted at macroscopic examination of pups at any dose level.
Incidentally, in the control group one pup had a sore in the thoraco-dorsal region, one further pup in this group had a missing tail tip. These findings were already recorded during the in life phase. At the high-dose level, one pup had a watery cyst in the left kidney.
No further findings were noted in pups at any dose level.

Dose descriptor:

NOAEL

Remarks:

for development

Generation:

F1

Effect level:

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

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 (100)
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 non pregnant females (A)	3	1	3	0
Numbers of pregnant females, which did not deliver any pups (B)	0	0	0	1
Number of females which reared their pups until day 4 post partum	8	10	8	10

(A) Female Nos. 45, 46, 55, 62, 74, 75 and 77

(B) Female No. 85 had implantations only

 

Conclusions:

Repeated oral-gavage administration of the test item to rats (according to OECD 422, GLP compliant) did not result in any adverse deveolpmental effects in rats. The test item was administered in vehicle (highly purified water) at dosages of 100, 300, and 1000 mg/kg body weight/day, animals in control groups received the vehicle only. Test item was administered to male rats for 28 days 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. The NOAEL was 1000 mg/kg bw.
Under the conditions of this study, no adverse effects were found in males or females up to the highest dose level of 1000 mg/kg bw/day.

All animals survived the scheduled study period.
During the treatment, faeces stained red with dose-dependent intensity of discoloration were noted in all males and females receiving test material. This observation was due to staining properties of the test item.

No effects on food consumption were noted in males at any dose level. Body weight gain was slightly but statistically significantly reduced in males at the dose level of 1000 mg/kg bw/day during the pre-pairing period. No differences in body weight gain were noted at any dose level during the remaining study period. Body weights of males in all dose groups were similar to the respective control values during the entire study period. Because lower body weight gain at the high-dose levels was reversible and did not cause significant changes in body weights, this effect was considered not to be adverse.

Food consumption, body weights and body weight gain of females were not affected by the treatment at any dose level.

No further test item-related observations were noted in males or females at any dose level during the live part of the study.

Terminal examinations revealed changes in motility of sperms in all dose groups. Statistically significant decrease in mean count of progressive sperms was noted at the dose levels of 100, 300 and 1000 mg/kg bw/day, statistically significant increase in mean count of stationary sperms was noted at the dose levels of 100 and 1000 mg/kg bw/day and statistically significant increase in mean count in not motile sperms was noted at the dose levels of 300 and 1000 mg/kg bw/day. However a significant dose dependent trend indicated by probability values of <0.05 was not established for any of these changes when performing a linear regression analysis (least squares).
No further effects on male reproductive system were noted during the study. Sperm morphology and sperm count at the high-dose level was similar to the control values. Weights of male reproductive organs, macroscopical and histopathological examination of testes and epididymides gave no indication of any treatment-related effect. Further, no indication of effects on reproduction was noted within this study up to and including the highest dose level. For this reason, changes in motility of sperms were considered not to be adverse in this study.


Reproduction and development were not affected by the treatment. Mating performance, fertility, duration of gestation, corpora lutea count, implantation rate, post implantation and postnatal loss or litter size were similar in the control and all dose groups. There were no test item-related findings in pups noted during the first litter check, the first 4 days post partum or during the necropsy, pups body weights and body weight gain were not affected by the treatment at any dose level.

Based on these results, the NOAEL (No Observed Adverse Effect Level) for general toxicity in males and females and for reproduction/developmental toxicity in this study was considered to be 1000 mg/kg bw/day, the highest dose level used.

Executive summary:

The purpose of this study was to generatepreliminaryinformation concerning the effects of the test material on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. In addition it provides information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

 

The test item was administered to male rats for 39 days 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.

 

The following dose levels were applied:

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 (highly purified water).

 

The following results were obtained:

 MORTALITY AND GENERAL TOLERABILITY OF PARENTAL ANIMALS

  All animals survived the scheduled study period.

Feces stained red with dose-dependent intensity of discoloration were noted in all males and females in all dose groups starting from day 2 of the treatment until completion of the study. This observation was due to staining properties of the test item.

  No further test item-related clinical signs or observations were noted in males or females at any dose level.

 

FUNCTIONAL OBSERVATIONAL BATTERY IN PARENTAL ANIMALS

  No test item-related findings were noted during the functional observational battery tests in males or females at any dose level.

 

FOOD CONSUMPTION OF PARENTAL ANIMALS

  No effects on food consumption were noted in males or females at any dose level.

 

BODY WEIGHTS OF PARENTAL ANIMALS

  In males at the dose level of 1000 mg/kg bw/day, a slight but statistically significant lower body weight gain was noted during the pre-pairing period. No differences in body weight gain were noted at any dose level during the remaining study period. Body weights of males in all dose groups were similar to the respective control values during the entire study period. Because reduction in body weight gain at the high-dose levels was reversible and did not cause significant changes in body weights, this effect was considered not to be adverse.

  Body weights and body weight gain of females were not affected by the treatment at any dose level.

 

CLINICAL LABORATORY INVESTIGATIONS IN PARENTAL ANIMALS

  No test item-related effects on hematology and clinical biochemistry parameters were noted in males or females at any dose level.

  No changes in urine parameters were noted in males at any dose level.

 

REPRODUCTION AND BREEDING DATA

  Mating performance, fertility, corpora lutea count, duration of gestation, implantation rate and post-implantation loss, litter size or postnatal loss were not affected by the treatment with the test item.

 

SEMINOLOGY AND SPERMATID COUNT IN PARENTAL MALES

Effects on sperm motility which might be test item-related were noted in all dose groups. Mean count of progressive sperms was statistically significantly reduced at the dose levels of 1000, 300 and 100 mg/kg bw/day, mean count of stationary sperms was statistically significantly increased at the dose levels of 1000 and 100 mg/kg bw/day and mean count of not motile sperms was statistically significantly increased at the dose level of 1000 and 300 mg/kg bw/day. But a significant dose dependent trend couldn’t be established.

In the absence of any findings during necropsy or histopathological examination of male reproductive organs as well as in the absence of any effects on reproduction, the differences in sperm motility were considered not to be adverse.

 

ORGAN WEIGHTS OF PARENTAL ANIMALS

  No changes in absolute organ weights or organ weights to body weights and to brain weights ratios were noted in males or females at any dose level.

 

MACROSCOPICAL FINDINGS AND HISTOPATHOLOGICAL EXAMINATIONS OF PARENTAL ANIMALS

  Type and distribution of findings noted during macroscopical examination did not indicate any test item-related effect.

  Treatment with test item did not cause pathological findings. All findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.

 

FINDINGS IN PUPS AT FIRST LITTER CHECK AND DURING LACTATION

  No test item-related findings were noted in pups during first litter check and during lactation at any dose level.

  Pups sex ratio was not affected by the exposure to the test item at any dose level.

 

PUP WEIGHTS TO DAY 4 POST PARTUM

  Body Weights and body weight gain of pups were not affected by the treatment with the test item at any dose level.

 

MACROSCOPICAL FINDINGS OF PUPS

  No test item-related findings were noted at macroscopic examination of pups at any dose level.

 

CONCLUSION

  Based on these results, the NOAEL (No Observed Adverse Effect Level) for general toxicity in males and females and for reproduction/developmental toxicity in this study was considered to be 1000 mg/kg bw/day, the highest dose level used.

Reference 6

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline 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

Details on species / strain selection:

Rat is the standard laboratory rodent species used for toxicity assessment and recommended by various regulatory authorities.
The Wistar rat was selected due to the large amount of background data available for this strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Hylasco Biotechnology Pvt. Ltd., Plot 4B, AKP, Turkapally Village, Shameerpet Mandal, RR Dist, Telangana 500078
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation: 13 weeks
Body weight range at the start of treatment: Males: 362.07 to 464.96 g & Females: 239.96 to 287.13C g
At the commencement of the treatment, the weight variation of rats used did not exceed ± 20 % of the mean body weight in each sex and group.
Conditions: Rats were housed in an environment controlled room. The temperature maintained during the experiment was between 21 to 24°C and relative humidity was between 49 to 68%. The photoperiod was 12 hours light and 12 hours dark cycle. Adequate fresh air supply of 12-15 air changes/hour
was maintained in the experimental room. The maximum and minimum temperature in the experimental room was recorded once daily. The relative humidity in the experimental room was calculated daily from dry and wet bulb temperature recordings.
Housing: Two rats of same sex were housed per cage in sterilized standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles with stainless steel sipper tubes. The last animal in recovery group of each sex was housed individually. Polycarbonate rat huts were provided to the animals as environmental enrichment objects and changed along with cage at least once a week. During the experimental period, animals were housed in a single experimental room of barrier area.
Bedding: Steam sterilized corn cob was used as bedding and changed along with the cage atleast twice a week.
Diet: Altromin Rat/Mice Maintenance diets manufactured by Altromin Spezialfutter GmbH & Co. KG, Im Seelenkamp 20, 32791 Lage, Germany, was provided ad libitum.
Water: Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd., Mumbai 400001,India, was provided ad libitum to rats in polycarbonate bottles with stainless steel sipper tubes.

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

Males: The dose formulation was administered orally by gavage to the rats of the specific groups once daily for 36 days at approximately the same time each day (varying by ± 3 hours) which includes 2 weeks prior to mating, during mating and post-mating, up to and including the day before scheduled sacrifice.
Females: The dose formulations were administered orally by gavage to the specific group of rats once daily at approximately the same time each day (varying by ± 3 hours). This includes 2 weeks prior to the mating and continued through mating, pregnancy and up to LD 13 (total 46-57 days), after which, pups were sacrificed on LD 13 and parental females (dams) were sacrificed on LD 14 after overnight fasting (water allowed).
The animals in the vehicle control group were handled in an identical manner to the treatment groups and were administered vehicle only.
The dose volume administered to each rat was 10 mL/kg body weight throughout the study. The dose volume was adjusted based on the most recent body weight of the individual rat.

Details on mating procedure:

One female was placed with one male from the same group in a 1:1 ratio. Cohabitation was continued until there was evidence of sperms in the vaginal smear and /or vaginal plug. All the females were successfully copulated within six days from the day of cohabitation. Subsequently, pregnant females were housed individually until LD 14. All the mated females were maintained till they littered. Not-littered females were sacrificed after 25 days from the day they were found sperm positive (by vaginal smear examination).
The day of confirmed mating was designated as Gestation Day ‘0’ (GD ‘0’). The pre-coital time (days) was calculated for each female.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For homogeneity and active ingredient (a.i.) concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 of the treatment period and during the 2nd month (day 37) of treatment and was analysed in-house. For each set, duplicate samples were drawn from the top, middle and bottom layers of each preparation and in case of the control duplicate samples from the middle layer were drawn.
The analysis was done as per the method validated under Eurofins Advinus Study No.: G19479. One set of samples were analysed for concentration (a.i) analysis and other set (second set) of samples were stored at ambient condition for reanalysis purpose as a backup. These second set of samples were disposed, as the analysis results of first set of samples were within the accepted limit as per study plan.
Formulations were considered acceptable as the mean results (calculated using all the replicate values) of all the layers and mean of each layer were within ±15.0 % of the claimed concentration and the relative standard deviation (% RSD) was equal to or less than 10.0 %.

Duration of treatment / exposure:

Males: The dose formulation was administered orally by gavage to the rats of the specific groups once daily for 36 days at approximately the same time each day (varying by ± 3 hours) which includes 2 weeks prior to mating, during mating and post-mating, up to and including the day before scheduled sacrifice.
Females: The dose formulations were administered orally by gavage to the specific group of rats once daily at approximately the same time each day (varying by ± 3 hours). This includes 2 weeks prior to the mating and continued through mating, pregnancy and up to LD 13 (total 46-57 days), after which, pups were sacrificed on LD 13 and parental females (dams) were sacrificed on LD 14 after overnight fasting (water allowed).
The animals in the vehicle control group were handled in an identical manner to the treatment groups and were administered vehicle only.
The dose volume administered to each rat was 10 mL/kg body weight throughout the study. The dose volume was adjusted based on the most recent body weight of the individual rat.

Frequency of treatment:

Daily

Details on study schedule:

Experimental starting date: 27 May 2020
Acclimatization: Start: 27 May 2020 & End: 31 May 2020
Pre-treatment period : Start: 01 June 2020 & End: 14 June 2020
Treatment start: Start: 15 June 2020 & End: 10 August 2020

Dose / conc.:

111 mg/kg bw/day (nominal)

Dose / conc.:

333 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes

Details on study design:

The test item was administered by oral gavage in graded doses to three groups of male and female rats. The males were dosed for six weeks, up to and including the day before scheduled sacrifice. This included a two-week period prior to mating, during the mating period and post mating.
Females were dosed throughout the treatment period. This included two weeks prior to mating (with the objective of covering at least two complete oestrous cycles), the variable time to conception, the duration of pregnancy and 13 days after delivery, up to and including the day before scheduled sacrifice.

Parental animals: Observations and examinations:

Parental animals: Observations and examinations
Clinical Signs, Morbidity and Mortality: All rats were observed for morbidity and mortalities twice daily i.e., once in the morning and once in the afternoon. As there were no clinical signs of toxicity, the observation for morbidity and mortality was carried out once in the morning during weekend and holidays. All rats were observed for clinical signs once daily during the treatment period. On the days of scheduled detailed clinical examination, clinical signs (after dosing) was included as a part of detailed clinical observations except on Day 1 wherein detailed clinical examination was done prior to the treatment and observations forgeneral clinical signs was done after dosing the animals.
Detailed Clinical Examination: Detailed clinical examination was done prior to the treatment on Day 1 and at weekly intervals thereafter (±1 day) during the treatment period. During detailed clinical examination, all rats were observed for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling or bizarre be haviour like self-mutilation, walking backwards). On the days of detailed clinical examination, observations for general clinical signs were not performed except post-dose observations on treatment Day 1.
Body Weights
i) Individual body weights of males were recorded on Day 1 and at weekly (±1 day) intervals the reafter. Individual body weights of females were recorded on Day 1 and at weekly intervals thereafter till cohabitation (till mating confirmation) with males.
ii) All dams were weighed on Gestation Day (GD) 0, 7, 14 and 20 and on Lactation Day (LD) 0, 4 and 13.

Food Consumption: Food consumption was calculated by using the food consumed at each interval per cage and dividing by the number of rats per cage and the number of days in the intervening period to determine the food consumption/rat/day. Food consumption was not measured during the cohabitation period.
Food consumption of pregnant dams was recorded on GD 7, 14 and 20 and on Days 4 and 13 of the lactation period.

Oestrous cyclicity (parental animals):

Vaginal smear was examined and the stage of oestrous cycle was recorded daily for two weeks before start of the treatment to select females with regular 4-5 days cyclicity for the study. The vaginal smear was also examined daily from the beginning of the treatment period until evidence of mating to determine the Day 0 of pregnancy/treatment-related effects on mating or precoital time. The time interval (in days) from the diestrus of an oestrous cycle to the next diestrus was
considered as the oestrous cycle length of an animal. Vaginal smears were also examined on the day of necropsy to determine the stage of the oestrous cycle.

Litter observations:

a. Each day in the morning, all the pups (both dead and alive) in a litter from each dam were observed for any external deformities and recorded.
b. The number of pups born (litter size), sex and individual pup body weight of male and female pups on LD 0 and 4 were recorded.
c. On Day 4 after birth, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four pups per sex per litter. Partial adjustment was done when the number of male or female pups prevents having four of each sex per litter. Pups were not el
iminated when the litter size drop below the culling target (8 pups/litter). Blood samples were collected from the available surplus pups of either sex, pooled, and used for determination of serum Thyroxine (T4) and Thyroid stimulating hormone (TSH) levels.
d. After standardization, the individual pup body weight was recorded on Day 7 and 13 of lactation.
e. The ano-genital distance (AGD) of each pup was measured on PND 0 and pup body weight was recorded. Ano-genital distance ratio was calculated by dividing the ano-genital distance from the cube root of body weight.
f. The number of nipples/areolae in male pups was counted on PND 13.
g. All the dead and sacrificed pups were examined for malformations and subjected to gross pathological examination.
h. The litters were observed daily to note the number of alive, dead and cannibalized pups.
i. In addition to daily clinical observations, any abnormal behaviour of the offspring was recorded.
j. Fertility index for dams, sires as well as the pup survival index until lactation day 4 was calculated.

Postmortem examinations (parental animals):

SACRIFICE
GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.] Yes
HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in [Section 9.8.2 in Main report] were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.] Yes
HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in [Section 9.8.2 in Main report] were prepared for microscopic examination and weighed, respectively.

Statistics:

Data captured using ProvantisTM: The body weight, organ weight and ano-genital distance data captured using Provantis™ was analyzed using built-in statistical tests. Derived data like net body weight gain, food consumption, ano-genital ratio, oestrous cycle length, organ weight ratios, post implantation loss (%), number of nipples/areolae in male pups, no. of implantations, pre-coital interval, mean litter size, sex ratio and gestation length (days), mating, fertil
ity and survival indices were analyzed using above mentioned methods. The statistical analysis of the hormonal data was carried out using licensed copies of SYSTAT Statistical package version 12.0. The data was tested for homogeneity of variances (Levene’s test) within the group before performing One-way analysis of variance (ANOVA). The non-optimal (non-normal or heteroschedastic) data was transformed, before ANOVA was performed. ANOVA was done using log
transformation. Comparison of means between treatment groups and control group was done using Dunnett’s t-test test when the overall treatment, ‘F’ test was found significant. All analyses and comparisons were evaluated at the 5% (p<0.05) level. Statistically significant differences (p<0.05), indicated by the aforementioned tests were designated throughout the report as stated below:
*: Significantly different from the vehicle control group

Reproductive indices:

Reproductive Performance Data of Parents
a. Male mating index (%)
Number of males with evidence of mating
=------------------------------------------------------------------- x 100
Number of males cohabited
b. Male fertility index (%)
Number of males siring a litter/impregnated a female
=----------------------------------------------------------------- x 100
Number of males cohabited
c. Female mating index (%)
Number of females mated
=------------------------------------------ x 100
Number of females cohabited
d. Female fertility index (%)
Number of pregnant females
= ------------------------------------------- x 100
Number of females used for mating
e. Mean number of implantations/group
Total number of implantations
= ---------------------------------------
Total number of pregnant animals
f. Post implantation loss (%)
Number of implantations - Number of live pups
= ------------------------------------------------------------------- x 100

Number of implantations

Offspring viability indices:

a. Mean litter size per group
Total Number of pups born
= -------------------------------------------------
Total Number of littered animals
b. Mean viable litter size
No. of viable pups
= -----------------------------------------
No. of females littered
c. Live birth index (%)
No. of viable pups born (at first observation)
= ----------------------------------------------------------x 100
Total no. of pups born (at first observation)
d. Day 4 survival index (%)
Number of viable pups on lactation Day 4
= -------------------------------------------------------- x 100
Number of viable pups born
e. Sex Ratio (%)
No. of male pups born
= -------------------------------- x 100
Total no. of pups born
f. Ano-genital Distance Ratio (mm/g1/3 )
Ano-genital distance
= --------------------------------
Cube root of body weight

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical signs and mortalities observed throughout the treatment period in either sex at all the doses tested. Incidence of dehydration and wound (right flank) was observed in one female (Rx6840) at 111mg/kg. This was considered not related to treatment because of isolated occurrence. However, yellow colour faeces was observed at at all the tested doses in either sex. This could be due to physical nature of the test item.
There were no abnormalities observed in pups.

Mortality:

no mortality observed

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:

no effects observed

Description (incidence and severity):

Hormonal Assay: Test item did not have any effects on effect on the thyroid hormone profile.

Reproductive function: oestrous cycle:

no effects observed

Reproductive performance:

no effects observed

To summarize, daily oral (gavage) administration of the test item “Novoperm-Gelb F2G (C.I. Pigment Yellow 194)” to Wistar rats at the dose levels of 111, 333 and 1000 mg/kg bwt/day for 2 weeks prior to mating, during mating, and post mating (males) or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery (females) had no effects on general health, food consumption, pre-coital time, gestation length, mating and fertility parameters. Male and female reproductive organs did not reveal any changes. There were no treatment-related effects on the uterine/implantation data and mean litter size. There were no external abnormalities in live or pups in any of the groups. No treatment-related changes in the ano-genital distance and ano-genital ratio were observed at any of the doses. The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested.

Treatment did not induce any test item-related adverse changes with respect to terminal fasting body weights, organ weights/ratios (including reproductive organs), thyroid hormone profile and histopathology in adult animals and pups at all dose levels tested. Grossly noted yellow contents in intestinal segments and stomach at ≥333 mg/kg/day were attributed was attributed to the physical appearance of the test item Novoperm-Gelb F2G (C.I. Pigment Yellow 194).

No Observed Adverse Effect Level
As there were no treatment related effects on reproduction and fertility parameters up to and including 1000 mg/kg bwt/day, the No Observed Adverse Effect Level (NOAEL) for Reproduction/Developmental Toxicity Screening Test for Novoperm-Gelb F2G (C.I. Pigment Yellow 194) is determined to be 1000 mg/kg bwt/day under the test conditions and doses employed.

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: No adverse effects were observed

Key result

Critical effects observed:

no

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were observed

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

To summarize, daily oral (gavage) administration of the test item C.I. Pigment Yellow 194 to Wistar rats at the dose levels of 111, 333 and 1000 mg/kg bwt/day for 2 weeks prior to mating, during mating, and post mating (males) or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery (females) had no effects on general health, food consumption, pre-coital time, gestation length, mating and fertility parameters. Male and female reproductive organs did not reveal any changes. There were no treatment-related effects on the uterine/implantation data and mean litter size. There were no external abnormalities in live or pups in any of the groups. No treatment-related changes in the ano-genital distance and ano-genital ratio were observed at any of the doses. The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested.

Treatment did not induce any test item-related adverse changes with respect to terminal fasting body weights, organ weights/ratios (including reproductive organs), thyroid hormone profile and histopathology in adult animals and pups at all dose levels tested. Grossly noted yellow contents in intestinal segments and stomach at ≥333 mg/kg/day were attributed was attributed to the physical appearance of the test item C.I. Pigment Yellow 194).

No Observed Adverse Effect Level
As there were no treatment related effects on reproduction and fertility parameters up to and including 1000 mg/kg bwt/day, the No Observed Adverse Effect Level (NOAEL) for Reproduction/Developmental Toxicity Screening Test for C.I. Pigment Yellow 194 is determined to be 1000 mg/kg bwt/day under the test conditions and doses employed.

Executive summary:

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

 

The test item, C.I. Pigment Yellow 194, was suspended in 0.5 % Carboxymethylcellulose Sodium salt (medium viscosity) in Milli-Q^®waterand administered by oral gavage at the dose levels of 111, 333 and 1000 mg/kg bwt/day to low (G2), mid (G3) and high dose (G4) group rats, respectively. A concurrent control group (G1) of rats received vehicle alone. The dose volume administered was 10 mL/kg bwt/day. Each group consisted of 10 male and 10 female rats. The dose formulations were administered once daily to a specific group of rats prior to mating, during mating and post-mating periods (for males), during pregnancy and up to Lactation Day (LD) 13 for females.

 

The identity of the test item was provided by the Sponsor by a Certificate of Analysis (CoA). The authenticity of the test item was not determined at the test facility. The stability of the test item in the vehicle was established separately under Eurofins Advinus Study No. G19479 at 1 and 100 mg/mL. Based on the results, the test item was found to be stable and homogeneous in the vehicle for up to 24 hours when stored at room temperature.

 

The dose formulations were analysed for homogeneity and active ingredient (a.i.) concentration on Day 1 and during 2^nd(Day 37) month of the treatment period. The results indicated that the analysed concentrations were within ± 15 % of variations from the claimed concentrations.

 

All rats were observed for clinical signs once daily. Body weight was recorded prior to the start of treatment on Day 1 and at weekly intervals thereafter. The body weights were also recorded at termination. Food consumption was recorded at weekly interval except during the cohabitation period.All dams were weighed onGestation Days (GD) 0, 7, 14 and 20 and on Lactation Days (LD) 0, 4 and 13. Food consumption was recorded on GD 7, 14 and 20 and on LD 4 and 13. The number, weight, survival and mortality of pups were observed during the lactation period. The ano-genital distance of each pup was measured on PND 0. All the surviving male pups were examined for the appearance of nipples/areolae on post-natal day (PND) 13. Thyroxine 4 (T4) and Thyroid Stimulating Hormone (TSH) analysis were performed in all males at termination, all dams on LD 14 (at termination) and available pups on LD 4 and 13. The animals were subjected to detailed necropsy at sacrifice after overnight fasting (water allowed) and study plan specified tissues were collected.

 

Tissues indicated for histopathology were collected and examined from all animals of control and high dose groups. Qualitative assessment of stages of spermatogenesis and evaluation of interstitial testicular cell structure were performed in testes sections. Gross lesions were examined in all the groups. The remaining tissues from lower dose groups were not examined as no target organs were identified at high dose

 

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

 

Clinical signs and Mortality:There were no clinical signs or mortalities observed at all the tested doses. The clinical sign of yellow coloured faecal matters was observed at all the tested doses. This could be due to physical nature of the test item.

Body weights:The mean body weights and Total body weight gainswere unaffected by the treatment at all the tested doses in both sexes.Food consumption:Treatment did not affect the food consumption at all the tested doses in either sex.

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

Fertility parameters:Treatment had no effect on pre-coital time or gestation length, oestrous cycle length, mating and fertility parameters in both sexes.

Litter parameters:There were no treatment-related effects on the uterine/implantation data, mean litter size and mean viable litter size. There were no external abnormalities in live or dead pups in any of the groups. No treatment-related changes in the ano-genital distance and ano-genital ratio were observed at any of the doses tested when compared to the control. The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested.

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

Terminal fasting body weights, organ weights and its ratios: There were no test item-related changes in terminal fasting body weights, organ weight and their ratios in adult male and female rats of all groups compared to the control group. There were no significant intergroup differences observed in the terminal body weights and thyroid gland weights in male/female pups.

Gross and histopathology: Grossly noted yellow contents in intestinal segments and stomach at ≥333 mg/kg/day were attributed was attributed to the physical appearance of the test item C.I. Pigment Yellow 194. There were no test item-related adverse histopathological changes observed either in parents or the offspring. The staging of spermatogenesis did not reveal any stage specific changes in testes and the spermatogenic cycles observed in the different seminiferous tubules.The qualitative assessment of stages of spermatogenesis and evaluation of interstitial testicular structures did not reveal any test item associated findings in parental rats.

 

In view of the results observed: As there were no treatment related effects on reproduction and fertility parameters up to and including 1000 mg/kg bwt/day, the No Observed Effect Level (NOEL) forReproduction/Developmental Toxicity Screening Test for C.I. Pigment Yellow 194 is determined to be 1000 mg/kg bwt/dayunder the test conditions and doses employed.

Reference 7

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

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

Adequacy of study:

key study

Study period:

2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

See read across justification document in chapter 13

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

Details on species / strain selection:

Rat is the standard laboratory rodent species used for toxicity assessment and recommended by various regulatory authorities.
The Wistar rat was selected due to the large amount of background data available for this strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Hylasco Biotechnology Pvt. Ltd., Plot 4B, AKP, Turkapally Village, Shameerpet Mandal, RR Dist, Telangana 500078
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Age at study initiation: 13 weeks
Body weight range at the start of treatment: Males: 362.07 to 464.96 g & Females: 239.96 to 287.13C g
At the commencement of the treatment, the weight variation of rats used did not exceed ± 20 % of the mean body weight in each sex and group.
Conditions: Rats were housed in an environment controlled room. The temperature maintained during the experiment was between 21 to 24°C and relative humidity was between 49 to 68%. The photoperiod was 12 hours light and 12 hours dark cycle. Adequate fresh air supply of 12-15 air changes/hour
was maintained in the experimental room. The maximum and minimum temperature in the experimental room was recorded once daily. The relative humidity in the experimental room was calculated daily from dry and wet bulb temperature recordings.
Housing: Two rats of same sex were housed per cage in sterilized standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles with stainless steel sipper tubes. The last animal in recovery group of each sex was housed individually. Polycarbonate rat huts were provided to the animals as environmental enrichment objects and changed along with cage at least once a week. During the experimental period, animals were housed in a single experimental room of barrier area.
Bedding: Steam sterilized corn cob was used as bedding and changed along with the cage atleast twice a week.
Diet: Altromin Rat/Mice Maintenance diets manufactured by Altromin Spezialfutter GmbH & Co. KG, Im Seelenkamp 20, 32791 Lage, Germany, was provided ad libitum.
Water: Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd., Mumbai 400001,India, was provided ad libitum to rats in polycarbonate bottles with stainless steel sipper tubes.

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

Males: The dose formulation was administered orally by gavage to the rats of the specific groups once daily for 36 days at approximately the same time each day (varying by ± 3 hours) which includes 2 weeks prior to mating, during mating and post-mating, up to and including the day before scheduled sacrifice.
Females: The dose formulations were administered orally by gavage to the specific group of rats once daily at approximately the same time each day (varying by ± 3 hours). This includes 2 weeks prior to the mating and continued through mating, pregnancy and up to LD 13 (total 46-57 days), after which, pups were sacrificed on LD 13 and parental females (dams) were sacrificed on LD 14 after overnight fasting (water allowed).
The animals in the vehicle control group were handled in an identical manner to the treatment groups and were administered vehicle only.
The dose volume administered to each rat was 10 mL/kg body weight throughout the study. The dose volume was adjusted based on the most recent body weight of the individual rat.

Details on mating procedure:

One female was placed with one male from the same group in a 1:1 ratio. Cohabitation was continued until there was evidence of sperms in the vaginal smear and /or vaginal plug. All the females were successfully copulated within six days from the day of cohabitation. Subsequently, pregnant females were housed individually until LD 14. All the mated females were maintained till they littered. Not-littered females were sacrificed after 25 days from the day they were found sperm positive (by vaginal smear examination).
The day of confirmed mating was designated as Gestation Day ‘0’ (GD ‘0’). The pre-coital time (days) was calculated for each female.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For homogeneity and active ingredient (a.i.) concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 of the treatment period and during the 2nd month (day 37) of treatment and was analysed in-house. For each set, duplicate samples were drawn from the top, middle and bottom layers of each preparation and in case of the control duplicate samples from the middle layer were drawn.
The analysis was done as per the method validated under Eurofins Advinus Study No.: G19479. One set of samples were analysed for concentration (a.i) analysis and other set (second set) of samples were stored at ambient condition for reanalysis purpose as a backup. These second set of samples were disposed, as the analysis results of first set of samples were within the accepted limit as per study plan.
Formulations were considered acceptable as the mean results (calculated using all the replicate values) of all the layers and mean of each layer were within ±15.0 % of the claimed concentration and the relative standard deviation (% RSD) was equal to or less than 10.0 %.

Duration of treatment / exposure:

Males: The dose formulation was administered orally by gavage to the rats of the specific groups once daily for 36 days at approximately the same time each day (varying by ± 3 hours) which includes 2 weeks prior to mating, during mating and post-mating, up to and including the day before scheduled sacrifice.
Females: The dose formulations were administered orally by gavage to the specific group of rats once daily at approximately the same time each day (varying by ± 3 hours). This includes 2 weeks prior to the mating and continued through mating, pregnancy and up to LD 13 (total 46-57 days), after which, pups were sacrificed on LD 13 and parental females (dams) were sacrificed on LD 14 after overnight fasting (water allowed).
The animals in the vehicle control group were handled in an identical manner to the treatment groups and were administered vehicle only.
The dose volume administered to each rat was 10 mL/kg body weight throughout the study. The dose volume was adjusted based on the most recent body weight of the individual rat.

Frequency of treatment:

Daily

Details on study schedule:

Experimental starting date: 27 May 2020
Acclimatization: Start: 27 May 2020 & End: 31 May 2020
Pre-treatment period : Start: 01 June 2020 & End: 14 June 2020
Treatment start: Start: 15 June 2020 & End: 10 August 2020

Dose / conc.:

111 mg/kg bw/day (nominal)

Dose / conc.:

333 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes

Details on study design:

The test item was administered by oral gavage in graded doses to three groups of male and female rats. The males were dosed for six weeks, up to and including the day before scheduled sacrifice. This included a two-week period prior to mating, during the mating period and post mating.
Females were dosed throughout the treatment period. This included two weeks prior to mating (with the objective of covering at least two complete oestrous cycles), the variable time to conception, the duration of pregnancy and 13 days after delivery, up to and including the day before scheduled sacrifice.

Parental animals: Observations and examinations:

Parental animals: Observations and examinations
Clinical Signs, Morbidity and Mortality: All rats were observed for morbidity and mortalities twice daily i.e., once in the morning and once in the afternoon. As there were no clinical signs of toxicity, the observation for morbidity and mortality was carried out once in the morning during weekend and holidays. All rats were observed for clinical signs once daily during the treatment period. On the days of scheduled detailed clinical examination, clinical signs (after dosing) was included as a part of detailed clinical observations except on Day 1 wherein detailed clinical examination was done prior to the treatment and observations forgeneral clinical signs was done after dosing the animals.
Detailed Clinical Examination: Detailed clinical examination was done prior to the treatment on Day 1 and at weekly intervals thereafter (±1 day) during the treatment period. During detailed clinical examination, all rats were observed for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling or bizarre be haviour like self-mutilation, walking backwards). On the days of detailed clinical examination, observations for general clinical signs were not performed except post-dose observations on treatment Day 1.
Body Weights
i) Individual body weights of males were recorded on Day 1 and at weekly (±1 day) intervals the reafter. Individual body weights of females were recorded on Day 1 and at weekly intervals thereafter till cohabitation (till mating confirmation) with males.
ii) All dams were weighed on Gestation Day (GD) 0, 7, 14 and 20 and on Lactation Day (LD) 0, 4 and 13.

Food Consumption: Food consumption was calculated by using the food consumed at each interval per cage and dividing by the number of rats per cage and the number of days in the intervening period to determine the food consumption/rat/day. Food consumption was not measured during the cohabitation period.
Food consumption of pregnant dams was recorded on GD 7, 14 and 20 and on Days 4 and 13 of the lactation period.

Oestrous cyclicity (parental animals):

Vaginal smear was examined and the stage of oestrous cycle was recorded daily for two weeks before start of the treatment to select females with regular 4-5 days cyclicity for the study. The vaginal smear was also examined daily from the beginning of the treatment period until evidence of mating to determine the Day 0 of pregnancy/treatment-related effects on mating or precoital time. The time interval (in days) from the diestrus of an oestrous cycle to the next diestrus was
considered as the oestrous cycle length of an animal. Vaginal smears were also examined on the day of necropsy to determine the stage of the oestrous cycle.

Litter observations:

a. Each day in the morning, all the pups (both dead and alive) in a litter from each dam were observed for any external deformities and recorded.
b. The number of pups born (litter size), sex and individual pup body weight of male and female pups on LD 0 and 4 were recorded.
c. On Day 4 after birth, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four pups per sex per litter. Partial adjustment was done when the number of male or female pups prevents having four of each sex per litter. Pups were not el
iminated when the litter size drop below the culling target (8 pups/litter). Blood samples were collected from the available surplus pups of either sex, pooled, and used for determination of serum Thyroxine (T4) and Thyroid stimulating hormone (TSH) levels.
d. After standardization, the individual pup body weight was recorded on Day 7 and 13 of lactation.
e. The ano-genital distance (AGD) of each pup was measured on PND 0 and pup body weight was recorded. Ano-genital distance ratio was calculated by dividing the ano-genital distance from the cube root of body weight.
f. The number of nipples/areolae in male pups was counted on PND 13.
g. All the dead and sacrificed pups were examined for malformations and subjected to gross pathological examination.
h. The litters were observed daily to note the number of alive, dead and cannibalized pups.
i. In addition to daily clinical observations, any abnormal behaviour of the offspring was recorded.
j. Fertility index for dams, sires as well as the pup survival index until lactation day 4 was calculated.

Postmortem examinations (parental animals):

SACRIFICE
GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.] Yes
HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in [Section 9.8.2 in Main report] were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.] Yes
HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in [Section 9.8.2 in Main report] were prepared for microscopic examination and weighed, respectively.

Statistics:

Data captured using ProvantisTM: The body weight, organ weight and ano-genital distance data captured using Provantis™ was analyzed using built-in statistical tests. Derived data like net body weight gain, food consumption, ano-genital ratio, oestrous cycle length, organ weight ratios, post implantation loss (%), number of nipples/areolae in male pups, no. of implantations, pre-coital interval, mean litter size, sex ratio and gestation length (days), mating, fertil
ity and survival indices were analyzed using above mentioned methods. The statistical analysis of the hormonal data was carried out using licensed copies of SYSTAT Statistical package version 12.0. The data was tested for homogeneity of variances (Levene’s test) within the group before performing One-way analysis of variance (ANOVA). The non-optimal (non-normal or heteroschedastic) data was transformed, before ANOVA was performed. ANOVA was done using log
transformation. Comparison of means between treatment groups and control group was done using Dunnett’s t-test test when the overall treatment, ‘F’ test was found significant. All analyses and comparisons were evaluated at the 5% (p<0.05) level. Statistically significant differences (p<0.05), indicated by the aforementioned tests were designated throughout the report as stated below:
*: Significantly different from the vehicle control group

Reproductive indices:

Reproductive Performance Data of Parents
a. Male mating index (%)
Number of males with evidence of mating
=------------------------------------------------------------------- x 100
Number of males cohabited
b. Male fertility index (%)
Number of males siring a litter/impregnated a female
=----------------------------------------------------------------- x 100
Number of males cohabited
c. Female mating index (%)
Number of females mated
=------------------------------------------ x 100
Number of females cohabited
d. Female fertility index (%)
Number of pregnant females
= ------------------------------------------- x 100
Number of females used for mating
e. Mean number of implantations/group
Total number of implantations
= ---------------------------------------
Total number of pregnant animals
f. Post implantation loss (%)
Number of implantations - Number of live pups
= ------------------------------------------------------------------- x 100

Number of implantations

Offspring viability indices:

a. Mean litter size per group
Total Number of pups born
= -------------------------------------------------
Total Number of littered animals
b. Mean viable litter size
No. of viable pups
= -----------------------------------------
No. of females littered
c. Live birth index (%)
No. of viable pups born (at first observation)
= ----------------------------------------------------------x 100
Total no. of pups born (at first observation)
d. Day 4 survival index (%)
Number of viable pups on lactation Day 4
= -------------------------------------------------------- x 100
Number of viable pups born
e. Sex Ratio (%)
No. of male pups born
= -------------------------------- x 100
Total no. of pups born
f. Ano-genital Distance Ratio (mm/g1/3 )
Ano-genital distance
= --------------------------------
Cube root of body weight

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clinical signs and mortalities observed throughout the treatment period in either sex at all the doses tested. Incidence of dehydration and wound (right flank) was observed in one female (Rx6840) at 111mg/kg. This was considered not related to treatment because of isolated occurrence. However, yellow colour faeces was observed at at all the tested doses in either sex. This could be due to physical nature of the test item.
There were no abnormalities observed in pups.

Mortality:

no mortality observed

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:

no effects observed

Description (incidence and severity):

Hormonal Assay: Test item did not have any effects on effect on the thyroid hormone profile.

Reproductive function: oestrous cycle:

no effects observed

Reproductive performance:

no effects observed

To summarize, daily oral (gavage) administration of the test item “Novoperm-Gelb F2G (C.I. Pigment Yellow 194)” to Wistar rats at the dose levels of 111, 333 and 1000 mg/kg bwt/day for 2 weeks prior to mating, during mating, and post mating (males) or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery (females) had no effects on general health, food consumption, pre-coital time, gestation length, mating and fertility parameters. Male and female reproductive organs did not reveal any changes. There were no treatment-related effects on the uterine/implantation data and mean litter size. There were no external abnormalities in live or pups in any of the groups. No treatment-related changes in the ano-genital distance and ano-genital ratio were observed at any of the doses. The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested.

Treatment did not induce any test item-related adverse changes with respect to terminal fasting body weights, organ weights/ratios (including reproductive organs), thyroid hormone profile and histopathology in adult animals and pups at all dose levels tested. Grossly noted yellow contents in intestinal segments and stomach at ≥333 mg/kg/day were attributed was attributed to the physical appearance of the test item Novoperm-Gelb F2G (C.I. Pigment Yellow 194).

No Observed Adverse Effect Level
As there were no treatment related effects on reproduction and fertility parameters up to and including 1000 mg/kg bwt/day, the No Observed Adverse Effect Level (NOAEL) for Reproduction/Developmental Toxicity Screening Test for Novoperm-Gelb F2G (C.I. Pigment Yellow 194) is determined to be 1000 mg/kg bwt/day under the test conditions and doses employed.

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: No adverse effects were observed

Key result

Critical effects observed:

no

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were observed

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

To summarize, daily oral (gavage) administration of the test item to Wistar rats at the dose levels of 111, 333 and 1000 mg/kg bwt/day for 2 weeks prior to mating, during mating, and post mating (males) or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery (females) had no effects on general health, food consumption, pre-coital time, gestation length, mating and fertility parameters. Male and female reproductive organs did not reveal any changes. There were no treatment-related effects on the uterine/implantation data and mean litter size. There were no external abnormalities in live or pups in any of the groups. No treatment-related changes in the ano-genital distance and ano-genital ratio were observed at any of the doses. The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested.

Treatment did not induce any test item-related adverse changes with respect to terminal fasting body weights, organ weights/ratios (including reproductive organs), thyroid hormone profile and histopathology in adult animals and pups at all dose levels tested. Grossly noted yellow contents in intestinal segments and stomach at ≥333 mg/kg/day were attributed was attributed to the physical appearance of the test item C.I. Pigment Yellow 194).

No Observed Adverse Effect Level
As there were no treatment related effects on reproduction and fertility parameters up to and including 1000 mg/kg bwt/day, the No Observed Adverse Effect Level (NOAEL) for Reproduction/Developmental Toxicity Screening Test for C.I. Pigment Yellow 194 is determined to be 1000 mg/kg bwt/day under the test conditions and doses employed.

Executive summary:

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

 

The test item was suspended in 0.5 % Carboxymethylcellulose Sodium salt (medium viscosity) in Milli-Q^®waterand administered by oral gavage at the dose levels of 111, 333 and 1000 mg/kg bwt/day to low (G2), mid (G3) and high dose (G4) group rats, respectively. A concurrent control group (G1) of rats received vehicle alone. The dose volume administered was 10 mL/kg bwt/day. Each group consisted of 10 male and 10 female rats. The dose formulations were administered once daily to a specific group of rats prior to mating, during mating and post-mating periods (for males), during pregnancy and up to Lactation Day (LD) 13 for females.

 

The identity of the test item was provided by the Sponsor by a Certificate of Analysis (CoA). The authenticity of the test item was not determined at the test facility. The stability of the test item in the vehicle was established separately under Eurofins Advinus Study No. G19479 at 1 and 100 mg/mL. Based on the results, the test item was found to be stable and homogeneous in the vehicle for up to 24 hours when stored at room temperature.

 

The dose formulations were analysed for homogeneity and active ingredient (a.i.) concentration on Day 1 and during 2^nd(Day 37) month of the treatment period. The results indicated that the analysed concentrations were within ± 15 % of variations from the claimed concentrations.

 

All rats were observed for clinical signs once daily. Body weight was recorded prior to the start of treatment on Day 1 and at weekly intervals thereafter. The body weights were also recorded at termination. Food consumption was recorded at weekly interval except during the cohabitation period.All dams were weighed onGestation Days (GD) 0, 7, 14 and 20 and on Lactation Days (LD) 0, 4 and 13. Food consumption was recorded on GD 7, 14 and 20 and on LD 4 and 13. The number, weight, survival and mortality of pups were observed during the lactation period. The ano-genital distance of each pup was measured on PND 0. All the surviving male pups were examined for the appearance of nipples/areolae on post-natal day (PND) 13. Thyroxine 4 (T4) and Thyroid Stimulating Hormone (TSH) analysis were performed in all males at termination, all dams on LD 14 (at termination) and available pups on LD 4 and 13. The animals were subjected to detailed necropsy at sacrifice after overnight fasting (water allowed) and study plan specified tissues were collected.

 

Tissues indicated for histopathology were collected and examined from all animals of control and high dose groups. Qualitative assessment of stages of spermatogenesis and evaluation of interstitial testicular cell structure were performed in testes sections. Gross lesions were examined in all the groups. The remaining tissues from lower dose groups were not examined as no target organs were identified at high dose

 

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

 

Clinical signs and Mortality:There were no clinical signs or mortalities observed at all the tested doses. The clinical sign of yellow coloured faecal matters was observed at all the tested doses. This could be due to physical nature of the test item.

Body weights:The mean body weights and Total body weight gainswere unaffected by the treatment at all the tested doses in both sexes.Food consumption:Treatment did not affect the food consumption at all the tested doses in either sex.

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

Fertility parameters:Treatment had no effect on pre-coital time or gestation length, oestrous cycle length, mating and fertility parameters in both sexes.

Litter parameters:There were no treatment-related effects on the uterine/implantation data, mean litter size and mean viable litter size. There were no external abnormalities in live or dead pups in any of the groups. No treatment-related changes in the ano-genital distance and ano-genital ratio were observed at any of the doses tested when compared to the control. The male pups did not exhibit areola/nipple retention on PND 13 at any of the doses tested.

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

Terminal fasting body weights, organ weights and its ratios: There were no test item-related changes in terminal fasting body weights, organ weight and their ratios in adult male and female rats of all groups compared to the control group. There were no significant intergroup differences observed in the terminal body weights and thyroid gland weights in male/female pups.

Gross and histopathology: Grossly noted yellow contents in intestinal segments and stomach at ≥333 mg/kg/day were attributed was attributed to the physical appearance of the test item C.I. Pigment Yellow 194. There were no test item-related adverse histopathological changes observed either in parents or the offspring. The staging of spermatogenesis did not reveal any stage specific changes in testes and the spermatogenic cycles observed in the different seminiferous tubules.The qualitative assessment of stages of spermatogenesis and evaluation of interstitial testicular structures did not reveal any test item associated findings in parental rats.

 

In view of the results observed: As there were no treatment related effects on reproduction and fertility parameters up to and including 1000 mg/kg bwt/day, the No Observed Effect Level (NOEL) forReproduction/Developmental Toxicity Screening Test for C.I. Pigment Yellow 194 is determined to be 1000 mg/kg bwt/dayunder the test conditions and doses employed.

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

The 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

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Species:

rabbit

Abnormalities:

not specified

Developmental effects observed:

not specified

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

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:

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 4

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:

The 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 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 5

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 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

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.

 

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

Close structural analogues of the registered substance did not cause any adverse reproductive or deveolpmental effects in rats.