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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

This OECD 422 study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item to rats. The test item was administered in vehicle (1.0% CMC / 0.05% Tween 80 in 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 32 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.


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.

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 09 FEB 2012 and 03 JUL 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD 422) and in compliance with GLP.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
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.
- Breeder: 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): 315 to 369 g (males) and 187 to 243 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.
- Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS

- Conditions: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). There was a 12 hour fluorescent light / 12-hour dark cycle with music during the light period.
- Accommodation: In groups of three to five animals 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, 100099 and S211008A63972). 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. Results of representative analyses for contaminants were included in the report as an Appendix.
- Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles. Results of bacteriological assay, chemical and contaminant analyses of representative samples were included in the report as an Appendix.
Route of administration:
oral: gavage
Vehicle:
other: 1.0% CMC / 0.05% Tween 80 in highly purified water
Details on exposure:
DOSE FORMULATIONS

The dose formulations were prepared weekly using the test item as supplied by the Sponsor.

Test item was weighed into a glass beaker on a tared precision balance. Appropriate amount of the vehicle was weighted and added to the test item (w/w). Dose formulations were mixed on a magnetic stirrer for approximately 2 hours until mixtures were homogeneous. 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 D44836 14-Day Oral Toxicity (Gavage) Study in the Wistar Rat, dose formulations are stable for at least 7 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 bw/day (control group), 100 mg/kg bw/day (group 2), 300 mg/kg bw/day (group 3) and 1000 mg/kg bw/day (Group 4)
- Rationale for Dose Level Selection: The dose levels were selected based on a previous dose range-finding toxicity study in Han Wistar rats, Harlan Laboratories Study D44836, using dose levels of 0, 100, 300 and 1000 mg/kg/day, where no adverse effects were observed up to the highest dose level.
- Dose Volume: 10 mL/kg body weight
- Dose Concentrations: 0 mg/mL (control group), 10 mg/mL (group 2), 30 mg/mL (group 3) and 100 mg/mL (group 4)
- Duration of Acclimatization Period: 7 days
- Duration of Treatment Period: 32 days males and approximately 7 weeks females
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 positive mating was determined (copulation plug or sperm) was designated day 0 post coitum.

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:
On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about of 2 g each concentration were taken prior to dosing for analysis of concentration and homogeneity. To confirm the stability (7 days) samples of about 2 g of each concentration were taken from the middle only of each aliquot used on day 7 of the treatment. During the sixth week of the treatment samples of about 0.5 g were taken to repeat the measurement of concentration, homogeneity and stability (7 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 stored at -20 ± 5 °C until analysis.

The samples were analyzed by UV-VIS measurement following an analytical procedure developed by Harlan Laboratories. The test item was used as the analytical standard.

The following results were obtained:

Blank samples showed no absorbance and, therefore, it was confirmed that only 1.0% CMC / 0.05% Tween 80 in highly purified water applied within the control experiment.

The application formulations investigated during the study were found to comprise test item in the range of 83.0% to 111.3% 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 9.8% (acceptance criterion: <15%) from the corresponding mean.

In addition, the test item was found to be stable in application formulations when kept seven days at room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean, except for group 2 and 3 prepared on 16-Feb-2012 that exceeded the acceptance criteria. In application formulations of group 2 (10 mg/mL), the maximum deviation of time-zero mean was found to be 11.8%, and in application formulations of group 3 (30 mg/mL), the maximum deviation of time-zero mean was found to be 35.8%. However, there are some hints of what could be the cause. On the first and 7th treatment day as well as backup samples (date of preparation 16-Feb-2012), erroneously high amount of samples (about 2 g instead 0.5 g) were taken for analytical measurements making it difficult to work up. That was considered to probably be the reason for the differences as a second time using small amount of sample (0.5 g) shows the homogeneity and stability of the application formulation.

In conclusion, the results indicate the accurate use of the test item and 1.0% CMC / 0.05% Tween 80 in highly purified water as vehicle during this study. Application formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.
Duration of treatment / exposure:
MALES

32 days

FEMALES

about 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 End: On day before sacrifice
- Blood Sampling: At Termination
- Necropsy: After 32 days of treatment, 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 End: On day 4 post partum
- Blood Sampling: On day 5 post partum
- Necropsy: On day 5 post partum (pups on day 4 post partum)
Remarks:
Doses / Concentrations:
0 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
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 4 post partum) relevant parameters were performed with 5 P generation males and 5 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 of each group. Blood samples from 5 lactating females of each group were obtained at the end of the pre-pairing period. Blood samples were drawn from the retro-orbital plexus of 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 five 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 was 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 was 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 was 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 32 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)

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 principal investigator for histopathology phase 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.

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 principal investigator. 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.

A histopathology peer review was performed. A histopathology phase report was provided by the principal investigator for inclusion in the main report as an appendix.
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 dead during the study, except those excessively cannibalized, were examined macroscopically.
Statistics:
The following statistical methods were used to analyze food consumption, body weights, reproduction data, grip strength, landing foot splay, body temperature, locomotor activity, hematology and biochemistry parameters, urinalysis, sperm analyses and organ weights:
- Means and standard deviations of various data were calculated and included in the report.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables can 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 cannot be assumed to follow a normal distribution.
- Fisher's exact-test was applied if the variables can 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):
Yellow 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):
Transient reduction in body weight gain was noted in males at the dose levels of 1000 and 300 mg/kg bw/day .
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Transient reduction in body weight gain was noted in males at the dose levels of 1000 and 300 mg/kg bw/day .
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):
Reduction in sperm motility at the dose levels of 300 and 1000 mg/kg bw/day.
Reproductive performance:
no effects observed
1. IN-LIVE DATA

VIABILITY / MORTALITY

All animals survived scheduled study period.

DAILY CLINICAL SIGNS OR OBSERVATIONS

Feces stained yellow were noted in all males and females in all dose groups starting from day 2 or 3 of the treatment until completion of the study. This observation was due to staining properties of the yellow coloured test item.

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

Incidentally, scabs on the neck were noted in one male in the control group.

FINDINGS AT DETAILED WEEKLY CLINICAL OBSERVATIONS

No test item-related findings were noted during weekly detailed clinical observations.

Incidentally, in one female at the dose level of 1000 mg/kg bw/day missing upper incisors on day 6 and 13 of the gestation period were recorded.

No further findings were noted in males or females in any dose group.

FUNCTIONAL OBSERVATIONAL BATTERY

None of the parameters under investigation during the functional observational battery gave an indication of a test item-related effect.

All findings recorded during the tests were considered not to be test item-related. Decreased or increased rearings, increased faeces balls and vocalization were noted in individual animals without dose dependency. At the dose level of 1000 mg/kg bw/day, one female was noted with multiple findings: reduced activity, ruffled fur, decreased rearings and salivation. Because of isolated occurrence, this observation was considered not to be related to the treatment.

Remaining parameters under investigation were similar in all groups.

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: 1079, 1198, 1252 and 1207 in males and 965, 917, 853 and 867 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: ±0.0%, -1.2% and -4.6% during the pre-pairing period and -2.0%, -2.0% and -0.8% during the after pairing period (percentages refer to the respective values in the control group).

FOOD CONSUMPTION OF FEMALES

No 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 on days 8 - 11 of the pre-pairing period and at the dose level of 300 mg/kg bw/day on days 7 - 14 of the gestation period. In the absence of any dose dependency, these differences were not related to the treatment.

Mean differences in food consumption at the dose levels 100, 300 and 1000 mg/kg bw/day were respectively: +8.0%, ±0.0%, and +3.4% during the pre-pairing period, +1.8%, +6.3% and +3.1% during the gestation period and +11.7%, -3.0% and +3.8% during the lactation period (percentages refer to the respective values in the control group).

BODY WEIGHTS OF MALES

At the dose levels of 1000 and 300 mg/kg bw/day, reduction in body weight gain was noted during the pre-pairing period; mean body weight gain within this period was +10% and +12% at the high- and mid-dose level respectively, compared to +14% in the control group. Reduction in body weight gain was statistically significant at the high-dose level starting from day 3 until the end of pre-pairing period and at the mid-dose level on days 7, 9, 11, 12 and 14 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.
Body weights of males in all dose groups were similar to the respective control values during the entire study period.
Because the reduction in body weight gain at the high- and mid-dose levels was reversible and did not result in any significant differences in body weights, this observation was considered not to be adverse.

Mean differences in body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were respectively: +14%, +13%, +12% and +10% during the pre-pairing period, +3%, +2%, +3% and 3% during the pairing period and +1%, +1%, +1% and +2% 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.

Mean differences in body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were respectively: 8%, 9%, 8% and 8% during the pre-pairing period, 53%, 53%, 59% and 57% during the gestation period and 6%, 7%, 6% and 6% 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.

At the dose level of 300 mg/kg bw/day in males, amount of large unstained cells (LUC) was statistically significantly lower when compared to the control value. No reduction of this value occurred at the dose level of 1000 mg/kg bw/day and therefore the difference at mid-dose level was not related to the treatment.

CLINICAL BIOCHEMISTRY

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

At the dose level of 300 mg/kg bw/day in males, potassium concentration was statistically significantly higher when compared to the control value. No increase of this value occurred at the dose level of 1000 mg/kg bw/day and therefore the difference at mid-dose level was not related to the treatment.

In females following parameters were statistically significantly lower if compared to the respective control values: concentration of albumin at the dose level of 100 mg/kg bw/day, concentration of albumin and albumin to globulin ratio at the dose level of 300 mg/kg bw/day and concentration of total protein and albumin at the dose level of 1000 mg/kg bw/day. Changes of these values were not dose depended and all values were within historical control range. Therefore these changes were considered not to be test item-related.

URINALYSIS

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

3. TERMINAL FINDINGS

SEMINOLOGY AND SPERMATID COUNT

At the dose levels of 1000 and 300 mg/kg bw/day, statistically significant changes in motility of sperms were noted. Mean count of progressive sperms was reduced at the high dose level and it was 60.4% compared to 75.6% in the control group. This value is within the range of a limited pool of historical controls for OECD 422 (n=4) extended by OECD 416 data (n=5). Mean count of not motile sperms was increased and was 35.5% and 34.2% at the high- and mid-dose dose levels, respectively, compared to 20.2% in the control group. 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). In the absence of any findings during necropsy or histopathological examination of male reproductive organs as well as absence of any effects on reproduction, the differences in sperm motility were considered not to be adverse.

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 (testis) or slightly higher (epididymidis) when compared to the respective control values.

ORGAN WEIGHTS

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

At the dose level of 100 mg/kg bw/day in males, statistically significantly lower weight of testis was noted. This effect was due to reduced testis weights of one male (no. 15, malformation of testis in this male correlated with histopathological findings). No significant changes of testis weights were noted at the mid- and high dose levels and therefore these changes were not related to the treatment with the test item.

No further significant differences in absolute or relative organ weights were noted in males or females at any dose level.

MACROSCOPICAL FINDINGS

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

HISTOPATHOLOGY FINDINGS

Under the conditions of this experiment, tets item did not induce histopathological lesions. All findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.
There were no abnormal lesions encountered during sperm staging regarding completeness of stages and maturation of cell populations. Individual lesions recorded were within the range of background alterations that may be recorded in this type of study, in rats of this strain and age.

During examination of reproduction organs of infertile males and females, bilateral total tubular degeneration of testes was found in male no. 15 at the dose level of 100 mg/kg bw/day. This lesion was considered to be the reason for infertility of male no.15. No further findings correlated to the infertility were noted in reproduction organs of the remaining infertile males or females.

4. REPRODUCTION AND BREEDING

MATING PERFORMANCE AND FERTILITY

Mating performance and fertility were not affected by the treatment at any dose level.

Percentage of mating was 100% in all groups. Mating of all females was recorded during the first seven days of the pairing period.

Mean (median) precoital times were 2.9 (3), 2.6 (3), 2.9 (3) and 2.8 (3) days at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.

Six females were not pregnant: two in the control group, one at each dose level 100 and 300 mg/kg bw/day and two at the dose level of 1000 mg/kg bw/day. 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 81.8%, 90.9%, 90.9% and 81.8% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.

One female at the high dose level had two implantation sites but delivered no pups. Consequently, gestation index (number of females with living pups as percentages of females pregnant) was 88.9% at the high-dose level and 100% in remaining groups.

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 13.6, 15.5, 17.4 and 14.4 in order of ascending dose levels.

At the dose level of 300 mg/kg bw/day, mean number of corpora lutea per dam was statistically significantly higher when compared to the control group. In the absence of any dose dependency, this observation was considered not to be related to the treatment.

DURATION OF GESTATION

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

Mean duration of gestation was 21.6, 21.8, 21.8 and 21.6 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 was 13.0, 13.0, 14.2 and 14.4 per dam whereas mean incidence of post-implantation loss was 1.0, 1.0, 1.4 and 0.4 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 12.0, 12.2, 12.8 and 14.1 in order of ascending dose levels.

Birth index (number of pups born alive as a percentage of implantations) was 92.3%, 92.2%, 90.1% and 97.0% at the dose level of 0, 100, 300 and 1000 mg/kg bw/day.
Key result
Dose descriptor:
NOAEL
Remarks:
for general toxicity
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: Transiently lower body weight gain noted at the dose levels of 1000 and 300 mg/kg bw/day was considered not to be adverse.
Key result
Dose descriptor:
NOAEL
Remarks:
for general toxicity
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: No toxicologically relevant findings were noted in females up to the dose level of 1000 mg/kg bw/day, the highest dose level used.
Key result
Dose descriptor:
NOAEL
Remarks:
for reproduction
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Reduced motility of sperms noted at the dose levels of 1000 and 300 mg/kg bw/day was considered not to be adverse.
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 eight pups (from 2 litters) were missing on day 4 and at the high dose level two pups (from two litters) were missing on day 2 of the lactation period. Mean postnatal loss during four days of lactation was 0.1, 0.1, 0.8 and 0.3% 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 99.1, 99.2, 93.8 and 98.2 in order of ascending dose levels.

At the dose level of 300 mg/kg bw/day, total number of pups lost was statistically significantly higher and viability index was statistically significantly lower when compared to the respective control values. These observations were due to a loss of seven pups in one litter. Because higher mortality of pups was noted only in one litter and in the absence of increased mortality of pups at the dose level of 1000 mg/kg bw/day, this observation was considered not to be related to the treatment.

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.

In the control group, missing tail tip was noted at first litter check and during lactation in one pup. At the dose level of 300 mg/kg bw/day, all pups in one litter had reduced temperature on day 3 and several pups from this litter had reduced temperature on day 4 of the lactation period. At the dose level of 1000 mg/kg bw/day, one pup, which was dead at first litter check had no milk in the stomach and was partially cannibalized.

No further findings were noted in pups at any dose level.

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 48%, 48%, 38% and 49% 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: 8.9 g, 9.2 g, 8.9 and 8.2 g and mean differences in body weights during lactation were +47.4%, +47.7%, 43.6% and +38.7%, at the dose levels of 0, 100, 300 and 1000 mg/kg/day, respectively.

At the dose level of 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 this dose level which was supported by observation that reduction of body weight gain was more pronounced in litters of higher size. Therefore this effect was not test item-related.

MACROSCOPICAL FINDINGS

No findings were found during necropsy of pups in any dose group.
Key result
Dose descriptor:
NOAEL
Remarks:
for development
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Under the condition of the study, post-implantation loss, body weights of pups and results of examination of pups did not indicate developmental effects up to the dose level of 1000 mg/kg bw/day, the highest dose level used.
Key result
Reproductive effects observed:
not specified

SUMMARY OF PERFORMANCE 

P Animals Breeding for F1 Litters

 

Group
(mg/kg/day)

1
(0)

2
(100)

3
(300)

4
(1000)

Female numbers

45-55

56-66

67-77

78-88

Number of females paired

11

11

11

11

Number of females mated

11

11

11

11

Number of non pregnant females (A)

2

1

1

2

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

9

10

10

8

(A) Female nos. 46,50, 59, 69, 83 and 85

(B) Female no. 87

Conclusions:
This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item to rats. The test item was administered in vehicle (1.0% CMC / 0.05% Tween 80 in 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 32 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.

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 yellow were noted in all males and females receiving test item. This observation was due to staining properties of the test item.

At the dose levels of 1000 and 300 mg/kg bw/day in males, statistically significantly reduced body weight gain was noted during the pre-pairing period. During the remaining study period body weight gain at the high- and mid-dose levels was similar to the control values. Body weights at these dose levels were similar to the control values during the entire study period. Because reduction of body weight gain was reversible and no statistically significant differences in body weights were noted at the high- and mid-dose levels, this effect was considered not to be adverse.

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 at the dose levels of 1000 and 300 mg/kg bw/day. Statistically significant reduction in mean count of progressive sperms and increase in mean count of not motile sperms were noted at the high dose level and statistically significant increase in mean count of not motile sperms was noted at the mid-dose level. 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.

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 was considered to be 1000 mg/kg bw/day, the highest dose level used.
Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of test item on 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 32 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 (1.0% CMC / 0.05% Tween 80 in highly purified water).

 

The following results were obtained:

 

MORTALITY AND GENERAL TOLERABILITY OF PARENTAL ANIMALS

 

All animals survived the scheduled study period.

 

During the treatment, yellow stained faeces was noted in all males and females in dose groups. This observation was due to staining properties of the test item.

 

No further test item-related observations were noted during clinical daily or detailed weekly observations in males or females at any dose level.

 

FUNCTIONAL OBSERVATIONAL BATTERY AND LOCOMOTOR ACTIVITY OF PARENTAL ANIMALS

 

None of the parameters under investigation during the functional observational battery gave an indication of a test item-related effect.

 

Locomotor activity was not affected by the treatment with test item 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 WEIGHT OF PARENTAL ANIMALS

 

In males at the dose levels of 1000 and 300 mg/kg bw/day, dose dependent and statistically significant reduction in 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- and mid-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 ANIMALS

 

At the dose level of 1000 mg/kg bw/day, reduction in mean count of progressive sperms and increase in mean count of not motile sperms were noted. At the dose level of 300 mg/kg bw/day, increase in mean count of not motile sperms was noted. 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 absence of any effects on reproduction, the differences in sperm motility were considered not to be adverse.

Sperm morphology and sperm count were not affected by the treatment at any dose level.

 

ORGAN WEIGHTS OF PARENTAL ANIMALS

 

No test item-related changes in organ weights 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.

All findings recorded during histopathological examination 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 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 IN PUPS

 

No findings were found during necropsy of pups in any dose group.

 

CONCLUSION

 

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

 

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:
other:
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

This OECD 422 study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item to rats. The test item was administered in vehicle (1.0% CMC / 0.05% Tween 80 in 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 32 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.


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.


 


In two studies according to OECD TG 414 the oral administration of test item  by gavage to presumed pregnant female New Zealand White Rabbits or rats during gestation did not produce any indication of maternal and developmental toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.
Based on the obtained results from this pre-natal developmental toxicity study conducted, the NOAEL (No observed adverse effect level) of the test item for both maternal and fetal toxicity in both species is estimated as 1000 mg/kg body weight/day under experimental conditions employed.


 


 

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Remarks:
Prenatal Developmental Toxicity Study
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
27 July 2021 to 04 April 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
OECD Test Guideline No. 414, “Prenatal Developmental Toxicity Study”, adopted on 25 June 2018
Justification for type of information:
See read across justification documkent in Chapter 13
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted on 25 June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Hylasco Biotechnology India Pvt. Ltd, Charles River Technology Licensee,
CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals) Registration No.: 1808/PO/RcBt/S/15/CPCSEA

- Age at study initiation: 11 weeks
- Weight at study initiation: Males: 240.25 to 298.41 g, Females: 200.65 to 249.44 g
- Housing: Sterilized standard polypropylene cage of Size: L 430 × B 285 × H 150 mm
- Diet (e.g. ad libitum): Altromin maintenance diet for rats and mice manufactured by Altromin Spezialfutter GmbH & Co. KG
- Water (e.g. ad libitum): Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period: Minimum of five days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1 to 23.2
- Humidity (%): 49 to 64%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark cycle and 12 hours fluorescent light

IN-LIFE DATES: From:30 July 2021 To:23 September 2021
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Justification for use and choice of vehicle : The test item was uniformly suspended in 1% w/v Carboxymethyl cellulose (CMC) at the concentration of 100 mg/mL the highest dose concentration selected for the study considering the dose volume of 10 mL/kg body weight as per in-house solubility/suspendibility test results. Hence, 1% w/v Carboxymethyl Cellulose was used as vehicle for test item formulations. 1% w/v Carboxymethyl Cellulose is a routinely used and universally accepted vehicle of choice for the oral toxicity studies.

- Concentration in vehicle: G2 (11.1 mg/mL), G3 (33.3 mg/mL) and G4 (100.0 mg/mL)
- Amount of vehicle: 10 mL/kg
- Lot no.: SLCH1520
- Purity: Analytical grade.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Sampling and analysis of formulations were performed during first week and last week of the treatment. Approximately, 5 mL of samples were collected in duplicates from top, middle and bottom layers from low, mid and high dose concentrations and in duplicates from single layer from vehicle control. The prepared test item formulations were stirred using magnetic stirrer during sampling.

The collected samples were transferred to Analytical Chemistry department of Bioneeds India Private Limited for dose formulation analysis. One set of aliquots of each formulation was analyzed. The second aliquot was stored as a backup purpose at established stability conditions. The second set of samples was discarded, as the analysis results of first set of samples were within the limits.

Formulations were considered acceptable, as the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) is ≤10% (treatment day 1: mean recovery (%) is 99.15%, 99.17% and 99.06% with % RSD of 0.06, 0.06 and 0.03 for groups G2, G3 and G4, respectively; treatment day 38: mean recovery (%) is 100.10%, 100.16% and 100.09% with % RSD of 0.06, 0.05 and 0.09 for groups G2, G3 and G4, respectively).
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:1:2 ratio
- M/F ratio per cage: 1:2 ratio (one male and two females)
- Length of cohabitation: 14 days, Females not mated within 14 days of pairing with the first male were placed with a second proven male for one week
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as gestation day 0 of pregnancy
- Any other deviations from standard protocol: No
Duration of treatment / exposure:
The test item/vehicle was administered to respective group of female rats once daily from gestation day 5 to 19.
Frequency of treatment:
Once Daily
Duration of test:
15 days per animal starting from implantation to one day prior to scheduled delivery (GD 5 to GD 19)
Dose / conc.:
111 mg/kg bw/day
Remarks:
Low dose
Dose / conc.:
333 mg/kg bw/day
Remarks:
Mid dose
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
High dose
No. of animals per sex per dose:
25 presumed pregnant females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: There was no information or data available from repeated dose/reproducti on toxicity and teratology studies for agrocer red 112, hence The dose levels of 0, 111, 333 and 1000 mg/kg body weight/day were selected as vehicle control, low, mid and high dose groups, respectively as consultation with sponsor.

- Rationale for animal assignment: The body weight of mated females on each day of gestation day ‘0’ (GD 0) was recorded and arranged in the ascending order of their body weight. These mated females were evenly distributed to all the groups based on their body weights so as to maintain comparable mean body weights for all groups, permanent identification numbers were assigned.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- Cage side observations checked in table [No.1] were included.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on gestation days (GD) 0, 3, 5, 8, 11, 14, 17, 19 and on 20 (day of caesarean section).

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined as g food/kg body weight/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: All visceral organs, along with uterus, ovaries and thyroid along with parathyroid.

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [ half per litter]
- Skeletal examinations: Yes: [ half per litter]
- Head examinations: Yes: [ half per litter]
Statistics:
Parametric: One-way ANOVA with Dunnett’s post test:
Maternal body weight,Percent change in maternal body weight, Corrected body weight for maternal increase, Gravid uterus weight, Maternal Feed consumption,Absolute / relative thyroid weights, Mean fetal weight per dam, Mean fetal crown rump length per dam.

Non-Parametric: Kruskal-Wallis test:
No. of corpora lutea per dam, No. of implantations per dam, Litter size per dam, No. of live fetuses per dam, Percent of live fetuses per dam, No. of early/late resorptions per dam, Percent of early/late resorptions per dam, Sex ratio (m/f) per dam, Pre/Post implantation losses (%) per dam, Fetal external / visceral / skeletal anomalies per dam.

Frequencies Comparison: Cross Tabs - Chi-square test: No. of Pregnant / Non-pregnant females (Pregnancy status), No. of dams with / without live fetuses, No. of dams with / without dead fetuses, No. of dams with / without resorptions.
Indices:
Corrected Body Weight (g) = (Gestation day 20 body weight - Gestation day 5 body weight) - Gravid uterus weight
Pre-implantation Loss (%) = Total Number of Corpora lutea - Number of Implantation Sites / Total Number of Corpora lutea x 100
Post-implantation Loss (%) = Number of Implantation sites - Number of Viable fetuses / Number of Implantation sites x 100
Historical control data:
In-house historical data available for the same species and strain and referred to end points where required in the study report.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs of toxicity noted in any of the animals from vehicle control and all the tested dose groups throughout the treatment period
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no changes noted in mean gestation (maternal) body weight and percent change in mean gestation (maternal) body weight gain in all the tested dose groups when compared with the vehicle control group. There were no changes noted in mean body weight change corrected for gravid uterus weight in all the tested dose groups when compared with the vehicle control group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no changes noted in mean gestation (maternal) feed consumption in all the tested dose groups when compared with the vehicle control group.
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related changes noted for mean serum T3 levels in any of the tested dose groups when compared to vehicle control group. The noted statistically significant increase in mean serum T3 levels (ng/mL) in group G3 is considered as incidental and un-related to treatment with test item as the obtained mean values are within normal range of same species and strain.

There were no changes noted for mean serum T4 levels in any of the tested dose groups when compared to vehicle control group.

There were no changes noted for mean serum TSH levels in any of the tested dose groups when compared to vehicle control group.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The thyroid along with parathyroid was collected, preserved and weighed post fixation from all the dams of each dose group. There were no changes noted for mean absolute and relative weight for this organ in all the tested dose groups when compared to the vehicle control group.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross pathological changes noted in any of the animals from all the tested dose groups and vehicle control group during conduct of the necropsy.

Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item related histopathological findings noted in thyroid and parathyroid glands of all treated animals.
However, ultimobranchial cysts and ectopic thymus in thyroid gland were noted in this study. These changes were considered as incidental findings as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rats.
Histopathological findings: neoplastic:
no effects observed
Number of abortions:
no effects observed
Description (incidence and severity):
No abortions were noted
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The mean percentage of pre-implantation loss per litter was 5.77, 7.29, 10.86 and 12.44 for groups G1, G2, G3 and G4 respectively.
There were no changes noted and no statistically significant differences noted for percentage of pre-implantation loss per litter in all the teste dose groups.

The mean percentage of post-implantation loss per litter was 9.61, 9.96, 6.94 and 11.62 for groups G1, G2, G3 and G4 respectively.
There were no changes or no statistically significant differences noted for mean percentage of post-implantation loss per litter in all the tested dose groups when compared to the vehicle control group.
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
Two incidental findings with total implantation loss (with total empty implantation sites/early resorptions) during conduct of necropsy was noted in 1 out of 25 females from group G1 and 1 out of 25 females from group G3.
Early or late resorptions:
no effects observed
Description (incidence and severity):
The mean number of early resorptions per litter was 0.71, 0.71, 0.62 and 0.95 with a percentage of early resorptions was 6.53, 9.20, 6.62 and 8.43 for groups G1, G2, G3 and G4 respectively. There were no changes or no statistically significant differences noted for mean number and percentage of early resorptions per dam across dose groups when compared to the vehicle control group.

The mean number of late resorptions per litter was 0.14, 0.00, 0.00 and 0.00 and the percentage of late resorptions was 1.30, 0.00, 0.00 and 0.00 for groups G1, G2, G3 and G4 respectively. There were no changes or no statistically significant differences noted for mean number and percentage of late resorptions per dam across dose groups when compared to the vehicle control group.
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
The mean number of dead fetuses per litter was 0.05, 0.10, 0.05 and 0.00 with a percentage of 0.79, 0.84, 0.34 and 0.00. There were no statistically significant differences noted for mean number of dead fetuses in all the tested dose groups when compared to the vehicle control group.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
No early deliveries noted before caesarean sectioning.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): A total of 21 (out of 25), 21 (out of 25), 21 (out of 25) and 22 (out of 25) females from group G1 (0 mg/kg body weight/day), G2 (111 mg/kg body weight/day),
G3 (333 mg/kg body weight/day) and G4 (1000 mg/kg body weight/day) were found with implantations with live fetuses yielding to pregnancy with rates of 84%, 84%, 84% and 88% respectively.

No female was littered during gestation period in all the dose groups and control group before caesarean sectioning.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
A total of 21 (out of 25), 21 (out of 25), 21 (out of 25) and 22 (out of 25) females from group G1 (0 mg/kg body weight/day), G2 (111 mg/kg body weight/day),
G3 (333 mg/kg body weight/day) and G4 (1000 mg/kg body weight/day) were found with implantations with live fetuses yielding to pregnancy with rates of 84%, 84%, 84% and 88% respectively. One incidental finding with total implantation loss (with empty implantation sites) during conduct of necropsy was noted in 1 out of 25 females from group G1 and 1 out of 25 females from group G3.
Details on maternal toxic effects:
The oral administration of test item Agrocer Red 112 by gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 did not produce any indication of maternal toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
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
histopathology: non-neoplastic
maternal abnormalities
mortality
necropsy findings
number of abortions
organ weights and organ / body weight ratios
pre and post implantation loss
total litter losses by resorption
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
effects observed, non-treatment-related
Localisation:
amniotic fluid
cervix
mammary gland
ovary
oviduct
placenta
umbilical cord
uterus
vagina
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no changes noted in mean fetal weight of either sex across the dose groups when compared with the vehicle control group.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): There were no changes noted in mean fetal weight of either sex across the dose groups when compared with the vehicle control group.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex ratio per litter calculated as the mean of male/female fetuses per litter was 1.49, 1.26, 1.21 and 0.92 for groups G1, G2, G3 and G4 respectively. There were changes or no statistically significant differences noted for mean male/female sex ratio across the dose groups when compared to the control group.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The mean litter sizes, assessed as the total number of fetuses in uteri [live plus dead] per dam, was 10.90, 10.05, 10.33 and 9.41 for groups G1, G2, G3 and G4, respectively.
There were no changes or no statistically significant differences noted in all the tested dose groups when compared with vehicle control group for mean litter size.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related fetal structural and developmental external malformations or variations noted for any of the fetuses examined from all the tested dose group litters and also no statistically significant differences occurred.

However, some of the sporadic fetal external alterations were noted across the dose groups and vehicle control group such as Subcutaneous hemorrhagic spot beneath the skin on different regions of the body, Skin discoloured / pale coloured, Bent tail, Kinked tail, Hyperextension (unilateral) of forelimb, Hyperextension (unilateral) of hind limb and Protrusion of tongue.These developmental and structural alterations are considered incidental as these observations are comparable with the vehicle control group and or also these developmental alterations are common for this species and strain.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There was no test item-related fetal structural malformations or variations noted for any of the fetuses examined from all the tested dose group litters during skeletal examination. The noted statistically significant decrease in number of incidences of unossified sternebra no. 6 in group G3 when compared with vehicle control group is considered as incidental and toxicologically in-signficant. These structural and skeletal alterations are considered incidental as these observations are comparable with the vehicle control group and also these alterations are common for this species and strain. Also, the noted alterations in ossifications, i.e. delayed ossification, incomplete ossification, dumbbell shaped ossification from various regions from all the tested dose groups and control group are considered as transient findings and are reported to resolve shortly with the development of fetus (refer table no. 16 in study report).
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related fetal structural and developmental visceral/soft tissue malformations or variations noted for any of the fetuses examined from all the tested dose group litters and also no statistically significant differences occurred. However, some of the sporadic fetal visceral/soft tissue alterations were noted across the dose groups and vehicle control group such as Pale colored liver, Renal pelvis dilation (bilateral) of kidneys, Dilatation of Ureters (bilateral), Dilated lateral ventricles of brain and Nasal conchae enlarged.These developmental soft tissue alterations are considered incidental as these observations are comparable with the vehicle control group and also these developmental alterations are common for this species and strain.
Other effects:
no effects observed
Description (incidence and severity):
There were no changes noted in mean fetal anogenital distance/mean fetal anogenital distance ratio and mean fetal crown rump length per litter of either across the dose groups when compared with the vehicle control group.
Details on embryotoxic / teratogenic effects:
The oral administration of test item Agrocer Red 112 by gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 did not produce any indication of fetal/developmental toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day
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
external malformations
skeletal malformations
visceral malformations
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
effects observed, non-treatment-related
Localisation:
external: ear
external: eye
external: face
external: limb
external: paw
external: tail
external: trunk
external: anogenital distance
external: anus
external: genital tubercle
external: large intestine
external: thorax
external: umbilicus
external: pelvic region
skeletal: skull
skeletal: skull, fontanelles
skeletal: skull sutures
skeletal: clavicle
skeletal: scapule
skeletal: forelimb
skeletal: sternum
skeletal: rib
skeletal: supernumerary rib
skeletal: vertebra
skeletal: pelvic girdle
skeletal: hindlimb
visceral/soft tissue: integumentary
visceral/soft tissue: gastrointestinal tract
visceral/soft tissue: hepatobiliary
visceral/soft tissue: urinary
visceral/soft tissue: cardiovascular
visceral/soft tissue: heamatopoietic
visceral/soft tissue: immune system
visceral/soft tissue: musculoskeletal system
visceral/soft tissue: nervous system
visceral/soft tissue: central nervous system
visceral/soft tissue: peripheral nervous system
visceral/soft tissue: somatic nervous system
visceral/soft tissue: autonomic nervous system
visceral/soft tissue: endocrine system
visceral/soft tissue: respiratory system
visceral/soft tissue: male reproductive system
visceral/soft tissue: female reproductive system
visceral/soft tissue: eye
visceral/soft tissue: ear
Key result
Developmental effects observed:
no
Conclusions:
In conclusion, the oral administration of test item by gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 did not produce any indication of maternal and developmental toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.
Based on the obtained results from this pre-natal developmental toxicity study conducted in Sprague Dawley Rats, the NOAEL (No observed adverse effect level) of the test item for both maternal and fetal toxicity is estimated as 1000 mg/kg body weight/day under experimental conditions employed.
Executive summary:

The objective of this prenatal developmental toxicity study inSprague Dawley rats conducted as per OECD test guidelines 414,wasto provide general information concerning the effects of prenatal exposure of test item on the pregnant females and in the developing organisms. This study was also conducted to assess the maternal toxicity in pregnant females and also structural abnormalities or altered growth in the fetuses. The aim ofthis study was to estimate no-observed-adverse-effect-level (NOAEL) of the test itemfor both maternal as well as fetal end points.


A total of 100 mated female Sprague Dawley rats were distributed to four groups. Each group (G1, G2, G3 and G4) consisted of 25 presumed pregnant females. The animals in group G1 were administered with vehicle [1% w/v Carboxymethyl cellulose], the animals in groups G2, G3 and G4 were administered with test item at the dose levels of111, 333 and 1000mg/kg body weight/dayfor low, mid and high dose groups respectively. The vehicle and test item formulations were administered orally by gavage at the dose volume of 10 mL/kg body weight to mated and presumed-pregnant females from Gestation Day [GD] 5 to 19. The end points of assessment for dams were maternal death, maternal body weight and clinical signs of maternal toxicity and the end points of assessment for fetuses were fetal weights, growth and development, structural variations and malformations or altered growth.


Homogeneity and dose formulation analysis for dose concentration verification was performed during the first and last week of the treatment. The analysis results of test samples were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) is ≤10%.


A total of 21 (out of 25), 21 (out of 25), 21 (out of 25) and 22 (out of 25) females from group G1, G2, G3 and G4 were found with implantations and live fetuses yielding to pregnancy with rates of 84%, 84%, 84% and 88% respectively. In groups G1 and G3, 1 out of 25 females each was noted with total implantation loss with no evidence of live fetuses.


All the dams from each group were observed for clinical signs of toxicity once daily, for mortalities twice daily during the experimental period. The body weight was recorded on Gestation Day (GD) 0, 3, 5, 8, 11, 14, 17, 19 and on 20 (day of caesarean section). The feed consumption was measured from GD 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17, 17 to 19 and 19 to 20. All the dams were euthanized on GD 20 by exposing to CO2asphyxiation and subjected to detailed gross pathological examination. All the females were observed for status of pregnancy and the gravid uterus weight for all the dams was recorded on the day of caesarean section. Each dam was observed for number of live fetuses, litter size, sex ratio, number of implantation sites and number of resorptions. The ovaries of all the dams were observed for number of corpora lutea. The pre-and post-implantation losses per dam were calculated based on number of corpora lutea and number of implantation sites. The weight of thyroid along with the parathyroid was recorded post-fixation for all the group animals. The assessment of thyroid hormones such as, thyroxine (T4), triiodothyronine (T3) and thyroid stimulating hormone (TSH) was conducted for all the group dams. All the dams from each group were evaluated for histopathology of thyroid along with the parathyroid.


All the fetuses collected from each litter were weighed and measured for its crown rump length and anogenital distance. The mean fetal weight, mean crown rump length, and mean anogenital distance measurement/ratio per litter was calculated. All the fetuses were subjected to external examination on the day of caesarean section. All the even numbered fetuses from each litter were subjected to fresh visceral (soft tissue) examination and fixed head sections examination. All the odd numbered fetuses from each litter were stained with Alizarin Red S stain and subjected to skeletal examination.


For maternal toxicity assessment, there were no clinical signs of toxicity and no mortality/morbidity noted in any of the dams in all the tested dose groups. There were no differences noted between the groups in mean maternal body weight, percent change in maternal body weight gain, body weight corrected for maternal increase and maternal feed consumption. There were no effects noted in mean number of live fetuses per litter, litter size, sex ratio, number of corpora lutea, number of implantation sites, number of incidences of resorptions, pre-and post-implantation losses per dam from all the tested dose groups. There were no differences noted between the dose groups in mean gravid uterus weight. The mean absolute and relative thyroid along with the parathyroid weight did not reveal any changes when weighed post-fixation and no test item-related changes were noted in mean thyroid hormonal levels (T3, T4 and TSH). There were no gross pathological changes noted in any of the dams during caesarean section and no test item-related microscopic changes were noted in thyroid along with the parathyroid of all dams during histopathological examination.


For fetal (pre-natal developmental) toxicity assessment, there were no test item-related changes noted in mean fetal weight, mean fetal crown rump length and mean fetal anogenital distance measurement / ratio per litter in either sex in all the tested dose groups. There was no test item-related fetal developmental and or structural alterations noted from all the tested dose group litters when subjected to fetal external, visceral and skeletal examinations. The observed fetal external/visceral/skeletal developmental variations and or malformations are considered as incidental and unrelated to treatment as these findings occurred infrequently or at a frequency similar to the vehicle control group and did not occur in a dose-dependent manner. Also, the observed mean litter/fetal proportions were within the in-house historical control range of this species and strain.

Endpoint:
developmental toxicity
Remarks:
Prenatal Developmental Toxicity Study, Adopted on 25 June 2018
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
27 July 2021 to 25 March 2022
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 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted on 25 June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Clariant Plastics & Coatings Deutschland
- Expiration date of the batch: 18-November-2027
- Purity test date: 95.5% (w/w)


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Stability under test conditions: for 6 hours and 48 hours at room temperature
- Solubility and stability of the test substance in the solvent/vehicle: uniformly suspended in 1% w/v Carboxymethyl cellulose (CMC) at the concentration of 100 mg/mL, the highest dose concentration.


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: formulation using 1% w/v Carboxymethyl cellulose (CMC) at the concentration 11.1, 33.3 and 100.0 of G2, G3 and G4 respectively
- Final preparation of a solid: formulation in vehicle


Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Adita Biosys Private Limited, Plot No. SPL 26, 2nd Stage, KSSIDC, Industrial Estate, Antharasanahalli, Tumakuru, Karnataka 572106. CPCSEA Registration No. 1868/PO/RcBt/S/16/CPCSEA
- Age at study initiation: 4 to 5 Months
- Weight at study initiation: Males: 2.15468 kg to 2.64927 kg;
Females: 2.00849 kg to 2.28642 kg
- Housing: stainless steel wire mesh cage (Size: L 24 x B 18 x H 18 inches)
- Diet (e.g. ad libitum): Altromin maintenance diet for rabbits (manufactured by Altromin Spezialfutter GmbH & Co. KG)
- Water (e.g. ad libitum): Deep bore-well water passed through reverse osmosis unit
- Acclimation period: (Minimum of five days) Start: 05 August 2021; End: 10 August 2021

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.8 to 20.9oC
- Humidity (%): 44 to 61%
- Air changes (per hr): 12 hours fluorescent light and 12 hours dark cycle


IN-LIFE DATES: From: 05 August 2021 To: 05 October 2021
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1% w/v in water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Justification for use and choice of vehicle: uniformly suspended in 1% w/v Carboxymethyl cellulose (CMC) at the concentration of 100 mg/mL, the highest dose concentration selected for the study considering the dose volume of 10 mL/kg body weight as per in-house suspendibility test results.
Hence, 1% w/v Carboxymethyl cellulose was used as vehicle for test item formulations. Carboxymethyl cellulose is a routinely used vehicle of choice for the oral toxicity studies.

- Concentration in vehicle: G2 (11.1 mg/mL), G3 (33.3 mg/mL) and G4 (100.0 mg/mL)
- Amount of vehicle (if gavage): 10 mL/kg
body weight
- Lot no. (if required): SLCH1520
- Purity: Analytical grade
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Sampling and analysis of formulations was performed during first week and last week of the treatment. Approximately, 5 mL of samples was collected in duplicates from top, middle and bottom layers from low, mid and high dose concentrations and in duplicates from single layer from vehicle control. The prepared test item formulations were stirred using magnetic stirrer during sampling.The collected samples were transferred to Analytical Chemistry department of Bioneeds India Private Limited for dose formulation analysis. One set of aliquots of each formulation was analyzed. The second aliquot was stored as a backup purpose at established stability conditions. The second set of samples were discarded, as the analysis results of first set of samples were within the limits. Formulations were considered acceptable, as the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) is ≤10%.
Details on mating procedure:
- M/F ratio per cage: 1:1 (M:F)
- Length of cohabitation: Until visual confirmation of mating
- Verification of same strain and source of both sexes: Yes
- Proof of pregnancy: [visual observation] referred to as [day 0] of pregnancy
- Any other deviations from standard protocol: no
Duration of treatment / exposure:
The test item/vehicle was administered by oral (gavage) route to respective group of animals once daily from gestation day (GD) 6 to 28.
Frequency of treatment:
once daily
Duration of test:
22 days per animal starting from implantation to one day prior to scheduled delivery (GD 6 to GD 28)
Dose / conc.:
111 mg/kg bw/day
Remarks:
Low dose
Dose / conc.:
333 mg/kg bw/day
Remarks:
Mid dose
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
High dose
No. of animals per sex per dose:
25 presumed pregnant females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: There was no information or data available from repeated dose/reproduction toxicity and teratology studies for agrocer red 112, hence The dose levels of 0, 111, 333 and 1000 mg/kg body weight/day were selected as vehicle control, low, mid and high dose groups, respectively as consultation with sponsor.
- Rationale for animal assignment (if not random):The body weight of mated females on each day of gestation day ‘0’ (GD 0) was recorded and arranged in the ascending order of their body weight. These mated females were evenly distributed to all the groups based on their body weights so as to maintain comparable mean body weights for all groups, permanent identification numbers were assigned.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- Cage side observations checked in table [No.1] were included.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on GD 0, 3, 6, 9, 12, 15, 18, 21, 24, 26, 28 and on 29 (day of caesarean section).

FOOD CONSUMPTION : Yes
- Food consumption for each animal determined as g food/kg body weight/day: Yes


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: all visceral organs along with uterus and ovaries

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [half per litter]
Statistics:
Parametric: One-way ANOVA with Dunnett’s post test:
Maternal body weight,Percent change in maternal body weight, Corrected body weight for maternal increase, Gravid uterus weight, Maternal Feed consumption,
Absolute / relative thyroid weights, Mean fetal weight per doe, Mean fetal crown rump length per doe

Non-Parametric: Kruskal-Wallis test:
No. of corpora lutea per doe, No. of implantations per doe, Litter size per doe, No. of live fetuses per doe, Percent of live fetuses per doe, No. of early/late resorptions per doe,
Percent of early/late resorptions per doe, Sex ratio (m/f) per doe, Pre/Post implantation losses (%) per doe, Fetal external / visceral / skeletal anomalies per doe.

Frequencies Comparison: Cross Tabs - Chi-square test:
No. of Pregnant / Non-pregnant females (Pregnancy status), No. of does with / without live fetuses, No. of does with / without dead fetuses, No. of does with / without resorptions

Indices:
Corrected Body Weight (g) = (Gestation day 29 body weight - Gestation day 6 body weight) - Gravid uterus weight
Pre-implantation Loss (%) = Total Number of Corpora lutea - Number of Implantation Sites / Total Number of Corpora lutea x 100
Post-implantation Loss (%) = Number of Implantation sites - Number of Viable fetuses / Number of Implantation sites x 100
Historical control data:
All the obtained Data within the historical control range
Clinical signs:
no effects observed
Description (incidence and severity):
There were no test item related clinical signs of toxicity noted at all the tested dose groups and vehicle control group animals during the experimental period.
Mortality:
no mortality observed
Description (incidence):
There were no morbidity/mortality noted at all the tested dose groups and vehicle control group animals during the experimental period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no changes noted in mean gestation (maternal) body weight and percent change in mean gestation (maternal) body weight gain in all the tested dose groups when compared with the vehicle control group.
There were no changes noted in mean body weight change corrected for gravid uterus weight in all the tested dose groups when compared with the vehicle control group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no changes noted in mean gestation (maternal) feed consumption in all the tested dose groups when compared with the vehicle control group.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The thyroid along with parathyroid was collected, preserved and weighed post fixation from all the does of each dose group. There were no changes noted for mean absolute and relative weight for this organ in all the tested dose groups when compared to the vehicle control group.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related histopathological findings in thyroid and parathyroid glands of all treated animals.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related histopathological findings in thyroid and parathyroid glands of all treated animals.
However, ultimobranchial cysts in thyroid gland was noted in this study. These changes were considered as incidental findings as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rabbits (Elizabeth McInnes, 2012).
Details on results:
The oral administration of test item Agrocer Red 112 by gavage to presumed pregnant female New Zealand White Rabbits from Gestation Day 6 to 28 did not produce any indication of maternal toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.

Number of abortions:
no effects observed
Description (incidence and severity):
No abortions were noted
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
The mean percentage of pre-implantation loss per litter was 13.78, 13.10, 13.58 and 15.76 for groups G1, G2, G3 and G4 respectively.
There were no statistically significant differences noted for percentage of pre-implantation loss per litter at all the tested dose groups when compared to the vehicle control group.
The mean percentage of post-implantation loss per litter was 0.00, 0.00, 0.00 and 4.05 for groups G1, G2, G3 and G4 respectively.
There were no statistically significant differences noted for percentage of post-implantation loss per litter in all the tested dose groups when compared to the vehicle control group.
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
One incidental finding with total implantation loss (with empty implantation sites) during conduct of necropsy was noted in 1 out of 25 females from group G2.
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
The mean number of early resorptions per litter was 0.00, 0.00, 0.00 and 0.10 and the percentage of early resorptions was 0.00, 0.00, 0.00 and 3.57 for groups G1, G2, G3 and G4 respectively. There were no statistically significant differences in the number and percentage of early resorptions per doe across dose groups when compared to the vehicle control group.
The mean number of late resorptions per litter was 0.00, 0.00, 0.00 and 0.05 and the percentage of late resorptions was 0.00, 0.00, 0.00 and 0.48 for groups G1, G2, G3 and G4 respectively. There were no statistically significant differences in the number and percentage of late resorptions per doe across dose groups when compared to the vehicle control group.
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no dead fetuses noted in any of the litters from all the tested dose and vehicle control groups during caesarean section.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
No early deliveries noted.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): A total of 21, 20, 21 and 21 females from groups G1 (0 mg/kg body weight/day),
G2 (111 mg/kg body weight/day), G3 (333 mg/kg body weight/day) and G4 (1000 mg/kg body weight/day) were found with implantations yielding to pregnancy with rates of 84%, 80%, 84% and 84% respectively.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
A total of 21(out of 25), 20(out of 25), 21(out of 25) and 21(out of 25) females from groups G1 (0 mg/kg body weight/day),
G2 (111 mg/kg body weight/day), G3 (333 mg/kg body weight/day) and G4 (1000 mg/kg body weight/day) were found with implantations yielding to pregnancy with rates of 84%, 80%, 84% and 84% respectively.One incidental finding with total implantation loss (with empty implantation sites) during conduct of necropsy was noted in 1 out of 25 females from group G2.
Details on maternal toxic effects:
The oral administration of Agrocer Red 112 by gavage to presumed pregnant female New Zealand White Rabbits from Gestation Day 6 to 28 did not produce any indication of maternal toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
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
histopathology: non-neoplastic
maternal abnormalities
mortality
necropsy findings
number of abortions
organ weights and organ / body weight ratios
pre and post implantation loss
total litter losses by resorption
Key result
Abnormalities:
effects observed, non-treatment-related
Localisation:
amniotic fluid
cervix
mammary gland
ovary
oviduct
placenta
umbilical cord
uterus
vagina
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no changes noted in mean fetal weight of either sex across the dose groups when compared with the vehicle control group.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): No such incidences noted in any of the tested dose groups.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No such incidences noted in any of the tested dose groups.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No such incidences noted in any of the tested dose groups.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no changes or no statistically significant differences noted in all the tested dose groups
when compared with vehicle control group for mean litter size.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related fetal external malformations or developmental variations for any of the fetuses examined from all the tested dose group litters.
General / developmental variations such as subcutaneous hemorrhagic spot/s on different regions of the body and noted external malformations such as hyperextension of forelimb and hyperextension of hindlimb across the tested dose group litters are considered incidental as these incidences are comparable with the vehicle control group and also these developmental alterations are common for this species and strain. Also, no remarkable differences or statistically significant changes noted for these alterations in any of the tested dose groups when compared with vehicle control group (refer table no. 10 in study report).
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There was no test item-related fetal visceral/soft tissue malformations or developmental variations for any of the fetuses examined from all the tested dose group litters.
These noted skeletal developmental variations and skeletal malformations are considered as incidental and un-related to treatment as these findings occurred infrequently or at a frequency similar to the vehicle control group and did not occur in a dose-dependent manner. Also, the occurred mean litter/fetal proportions were within the in-house historical control range of this species and strain (refer table no. 12 in study report).
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There was no test item-related fetal visceral/soft tissue malformations or developmental variations for any of the fetuses examined from all the tested dose group litters.
Developmental variations such as discoloured liver/thymus/lungs/spleen, dilatation of renal pelvis and dilatation of ureters across the tested dose group litters are considered incidental as these incidences are comparable with the vehicle control group and also these developmental variations are common for this species and strain.
developmental malformations lateral/third ventricular dilatation of brain, retinal fold, misshapen liver lobes and retrocaval ureters across the tested dose group litters are considered incidental as these incidences are comparable with the vehicle control group and also these developmental alterations are common for this species and strain(refer table no. 11 in study report).
Other effects:
no effects observed
Description (incidence and severity):
There were no changes noted in mean fetal crown rump length per litter of either across the dose groups when compared with the vehicle control group.
Details on embryotoxic / teratogenic effects:
oral administration of test item Agrocer Red 112 by gavage to presumed pregnant female New Zealand White Rabbits from Gestation Day 6 to 28 did not produce any indication of developmental toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day
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
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
effects observed, non-treatment-related
Localisation:
external: cranium
external: ear
external: eye
external: face
external: limb
external: paw
external: tail
external: trunk
external: anogenital distance
external: anus
external: genital tubercle
external: large intestine
external: thorax
external: umbilicus
external: pelvic region
skeletal: skull
skeletal: skull, fontanelles
skeletal: skull sutures
skeletal: clavicle
skeletal: scapule
skeletal: forelimb
skeletal: sternum
skeletal: rib
skeletal: supernumerary rib
skeletal: vertebra
skeletal: pelvic girdle
skeletal: hindlimb
visceral/soft tissue: integumentary
visceral/soft tissue: gastrointestinal tract
visceral/soft tissue: hepatobiliary
visceral/soft tissue: urinary
visceral/soft tissue: cardiovascular
visceral/soft tissue: heamatopoietic
visceral/soft tissue: immune system
visceral/soft tissue: musculoskeletal system
visceral/soft tissue: nervous system
visceral/soft tissue: central nervous system
visceral/soft tissue: peripheral nervous system
visceral/soft tissue: somatic nervous system
visceral/soft tissue: autonomic nervous system
visceral/soft tissue: endocrine system
visceral/soft tissue: respiratory system
visceral/soft tissue: male reproductive system
visceral/soft tissue: female reproductive system
visceral/soft tissue: eye
visceral/soft tissue: ear
Key result
Developmental effects observed:
no
Conclusions:
In conclusion, the oral administration of test item by gavage to presumed pregnant female New Zealand White Rabbits from Gestation Day 6 to 28 did not produce any indication of maternal and developmental toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.
Based on the obtained results from this pre-natal developmental toxicity study conducted in New Zealand White Rabbits, the NOAEL (No observed adverse effect level) for both maternal and fetal toxicity is estimated as 1000 mg/kg body weight/day under experimental conditions employed.
Executive summary:

The objective of this study was to provide general information concerning the effects of prenatal exposure of test item on the pregnant rabbits and in the developing organisms; this included the assessment of maternal toxicity as well as death, structural abnormalities, or altered growth in the fetuses.


A total of 100 mated female New Zealand White rabbits distributed to four groups. Each group (G1, G2, G3 and G4) consisted of 25 presumed pregnant rabbits. The animals in group G1 were administered with vehicle [1% w/v Carboxymethyl cellulose], the animals in groups G2, G3 and G4 were administered with test item at the dose levels of 111, 333 and 1000 mg/kg body weight for low, mid and high dose groups respectively. The vehicle and test item formulations were administered orally by gavage at the dose volume of 10 mL/kg body weight to mated and presumed-pregnant females from Gestation Day [GD] 6 to 28. The end points of assessment for does were maternal death, maternal body weight and clinical signs of maternal toxicity and the end points of assessment for fetuses were fetal weights, growth and development, structural variations and malformations or altered growth.


Homogeneity and dose formulation analysis for dose concentration verification were performed during the first and last week of treatment. The prepared formulations were considered acceptable and the mean results were within the range of 85 to 115% of nominal concentration and the relative standard deviation (% RSD) is less than 10%(mean recovery was between 98.62% and 99.97% at all measurements).


 A total of 21, 20, 21 and 21 females from group G1, G2, G3 and G4 were found with implantations yielding pregnancy rates of 84%, 80%, 84% and 84% respectively.


All the animals from each group were observed for clinical signs of toxicity once daily, for mortalities twice daily during the experimental period. The body weight was recorded on Gestation Day (GD) 0, 3, 6, 9, 12, 15, 18, 21, 24, 26, 28 and on 29 (day of caesarean section). The feed consumption was measured from GD 0 to 3, 3 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18, 18 to 21, 21 to 24, 24 to 26, 26 to 28 and 28 to 29. All the animals were euthanized on gestation Day 29 by intravenous injection of sodium thiopentone and subjected to detailed gross pathological examination. All the females were observed for status of pregnancy and the gravid uterus weight for all the does was recorded on the day of caesarean section. Each doe was observed for number of live or dead fetuses, litter size, sex ratio, number of implantation sites and number of resorptions. The ovaries of all the does were observed for number of corpora lutea. The pre-and post-implantation losses per doe were calculated based on number of corpora lutea and number of implantation sites. The weight of thyroid along with the parathyroid was recorded post-fixation for all the animals. All the does were evaluated for histopathology of thyroid along with the parathyroid.


All the fetuses collected from each litter were weighed and measured for their crown rump length. The mean fetal weight and mean crown rump length per litter was calculated. All the fetuses were subjected to external examination on the day of caesarean section. All the fetuses from each litter were subjected for fresh soft tissue (visceral) examination and fixed head sections examination, except the heads of fetuses which were allotted for skeletal evaluation. The bodies of fetuses without heads and all other intact fetuses from each litter were double stained with alcian blue for cartilage and Alizarin Red S for bones and subjected to skeletal examination.


For maternal toxicity assessment, there were no clinical signs of toxicity and no mortality/morbidity noted during the experimental period at all the tested dose and vehicle control group animals. No changes were noted in mean maternal body weight, percent change in mean maternal body weight gain and mean maternal feed consumption at all the tested dose groups. There were no gross pathological changes noted during caesarean section of all the tested dose and vehicle control group animals. There were no effects noted in pregnancy rate and mean gravid uterus weight in all the tested dose groups. There were no effects noted in mean number of live fetuses, mean litter size, mean sex ratio, mean number of implantation sites, mean number of resorptions, mean number of corpora lutea at all the tested dose groups. The mean pre- and post-implantation losses per doe were unaffected at all the tested dose groups. The mean absolute and relative thyroid along with the parathyroid weight did not reveal any changes when weighed post-fixation at all the tested dose groups. No test item-related microscopic changes were noted in thyroid along with the parathyroid of all the does during histopathological examination from all the tested dose groups.


For fetal (pre-natal developmental) toxicity assessment, there were no test item-related changes noted in mean fetal weight and mean fetal crown rump length per litter in either sex at all the tested dose groups. There was no test item-related fetal developmental and or structural alterations noted from all the tested dose group litters when subjected to fetal external, visceral and skeletal examinations. The observed fetal external/visceral/skeletal developmental variations and or malformations are considered as incidental and unrelated to treatment as these findings occurred infrequently or at a frequency similar to the vehicle control group and did not occur in a dose-dependant manner.

Endpoint:
developmental toxicity
Remarks:
Prenatal Developmental Toxicity Study, Adopted on 25 June 2018
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 July 2021 to 25 March 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted on 25 June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Clariant Plastics & Coatings Deutschland
- Expiration date of the batch: 18-November-2027
- Purity test date: 95.5% (w/w)


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (21 to 29°C)
- Stability under test conditions: for 6 hours and 48 hours at room temperature
- Solubility and stability of the test substance in the solvent/vehicle: uniformly suspended in 1% w/v Carboxymethyl cellulose (CMC) at the concentration of 100 mg/mL, the highest dose concentration.


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: formulation using 1% w/v Carboxymethyl cellulose (CMC) at the concentration 11.1, 33.3 and 100.0 of G2, G3 and G4 respectively
- Final preparation of a solid: formulation in vehicle


Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Adita Biosys Private Limited, Plot No. SPL 26, 2nd Stage, KSSIDC, Industrial Estate, Antharasanahalli, Tumakuru, Karnataka 572106. CPCSEA Registration No. 1868/PO/RcBt/S/16/CPCSEA
- Age at study initiation: 4 to 5 Months
- Weight at study initiation: Males: 2.15468 kg to 2.64927 kg;
Females: 2.00849 kg to 2.28642 kg
- Housing: stainless steel wire mesh cage (Size: L 24 x B 18 x H 18 inches)
- Diet (e.g. ad libitum): Altromin maintenance diet for rabbits (manufactured by Altromin Spezialfutter GmbH & Co. KG)
- Water (e.g. ad libitum): Deep bore-well water passed through reverse osmosis unit
- Acclimation period: (Minimum of five days) Start: 05 August 2021; End: 10 August 2021

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.8 to 20.9oC
- Humidity (%): 44 to 61%
- Air changes (per hr): 12 hours fluorescent light and 12 hours dark cycle


IN-LIFE DATES: From: 05 August 2021 To: 05 October 2021
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1% w/v in water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Justification for use and choice of vehicle: uniformly suspended in 1% w/v Carboxymethyl cellulose (CMC) at the concentration of 100 mg/mL, the highest dose concentration selected for the study considering the dose volume of 10 mL/kg body weight as per in-house suspendibility test results.
Hence, 1% w/v Carboxymethyl cellulose was used as vehicle for test item formulations. Carboxymethyl cellulose is a routinely used vehicle of choice for the oral toxicity studies.

- Concentration in vehicle: G2 (11.1 mg/mL), G3 (33.3 mg/mL) and G4 (100.0 mg/mL)
- Amount of vehicle (if gavage): 10 mL/kg
body weight
- Lot no. (if required): SLCH1520
- Purity: Analytical grade
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Sampling and analysis of formulations was performed during first week and last week of the treatment. Approximately, 5 mL of samples was collected in duplicates from top, middle and bottom layers from low, mid and high dose concentrations and in duplicates from single layer from vehicle control. The prepared test item formulations were stirred using magnetic stirrer during sampling.The collected samples were transferred to Analytical Chemistry department of Bioneeds India Private Limited for dose formulation analysis. One set of aliquots of each formulation was analyzed. The second aliquot was stored as a backup purpose at established stability conditions. The second set of samples were discarded, as the analysis results of first set of samples were within the limits. Formulations were considered acceptable, as the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) is ≤10%.
Details on mating procedure:
- M/F ratio per cage: 1:1 (M:F)
- Length of cohabitation: Until visual confirmation of mating
- Verification of same strain and source of both sexes: Yes
- Proof of pregnancy: [visual observation] referred to as [day 0] of pregnancy
- Any other deviations from standard protocol: no
Duration of treatment / exposure:
The test item/vehicle was administered by oral (gavage) route to respective group of animals once daily from gestation day (GD) 6 to 28.
Frequency of treatment:
once daily
Duration of test:
22 days per animal starting from implantation to one day prior to scheduled delivery (GD 6 to GD 28)
Dose / conc.:
111 mg/kg bw/day
Remarks:
Low dose
Dose / conc.:
333 mg/kg bw/day
Remarks:
Mid dose
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
High dose
No. of animals per sex per dose:
25 presumed pregnant females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: There was no information or data available from repeated dose/reproduction toxicity and teratology studies for agrocer red 112, hence The dose levels of 0, 111, 333 and 1000 mg/kg body weight/day were selected as vehicle control, low, mid and high dose groups, respectively as consultation with sponsor.
- Rationale for animal assignment (if not random):The body weight of mated females on each day of gestation day ‘0’ (GD 0) was recorded and arranged in the ascending order of their body weight. These mated females were evenly distributed to all the groups based on their body weights so as to maintain comparable mean body weights for all groups, permanent identification numbers were assigned.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- Cage side observations checked in table [No.1] were included.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on GD 0, 3, 6, 9, 12, 15, 18, 21, 24, 26, 28 and on 29 (day of caesarean section).

FOOD CONSUMPTION : Yes
- Food consumption for each animal determined as g food/kg body weight/day: Yes


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: all visceral organs along with uterus and ovaries

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [half per litter]
Statistics:
Parametric: One-way ANOVA with Dunnett’s post test:
Maternal body weight,Percent change in maternal body weight, Corrected body weight for maternal increase, Gravid uterus weight, Maternal Feed consumption,
Absolute / relative thyroid weights, Mean fetal weight per doe, Mean fetal crown rump length per doe

Non-Parametric: Kruskal-Wallis test:
No. of corpora lutea per doe, No. of implantations per doe, Litter size per doe, No. of live fetuses per doe, Percent of live fetuses per doe, No. of early/late resorptions per doe,
Percent of early/late resorptions per doe, Sex ratio (m/f) per doe, Pre/Post implantation losses (%) per doe, Fetal external / visceral / skeletal anomalies per doe.

Frequencies Comparison: Cross Tabs - Chi-square test:
No. of Pregnant / Non-pregnant females (Pregnancy status), No. of does with / without live fetuses, No. of does with / without dead fetuses, No. of does with / without resorptions

Indices:
Corrected Body Weight (g) = (Gestation day 29 body weight - Gestation day 6 body weight) - Gravid uterus weight
Pre-implantation Loss (%) = Total Number of Corpora lutea - Number of Implantation Sites / Total Number of Corpora lutea x 100
Post-implantation Loss (%) = Number of Implantation sites - Number of Viable fetuses / Number of Implantation sites x 100
Historical control data:
All the obtained Data within the historical control range
Clinical signs:
no effects observed
Description (incidence and severity):
There were no test item related clinical signs of toxicity noted at all the tested dose groups and vehicle control group animals during the experimental period.
Mortality:
no mortality observed
Description (incidence):
There were no morbidity/mortality noted at all the tested dose groups and vehicle control group animals during the experimental period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no changes noted in mean gestation (maternal) body weight and percent change in mean gestation (maternal) body weight gain in all the tested dose groups when compared with the vehicle control group.
There were no changes noted in mean body weight change corrected for gravid uterus weight in all the tested dose groups when compared with the vehicle control group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no changes noted in mean gestation (maternal) feed consumption in all the tested dose groups when compared with the vehicle control group.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The thyroid along with parathyroid was collected, preserved and weighed post fixation from all the does of each dose group. There were no changes noted for mean absolute and relative weight for this organ in all the tested dose groups when compared to the vehicle control group.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related histopathological findings in thyroid and parathyroid glands of all treated animals.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related histopathological findings in thyroid and parathyroid glands of all treated animals.
However, ultimobranchial cysts in thyroid gland was noted in this study. These changes were considered as incidental findings as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rabbits (Elizabeth McInnes, 2012).
Details on results:
The oral administration of test item Agrocer Red 112 by gavage to presumed pregnant female New Zealand White Rabbits from Gestation Day 6 to 28 did not produce any indication of maternal toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.

Number of abortions:
no effects observed
Description (incidence and severity):
No abortions were noted
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
The mean percentage of pre-implantation loss per litter was 13.78, 13.10, 13.58 and 15.76 for groups G1, G2, G3 and G4 respectively.
There were no statistically significant differences noted for percentage of pre-implantation loss per litter at all the tested dose groups when compared to the vehicle control group.
The mean percentage of post-implantation loss per litter was 0.00, 0.00, 0.00 and 4.05 for groups G1, G2, G3 and G4 respectively.
There were no statistically significant differences noted for percentage of post-implantation loss per litter in all the tested dose groups when compared to the vehicle control group.
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
One incidental finding with total implantation loss (with empty implantation sites) during conduct of necropsy was noted in 1 out of 25 females from group G2.
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
The mean number of early resorptions per litter was 0.00, 0.00, 0.00 and 0.10 and the percentage of early resorptions was 0.00, 0.00, 0.00 and 3.57 for groups G1, G2, G3 and G4 respectively. There were no statistically significant differences in the number and percentage of early resorptions per doe across dose groups when compared to the vehicle control group.
The mean number of late resorptions per litter was 0.00, 0.00, 0.00 and 0.05 and the percentage of late resorptions was 0.00, 0.00, 0.00 and 0.48 for groups G1, G2, G3 and G4 respectively. There were no statistically significant differences in the number and percentage of late resorptions per doe across dose groups when compared to the vehicle control group.
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no dead fetuses noted in any of the litters from all the tested dose and vehicle control groups during caesarean section.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
No early deliveries noted.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): A total of 21, 20, 21 and 21 females from groups G1 (0 mg/kg body weight/day),
G2 (111 mg/kg body weight/day), G3 (333 mg/kg body weight/day) and G4 (1000 mg/kg body weight/day) were found with implantations yielding to pregnancy with rates of 84%, 80%, 84% and 84% respectively.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
A total of 21(out of 25), 20(out of 25), 21(out of 25) and 21(out of 25) females from groups G1 (0 mg/kg body weight/day),
G2 (111 mg/kg body weight/day), G3 (333 mg/kg body weight/day) and G4 (1000 mg/kg body weight/day) were found with implantations yielding to pregnancy with rates of 84%, 80%, 84% and 84% respectively.One incidental finding with total implantation loss (with empty implantation sites) during conduct of necropsy was noted in 1 out of 25 females from group G2.
Details on maternal toxic effects:
The oral administration of Agrocer Red 112 by gavage to presumed pregnant female New Zealand White Rabbits from Gestation Day 6 to 28 did not produce any indication of maternal toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
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
histopathology: non-neoplastic
maternal abnormalities
mortality
necropsy findings
number of abortions
organ weights and organ / body weight ratios
pre and post implantation loss
total litter losses by resorption
Key result
Abnormalities:
effects observed, non-treatment-related
Localisation:
amniotic fluid
cervix
mammary gland
ovary
oviduct
placenta
umbilical cord
uterus
vagina
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no changes noted in mean fetal weight of either sex across the dose groups when compared with the vehicle control group.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): No such incidences noted in any of the tested dose groups.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
No such incidences noted in any of the tested dose groups.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No such incidences noted in any of the tested dose groups.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no changes or no statistically significant differences noted in all the tested dose groups
when compared with vehicle control group for mean litter size.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related fetal external malformations or developmental variations for any of the fetuses examined from all the tested dose group litters.
General / developmental variations such as subcutaneous hemorrhagic spot/s on different regions of the body and noted external malformations such as hyperextension of forelimb and hyperextension of hindlimb across the tested dose group litters are considered incidental as these incidences are comparable with the vehicle control group and also these developmental alterations are common for this species and strain. Also, no remarkable differences or statistically significant changes noted for these alterations in any of the tested dose groups when compared with vehicle control group (refer table no. 10 in study report).
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There was no test item-related fetal visceral/soft tissue malformations or developmental variations for any of the fetuses examined from all the tested dose group litters.
These noted skeletal developmental variations and skeletal malformations are considered as incidental and un-related to treatment as these findings occurred infrequently or at a frequency similar to the vehicle control group and did not occur in a dose-dependent manner. Also, the occurred mean litter/fetal proportions were within the in-house historical control range of this species and strain (refer table no. 12 in study report).
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There was no test item-related fetal visceral/soft tissue malformations or developmental variations for any of the fetuses examined from all the tested dose group litters.
Developmental variations such as discoloured liver/thymus/lungs/spleen, dilatation of renal pelvis and dilatation of ureters across the tested dose group litters are considered incidental as these incidences are comparable with the vehicle control group and also these developmental variations are common for this species and strain.
developmental malformations lateral/third ventricular dilatation of brain, retinal fold, misshapen liver lobes and retrocaval ureters across the tested dose group litters are considered incidental as these incidences are comparable with the vehicle control group and also these developmental alterations are common for this species and strain(refer table no. 11 in study report).
Other effects:
no effects observed
Description (incidence and severity):
There were no changes noted in mean fetal crown rump length per litter of either across the dose groups when compared with the vehicle control group.
Details on embryotoxic / teratogenic effects:
oral administration of test item Agrocer Red 112 by gavage to presumed pregnant female New Zealand White Rabbits from Gestation Day 6 to 28 did not produce any indication of developmental toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day
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
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
effects observed, non-treatment-related
Localisation:
external: cranium
external: ear
external: eye
external: face
external: limb
external: paw
external: tail
external: trunk
external: anogenital distance
external: anus
external: genital tubercle
external: large intestine
external: thorax
external: umbilicus
external: pelvic region
skeletal: skull
skeletal: skull, fontanelles
skeletal: skull sutures
skeletal: clavicle
skeletal: scapule
skeletal: forelimb
skeletal: sternum
skeletal: rib
skeletal: supernumerary rib
skeletal: vertebra
skeletal: pelvic girdle
skeletal: hindlimb
visceral/soft tissue: integumentary
visceral/soft tissue: gastrointestinal tract
visceral/soft tissue: hepatobiliary
visceral/soft tissue: urinary
visceral/soft tissue: cardiovascular
visceral/soft tissue: heamatopoietic
visceral/soft tissue: immune system
visceral/soft tissue: musculoskeletal system
visceral/soft tissue: nervous system
visceral/soft tissue: central nervous system
visceral/soft tissue: peripheral nervous system
visceral/soft tissue: somatic nervous system
visceral/soft tissue: autonomic nervous system
visceral/soft tissue: endocrine system
visceral/soft tissue: respiratory system
visceral/soft tissue: male reproductive system
visceral/soft tissue: female reproductive system
visceral/soft tissue: eye
visceral/soft tissue: ear
Key result
Developmental effects observed:
no
Conclusions:
In conclusion, the oral administration of test item Agrocer Red 112 by gavage to presumed pregnant female New Zealand White Rabbits from Gestation Day 6 to 28 did not produce any indication of maternal and developmental toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.
Based on the obtained results from this pre-natal developmental toxicity study conducted in New Zealand White Rabbits, the NOAEL (No observed adverse effect level) of Agrocer Red 112 for both maternal and fetal toxicity is estimated as 1000 mg/kg body weight/day under experimental conditions employed.
Executive summary:

The objective of this study was to provide general information concerning the effects of prenatal exposure of test itemAgrocer Red 112on the pregnant rabbits and in the developing organisms; this included the assessment of maternal toxicity as well as death, structural abnormalities, or altered growth in the fetuses.

A total of 100 mated female New Zealand White rabbits distributed to four groups. Each group (G1, G2, G3 and G4) consisted of 25 presumed pregnant rabbits. The animals in group G1 were administered with vehicle [1% w/v Carboxymethyl cellulose], the animals in groups G2, G3 and G4 were administered with test item at the dose levels of 111, 333 and 1000 mg/kg body weight for low, mid and high dose groups respectively. The vehicle and test item formulations were administered orally by gavage at the dose volume of 10 mL/kg body weight to mated and presumed-pregnant females from Gestation Day [GD] 6 to 28. The end points of assessment for does were maternal death, maternal body weight and clinical signs of maternal toxicity and the end points of assessment for fetuses were fetal weights, growth and development, structural variations and malformations or altered growth.

Homogeneity and dose formulation analysis for dose concentration verification were performed during the first and last week of treatment. The prepared formulations were considered acceptable and the mean results were within the range of 85 to 115% of nominal concentration and the relative standard deviation (% RSD) is less than 10%(mean recovery was between 98.62% and 99.97% at all measurements).

 A total of 21, 20, 21 and 21 females from group G1, G2, G3 and G4 were found with implantations yielding pregnancy rates of 84%, 80%, 84% and 84% respectively.

All the animals from each group were observed for clinical signs of toxicity once daily, for mortalities twice daily during the experimental period. The body weight was recorded on Gestation Day (GD) 0, 3, 6, 9, 12, 15, 18, 21, 24, 26, 28 and on 29 (day of caesarean section). The feed consumption was measured from GD 0 to 3, 3 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 18, 18 to 21, 21 to 24, 24 to 26, 26 to 28 and 28 to 29. All the animals were euthanized on gestation Day 29 by intravenous injection of sodium thiopentone and subjected to detailed gross pathological examination. All the females were observed for status of pregnancy and the gravid uterus weight for all the does was recorded on the day of caesarean section. Each doe was observed for number of live or dead fetuses, litter size, sex ratio, number of implantation sites and number of resorptions. The ovaries of all the does were observed for number of corpora lutea. The pre-and post-implantation losses per doe were calculated based on number of corpora lutea and number of implantation sites. The weight of thyroid along with the parathyroid was recorded post-fixation for all the animals. All the does were evaluated for histopathology of thyroid along with the parathyroid.

All the fetuses collected from each litter were weighed and measured for their crown rump length. The mean fetal weight and mean crown rump length per litter was calculated. All the fetuses were subjected to external examination on the day of caesarean section. All the fetuses from each litter were subjected for fresh soft tissue (visceral) examination and fixed head sections examination, except the heads of fetuses which were allotted for skeletal evaluation. The bodies of fetuses without heads and all other intact fetuses from each litter were double stained with alcian blue for cartilage and Alizarin Red S for bones and subjected to skeletal examination.

For maternal toxicity assessment, there were no clinical signs of toxicity and no mortality/morbidity noted during the experimental period at all the tested dose and vehicle control group animals. No changes were noted in mean maternal body weight, percent change in mean maternal body weight gain and mean maternal feed consumption at all the tested dose groups. There were no gross pathological changes noted during caesarean section of all the tested dose and vehicle control group animals. There were no effects noted in pregnancy rate and mean gravid uterus weight in all the tested dose groups. There were no effects noted in mean number of live fetuses, mean litter size, mean sex ratio, mean number of implantation sites, mean number of resorptions, mean number of corpora lutea at all the tested dose groups. The mean pre- and post-implantation losses per doe were unaffected at all the tested dose groups. The mean absolute and relative thyroid along with the parathyroid weight did not reveal any changes when weighed post-fixation at all the tested dose groups. No test item-related microscopic changes were noted in thyroid along with the parathyroid of all the does during histopathological examination from all the tested dose groups.

For fetal (pre-natal developmental) toxicity assessment, there were no test item-related changes noted in mean fetal weight and mean fetal crown rump length per litter in either sex at all the tested dose groups. There was no test item-related fetal developmental and or structural alterations noted from all the tested dose group litters when subjected to fetal external, visceral and skeletal examinations. The observed fetal external/visceral/skeletal developmental variations and or malformations are considered as incidental and unrelated to treatment as these findings occurred infrequently or at a frequency similar to the vehicle control group and did not occur in a dose-dependant manner.

Endpoint:
developmental toxicity
Remarks:
Prenatal Developmental Toxicity Study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 July 2021 to 04 April 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
OECD Test Guideline No. 414, “Prenatal Developmental Toxicity Study”, adopted on 25 June 2018
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
adopted on 25 June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Hylasco Biotechnology India Pvt. Ltd, Charles River Technology Licensee,
CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals) Registration No.: 1808/PO/RcBt/S/15/CPCSEA

- Age at study initiation: 11 weeks
- Weight at study initiation: Males: 240.25 to 298.41 g, Females: 200.65 to 249.44 g
- Housing: Sterilized standard polypropylene cage of Size: L 430 × B 285 × H 150 mm
- Diet (e.g. ad libitum): Altromin maintenance diet for rats and mice manufactured by Altromin Spezialfutter GmbH & Co. KG
- Water (e.g. ad libitum): Deep bore-well water passed through reverse osmosis unit was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period: Minimum of five days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1 to 23.2
- Humidity (%): 49 to 64%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark cycle and 12 hours fluorescent light

IN-LIFE DATES: From:30 July 2021 To:23 September 2021
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Justification for use and choice of vehicle : The test item was uniformly suspended in 1% w/v Carboxymethyl cellulose (CMC) at the concentration of 100 mg/mL the highest dose concentration selected for the study considering the dose volume of 10 mL/kg body weight as per in-house solubility/suspendibility test results. Hence, 1% w/v Carboxymethyl Cellulose was used as vehicle for test item formulations. 1% w/v Carboxymethyl Cellulose is a routinely used and universally accepted vehicle of choice for the oral toxicity studies.

- Concentration in vehicle: G2 (11.1 mg/mL), G3 (33.3 mg/mL) and G4 (100.0 mg/mL)
- Amount of vehicle: 10 mL/kg
- Lot no.: SLCH1520
- Purity: Analytical grade.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Sampling and analysis of formulations were performed during first week and last week of the treatment. Approximately, 5 mL of samples were collected in duplicates from top, middle and bottom layers from low, mid and high dose concentrations and in duplicates from single layer from vehicle control. The prepared test item formulations were stirred using magnetic stirrer during sampling.

The collected samples were transferred to Analytical Chemistry department of Bioneeds India Private Limited for dose formulation analysis. One set of aliquots of each formulation was analyzed. The second aliquot was stored as a backup purpose at established stability conditions. The second set of samples was discarded, as the analysis results of first set of samples were within the limits.

Formulations were considered acceptable, as the mean results were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) is ≤10% (treatment day 1: mean recovery (%) is 99.15%, 99.17% and 99.06% with % RSD of 0.06, 0.06 and 0.03 for groups G2, G3 and G4, respectively; treatment day 38: mean recovery (%) is 100.10%, 100.16% and 100.09% with % RSD of 0.06, 0.05 and 0.09 for groups G2, G3 and G4, respectively).
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:1:2 ratio
- M/F ratio per cage: 1:2 ratio (one male and two females)
- Length of cohabitation: 14 days, Females not mated within 14 days of pairing with the first male were placed with a second proven male for one week
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as gestation day 0 of pregnancy
- Any other deviations from standard protocol: No
Duration of treatment / exposure:
The test item/vehicle was administered to respective group of female rats once daily from gestation day 5 to 19.
Frequency of treatment:
Once Daily
Duration of test:
15 days per animal starting from implantation to one day prior to scheduled delivery (GD 5 to GD 19)
Dose / conc.:
111 mg/kg bw/day
Remarks:
Low dose
Dose / conc.:
333 mg/kg bw/day
Remarks:
Mid dose
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
High dose
No. of animals per sex per dose:
25 presumed pregnant females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: There was no information or data available from repeated dose/reproducti on toxicity and teratology studies for agrocer red 112, hence The dose levels of 0, 111, 333 and 1000 mg/kg body weight/day were selected as vehicle control, low, mid and high dose groups, respectively as consultation with sponsor.

- Rationale for animal assignment: The body weight of mated females on each day of gestation day ‘0’ (GD 0) was recorded and arranged in the ascending order of their body weight. These mated females were evenly distributed to all the groups based on their body weights so as to maintain comparable mean body weights for all groups, permanent identification numbers were assigned.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- Cage side observations checked in table [No.1] were included.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on gestation days (GD) 0, 3, 5, 8, 11, 14, 17, 19 and on 20 (day of caesarean section).

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined as g food/kg body weight/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: All visceral organs, along with uterus, ovaries and thyroid along with parathyroid.

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [ half per litter]
- Skeletal examinations: Yes: [ half per litter]
- Head examinations: Yes: [ half per litter]
Statistics:
Parametric: One-way ANOVA with Dunnett’s post test:
Maternal body weight,Percent change in maternal body weight, Corrected body weight for maternal increase, Gravid uterus weight, Maternal Feed consumption,Absolute / relative thyroid weights, Mean fetal weight per dam, Mean fetal crown rump length per dam.

Non-Parametric: Kruskal-Wallis test:
No. of corpora lutea per dam, No. of implantations per dam, Litter size per dam, No. of live fetuses per dam, Percent of live fetuses per dam, No. of early/late resorptions per dam, Percent of early/late resorptions per dam, Sex ratio (m/f) per dam, Pre/Post implantation losses (%) per dam, Fetal external / visceral / skeletal anomalies per dam.

Frequencies Comparison: Cross Tabs - Chi-square test: No. of Pregnant / Non-pregnant females (Pregnancy status), No. of dams with / without live fetuses, No. of dams with / without dead fetuses, No. of dams with / without resorptions.
Indices:
Corrected Body Weight (g) = (Gestation day 20 body weight - Gestation day 5 body weight) - Gravid uterus weight
Pre-implantation Loss (%) = Total Number of Corpora lutea - Number of Implantation Sites / Total Number of Corpora lutea x 100
Post-implantation Loss (%) = Number of Implantation sites - Number of Viable fetuses / Number of Implantation sites x 100
Historical control data:
In-house historical data available for the same species and strain and referred to end points where required in the study report.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs of toxicity noted in any of the animals from vehicle control and all the tested dose groups throughout the treatment period
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no changes noted in mean gestation (maternal) body weight and percent change in mean gestation (maternal) body weight gain in all the tested dose groups when compared with the vehicle control group. There were no changes noted in mean body weight change corrected for gravid uterus weight in all the tested dose groups when compared with the vehicle control group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no changes noted in mean gestation (maternal) feed consumption in all the tested dose groups when compared with the vehicle control group.
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related changes noted for mean serum T3 levels in any of the tested dose groups when compared to vehicle control group. The noted statistically significant increase in mean serum T3 levels (ng/mL) in group G3 is considered as incidental and un-related to treatment with test item as the obtained mean values are within normal range of same species and strain.

There were no changes noted for mean serum T4 levels in any of the tested dose groups when compared to vehicle control group.

There were no changes noted for mean serum TSH levels in any of the tested dose groups when compared to vehicle control group.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The thyroid along with parathyroid was collected, preserved and weighed post fixation from all the dams of each dose group. There were no changes noted for mean absolute and relative weight for this organ in all the tested dose groups when compared to the vehicle control group.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross pathological changes noted in any of the animals from all the tested dose groups and vehicle control group during conduct of the necropsy.

Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item related histopathological findings noted in thyroid and parathyroid glands of all treated animals.
However, ultimobranchial cysts and ectopic thymus in thyroid gland were noted in this study. These changes were considered as incidental findings as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rats.
Histopathological findings: neoplastic:
no effects observed
Number of abortions:
no effects observed
Description (incidence and severity):
No abortions were noted
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The mean percentage of pre-implantation loss per litter was 5.77, 7.29, 10.86 and 12.44 for groups G1, G2, G3 and G4 respectively.
There were no changes noted and no statistically significant differences noted for percentage of pre-implantation loss per litter in all the teste dose groups.

The mean percentage of post-implantation loss per litter was 9.61, 9.96, 6.94 and 11.62 for groups G1, G2, G3 and G4 respectively.
There were no changes or no statistically significant differences noted for mean percentage of post-implantation loss per litter in all the tested dose groups when compared to the vehicle control group.
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
Two incidental findings with total implantation loss (with total empty implantation sites/early resorptions) during conduct of necropsy was noted in 1 out of 25 females from group G1 and 1 out of 25 females from group G3.
Early or late resorptions:
no effects observed
Description (incidence and severity):
The mean number of early resorptions per litter was 0.71, 0.71, 0.62 and 0.95 with a percentage of early resorptions was 6.53, 9.20, 6.62 and 8.43 for groups G1, G2, G3 and G4 respectively. There were no changes or no statistically significant differences noted for mean number and percentage of early resorptions per dam across dose groups when compared to the vehicle control group.

The mean number of late resorptions per litter was 0.14, 0.00, 0.00 and 0.00 and the percentage of late resorptions was 1.30, 0.00, 0.00 and 0.00 for groups G1, G2, G3 and G4 respectively. There were no changes or no statistically significant differences noted for mean number and percentage of late resorptions per dam across dose groups when compared to the vehicle control group.
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
The mean number of dead fetuses per litter was 0.05, 0.10, 0.05 and 0.00 with a percentage of 0.79, 0.84, 0.34 and 0.00. There were no statistically significant differences noted for mean number of dead fetuses in all the tested dose groups when compared to the vehicle control group.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
No early deliveries noted before caesarean sectioning.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): A total of 21 (out of 25), 21 (out of 25), 21 (out of 25) and 22 (out of 25) females from group G1 (0 mg/kg body weight/day), G2 (111 mg/kg body weight/day),
G3 (333 mg/kg body weight/day) and G4 (1000 mg/kg body weight/day) were found with implantations with live fetuses yielding to pregnancy with rates of 84%, 84%, 84% and 88% respectively.

No female was littered during gestation period in all the dose groups and control group before caesarean sectioning.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
A total of 21 (out of 25), 21 (out of 25), 21 (out of 25) and 22 (out of 25) females from group G1 (0 mg/kg body weight/day), G2 (111 mg/kg body weight/day),
G3 (333 mg/kg body weight/day) and G4 (1000 mg/kg body weight/day) were found with implantations with live fetuses yielding to pregnancy with rates of 84%, 84%, 84% and 88% respectively. One incidental finding with total implantation loss (with empty implantation sites) during conduct of necropsy was noted in 1 out of 25 females from group G1 and 1 out of 25 females from group G3.
Details on maternal toxic effects:
The oral administration of test item Agrocer Red 112 by gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 did not produce any indication of maternal toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
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
histopathology: non-neoplastic
maternal abnormalities
mortality
necropsy findings
number of abortions
organ weights and organ / body weight ratios
pre and post implantation loss
total litter losses by resorption
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
effects observed, non-treatment-related
Localisation:
amniotic fluid
cervix
mammary gland
ovary
oviduct
placenta
umbilical cord
uterus
vagina
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no changes noted in mean fetal weight of either sex across the dose groups when compared with the vehicle control group.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): There were no changes noted in mean fetal weight of either sex across the dose groups when compared with the vehicle control group.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex ratio per litter calculated as the mean of male/female fetuses per litter was 1.49, 1.26, 1.21 and 0.92 for groups G1, G2, G3 and G4 respectively. There were changes or no statistically significant differences noted for mean male/female sex ratio across the dose groups when compared to the control group.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The mean litter sizes, assessed as the total number of fetuses in uteri [live plus dead] per dam, was 10.90, 10.05, 10.33 and 9.41 for groups G1, G2, G3 and G4, respectively.
There were no changes or no statistically significant differences noted in all the tested dose groups when compared with vehicle control group for mean litter size.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related fetal structural and developmental external malformations or variations noted for any of the fetuses examined from all the tested dose group litters and also no statistically significant differences occurred.

However, some of the sporadic fetal external alterations were noted across the dose groups and vehicle control group such as Subcutaneous hemorrhagic spot beneath the skin on different regions of the body, Skin discoloured / pale coloured, Bent tail, Kinked tail, Hyperextension (unilateral) of forelimb, Hyperextension (unilateral) of hind limb and Protrusion of tongue.These developmental and structural alterations are considered incidental as these observations are comparable with the vehicle control group and or also these developmental alterations are common for this species and strain.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There was no test item-related fetal structural malformations or variations noted for any of the fetuses examined from all the tested dose group litters during skeletal examination. The noted statistically significant decrease in number of incidences of unossified sternebra no. 6 in group G3 when compared with vehicle control group is considered as incidental and toxicologically in-signficant. These structural and skeletal alterations are considered incidental as these observations are comparable with the vehicle control group and also these alterations are common for this species and strain. Also, the noted alterations in ossifications, i.e. delayed ossification, incomplete ossification, dumbbell shaped ossification from various regions from all the tested dose groups and control group are considered as transient findings and are reported to resolve shortly with the development of fetus (refer table no. 16 in study report).
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related fetal structural and developmental visceral/soft tissue malformations or variations noted for any of the fetuses examined from all the tested dose group litters and also no statistically significant differences occurred. However, some of the sporadic fetal visceral/soft tissue alterations were noted across the dose groups and vehicle control group such as Pale colored liver, Renal pelvis dilation (bilateral) of kidneys, Dilatation of Ureters (bilateral), Dilated lateral ventricles of brain and Nasal conchae enlarged.These developmental soft tissue alterations are considered incidental as these observations are comparable with the vehicle control group and also these developmental alterations are common for this species and strain.
Other effects:
no effects observed
Description (incidence and severity):
There were no changes noted in mean fetal anogenital distance/mean fetal anogenital distance ratio and mean fetal crown rump length per litter of either across the dose groups when compared with the vehicle control group.
Details on embryotoxic / teratogenic effects:
The oral administration of test item Agrocer Red 112 by gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 did not produce any indication of fetal/developmental toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day
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
external malformations
skeletal malformations
visceral malformations
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
effects observed, non-treatment-related
Localisation:
external: ear
external: eye
external: face
external: limb
external: paw
external: tail
external: trunk
external: anogenital distance
external: anus
external: genital tubercle
external: large intestine
external: thorax
external: umbilicus
external: pelvic region
skeletal: skull
skeletal: skull, fontanelles
skeletal: skull sutures
skeletal: clavicle
skeletal: scapule
skeletal: forelimb
skeletal: sternum
skeletal: rib
skeletal: supernumerary rib
skeletal: vertebra
skeletal: pelvic girdle
skeletal: hindlimb
visceral/soft tissue: integumentary
visceral/soft tissue: gastrointestinal tract
visceral/soft tissue: hepatobiliary
visceral/soft tissue: urinary
visceral/soft tissue: cardiovascular
visceral/soft tissue: heamatopoietic
visceral/soft tissue: immune system
visceral/soft tissue: musculoskeletal system
visceral/soft tissue: nervous system
visceral/soft tissue: central nervous system
visceral/soft tissue: peripheral nervous system
visceral/soft tissue: somatic nervous system
visceral/soft tissue: autonomic nervous system
visceral/soft tissue: endocrine system
visceral/soft tissue: respiratory system
visceral/soft tissue: male reproductive system
visceral/soft tissue: female reproductive system
visceral/soft tissue: eye
visceral/soft tissue: ear
Key result
Developmental effects observed:
no
Conclusions:
In conclusion, the oral administration of test item Agrocer Red 112 by gavage to presumed pregnant female Sprague Dawley Rats from Gestation Day 5 to 19 did not produce any indication of maternal and developmental toxicity at the dose levels of 111, 333 and 1000 mg/kg body weight/day under experimental conditions employed.
Based on the obtained results from this pre-natal developmental toxicity study conducted in Sprague Dawley Rats, the NOAEL (No observed adverse effect level) of Agrocer Red 112 for both maternal and fetal toxicity is estimated as 1000 mg/kg body weight/day under experimental conditions employed.
Executive summary:

The objective of this prenatal developmental toxicity study inSprague Dawley rats conducted as per OECD test guidelines 414,wasto provide general information concerning the effects of prenatal exposure of test itemAgrocer Red 112on the pregnant females and in the developing organisms. This study was also conducted to assess the maternal toxicity in pregnant females and also structural abnormalities or altered growth in the fetuses. The aim ofthis study was to estimate no-observed-adverse-effect-level (NOAEL) of the test itemfor both maternal as well as fetal end points.

A total of 100 mated female Sprague Dawley rats were distributed to four groups. Each group (G1, G2, G3 and G4) consisted of 25 presumed pregnant females. The animals in group G1 were administered with vehicle [1% w/v Carboxymethyl cellulose], the animals in groups G2, G3 and G4 were administered with test item at the dose levels of111, 333 and 1000mg/kg body weight/dayfor low, mid and high dose groups respectively. The vehicle and test item formulations were administered orally by gavage at the dose volume of 10 mL/kg body weight to mated and presumed-pregnant females from Gestation Day [GD] 5 to 19. The end points of assessment for dams were maternal death, maternal body weight and clinical signs of maternal toxicity and the end points of assessment for fetuses were fetal weights, growth and development, structural variations and malformations or altered growth.

Homogeneity and dose formulation analysis for dose concentration verification was performed during the first and last week of the treatment. The analysis results of test samples were within the range of 85 to 115% of the nominal concentration and the relative standard deviation (% RSD) is ≤10%.

A total of 21 (out of 25), 21 (out of 25), 21 (out of 25) and 22 (out of 25) females from group G1, G2, G3 and G4 were found with implantations and live fetuses yielding to pregnancy with rates of 84%, 84%, 84% and 88% respectively. In groups G1 and G3, 1 out of 25 females each was noted with total implantation loss with no evidence of live fetuses.

All the dams from each group were observed for clinical signs of toxicity once daily, for mortalities twice daily during the experimental period. The body weight was recorded on Gestation Day (GD) 0, 3, 5, 8, 11, 14, 17, 19 and on 20 (day of caesarean section). The feed consumption was measured from GD 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17, 17 to 19 and 19 to 20. All the dams were euthanized on GD 20 by exposing to CO2asphyxiation and subjected to detailed gross pathological examination. All the females were observed for status of pregnancy and the gravid uterus weight for all the dams was recorded on the day of caesarean section. Each dam was observed for number of live fetuses, litter size, sex ratio, number of implantation sites and number of resorptions. The ovaries of all the dams were observed for number of corpora lutea. The pre-and post-implantation losses per dam were calculated based on number of corpora lutea and number of implantation sites. The weight of thyroid along with the parathyroid was recorded post-fixation for all the group animals. The assessment of thyroid hormones such as, thyroxine (T4), triiodothyronine (T3) and thyroid stimulating hormone (TSH) was conducted for all the group dams. All the dams from each group were evaluated for histopathology of thyroid along with the parathyroid.

All the fetuses collected from each litter were weighed and measured for its crown rump length and anogenital distance. The mean fetal weight, mean crown rump length, and mean anogenital distance measurement/ratio per litter was calculated. All the fetuses were subjected to external examination on the day of caesarean section. All the even numbered fetuses from each litter were subjected to fresh visceral (soft tissue) examination and fixed head sections examination. All the odd numbered fetuses from each litter were stained with Alizarin Red S stain and subjected to skeletal examination.

For maternal toxicity assessment, there were no clinical signs of toxicity and no mortality/morbidity noted in any of the dams in all the tested dose groups. There were no differences noted between the groups in mean maternal body weight, percent change in maternal body weight gain, body weight corrected for maternal increase and maternal feed consumption. There were no effects noted in mean number of live fetuses per litter, litter size, sex ratio, number of corpora lutea, number of implantation sites, number of incidences of resorptions, pre-and post-implantation losses per dam from all the tested dose groups. There were no differences noted between the dose groups in mean gravid uterus weight. The mean absolute and relative thyroid along with the parathyroid weight did not reveal any changes when weighed post-fixation and no test item-related changes were noted in mean thyroid hormonal levels (T3, T4 and TSH). There were no gross pathological changes noted in any of the dams during caesarean section and no test item-related microscopic changes were noted in thyroid along with the parathyroid of all dams during histopathological examination.

For fetal (pre-natal developmental) toxicity assessment, there were no test item-related changes noted in mean fetal weight, mean fetal crown rump length and mean fetal anogenital distance measurement / ratio per litter in either sex in all the tested dose groups. There was no test item-related fetal developmental and or structural alterations noted from all the tested dose group litters when subjected to fetal external, visceral and skeletal examinations. The observed fetal external/visceral/skeletal developmental variations and or malformations are considered as incidental and unrelated to treatment as these findings occurred infrequently or at a frequency similar to the vehicle control group and did not occur in a dose-dependent manner. Also, the observed mean litter/fetal proportions were within the in-house historical control range of this species and strain.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 09 FEB 2012 and 03 JUL 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD 422) and in compliance with GLP.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxocity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS

- Animals: Rat, RccHanTM: WIST(SPF)
- Rationale: Recognized by international guidelines as a recommended test system.
- Breeder: 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): 315 to 369 g (males) and 187 to 243 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.
- Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS

- Conditions: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). There was a 12 hour fluorescent light / 12-hour dark cycle with music during the light period.
- Accommodation: In groups of three to five animals 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, 100099 and S211008A63972). 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. Results of representative analyses for contaminants were included in the report as an Appendix.
- Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles. Results of bacteriological assay, chemical and contaminant analyses of representative samples were included in the report as an Appendix.
Route of administration:
oral: gavage
Vehicle:
other: 1.0% CMC / 0.05% Tween 80 in highly purified water
Details on exposure:
DOSE FORMULATIONS

The dose formulations were prepared weekly using the test item as supplied by the Sponsor.

Test item was weighed into a glass beaker on a tared precision balance. Appropriate amount of the vehicle was weighted and added to the test item (w/w). Dose formulations were mixed on a magnetic stirrer for approximately 2 hours until mixtures were homogeneous. 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 D44836 14-Day Oral Toxicity (Gavage) Study in the Wistar Rat, dose formulations are stable for at least 7 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 bw/day (control group), 100 mg/kg bw/day (group 2), 300 mg/kg bw/day (group 3) and 1000 mg/kg bw/day (Group 4)
- Rationale for Dose Level Selection: The dose levels were selected based on a previous dose range-finding toxicity study in Han Wistar rats, Harlan Laboratories Study D44836, using dose levels of 0, 100, 300 and 1000 mg/kg/day, where no adverse effects were observed up to the highest dose level.
- Dose Volume: 10 mL/kg body weight
- Dose Concentrations: 0 mg/mL (control group), 10 mg/mL (group 2), 30 mg/mL (group 3) and 100 mg/mL (group 4)
- Duration of Acclimatization Period: 7 days
- Duration of Treatment Period: 32 days males and approximately 7 weeks females
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about of 2 g each concentration were taken prior to dosing for analysis of concentration and homogeneity. To confirm the stability (7 days) samples of about 2 g of each concentration were taken from the middle only of each aliquot used on day 7 of the treatment. During the sixth week of the treatment samples of about 0.5 g were taken to repeat the measurement of concentration, homogeneity and stability (7 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 stored at -20 ± 5 °C until analysis.

The samples were analyzed by UV-VIS measurement following an analytical procedure developed by Harlan Laboratories. The test item was used as the analytical standard.

Following results were obtained:

Blank samples showed no absorbance and, therefore, it was confirmed that only 1.0% CMC / 0.05% Tween 80 in highly purified water applied within the control experiment.

The application formulations investigated during the study were found to comprise test item in the range of 83.0% to 111.3% and, thus, the required content limit of ±20% with reference to the nominal content was met. The homogeneous distribution of the test item in the preparations was approved because single results found did not deviate more than 9.8% (acceptance criterion: <15%) from the corresponding mean.

In addition, the test item was found to be stable in application formulations when kept seven days at room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean, except for group 2 and 3 prepared on 16-Feb-2012 that exceeded the acceptance criteria. In application formulations of group 2 (10 mg/mL), the maximum deviation of time-zero mean was found to be 11.8%, and in application formulations of group 3 (30 mg/mL), the maximum deviation of time-zero mean was found to be 35.8%. However, there are some hints of what could be the cause. On the first and 7th treatment day as well as backup samples (date of preparation 16-Feb-2012), erroneously high amount of samples (about 2 g instead 0.5 g) were taken for analytical measurements making it difficult to work up. That was considered to probably be the reason for the differences as a second time using small amount of sample (0.5 g) shows the homogeneity and stability of the application formulation.

In conclusion, the results indicate the accurate use of the test item and 1.0% CMC / 0.05% Tween 80 in highly purified water as vehicle during this study. Application formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.
Details on mating procedure:
During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed. The females were removed and housed individually if: the daily vaginal smear was sperm positive or a copulation plug was observed. The day on which positive mating was determined (copulation plug or sperm) was designated day 0 post coitum.

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

32 days

FEMALES

about 7 weeks
Frequency of treatment:
once daily
Duration of test:
MALES

- Acclimatization: 7 days
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum
- Treatment End: On day before sacrifice
- Blood Sampling: At Termination
- Necropsy: After 32 days of treatment, 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 End: On day 4 post partum
- Blood Sampling: On day 5 post partum
- Necropsy: On day 5 post partum (pups on day 4 post partum)
No. of animals per sex per dose:
11
Control animals:
yes, concurrent vehicle
Maternal examinations:
VIABILITY/MORTALITY

Twice daily

CLINICAL SIGNS

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

FOOD CONSUMPTION

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 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, on day 4 post partum, relevant parameters were performed with 5 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 from 5 lactating females of each group at the end of the pre-pairing period. Blood samples were drawn from the retro-orbital plexus of 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

TERMINATION AND NECROPSY

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, from 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 females were preserved in neutral phosphate buffered 4% formaldehyde solution:
- Ovaries

In addition, from 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 principal investigator for histopathology phase 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.

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 principal investigator. The same applied to all occurring gross lesions.

Histological examination of ovaries was carried out on any females that did not give birth.

A histopathology peer review was performed. A histopathology phase report was provided by the principal investigator for inclusion in the main report as an appendix.
Ovaries and uterine content:
The ovaries and uterus were examined after termination on day 5 post partum. Examinations included number of corpora lutea and number of implantation sites.
Fetal examinations:
Not performed.
Statistics:
The following statistical methods were used to analyze food consumption, body weights, reproduction data, grip strength, landing foot splay, body temperature, locomotor activity, hematology and biochemistry parameters, urinalysis, sperm analyses and organ weights:
- Means and standard deviations of various data were calculated and included in the report.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables can 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 cannot be assumed to follow a normal distribution.
- Fisher's exact-test was applied if the variables can be dichotomized without loss of information.
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, dead/live pups at first litter check, pups sex ratio and viability index.
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
1. IN-LIVE DATA

VIABILITY / MORTALITY

All animals survived scheduled study period.

DAILY CLINICAL SIGNS OR OBSERVATIONS

Feces stained yellow were noted in all females in all dose groups starting from day 2 or 3 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 females at any dose level.

FINDINGS AT DETAILED WEEKLY CLINICAL OBSERVATIONS

No test item-related findings were noted during weekly detailed clinical observations.

Incidentally, in one female at the dose level of 1000 mg/kg bw/day missing upper incisors on day 6 and 13 of the gestation period were recorded.

No further findings were noted in males or females in any dose group.

FUNCTIONAL OBSERVATIONAL BATTERY

None of the parameters under investigation during the functional observational battery gave an indication of a test item-related effect.

All findings recorded during the tests were considered not to be test item-related. Decreased or increased rearings, increased faeces balls and vocalization were noted in individual animals without dose dependency. At the dose level of 1000 mg/kg bw/day, one female was noted with multiple findings: reduced activity, ruffled fur, decreased rearings and salivation. Because of isolated occurrence, this observation was considered not to be related to the treatment.

Remaining parameters under investigation were similar in all groups.

LOCOMOTOR ACTIVITY

No effects on locomotor activity were noted in 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: 965, 917, 853 and 867 in females.

FOOD CONSUMPTION

No 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 on days 8 - 11 of the pre-pairing period and at the dose level of 300 mg/kg bw/day on days 7 - 14 of the gestation period. In the absence of any dose dependency, these differences were not related to the treatment.

Mean differences in food consumption at the dose levels 100, 300 and 1000 mg/kg bw/day were respectively: +8.0%, ±0.0%, and +3.4% during the pre-pairing period, +1.8%, +6.3% and +3.1% during the gestation period and +11.7%, -3.0% and +3.8% during the lactation period (percentages refer to the respective values in the control group).

BODY WEIGHTS

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

Mean differences in body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were respectively: 8%, 9%, 8% and 8% during the pre-pairing period, 53%, 53%, 59% and 57% during the gestation period and 6%, 7%, 6% and 6% during the lactation period (percentages refer to the body weight change within the respective period).

2. CLINICAL LABORATORY INVESTIGATIONS

HEMATOLOGY

No effects on hematology parameters were noted in females at any dose level.

CLINICAL BIOCHEMISTRY

No test item-related effects on biochemistry parameters were noted in females at any dose level.

Following parameters were statistically significantly lower if compared to the respective control values: concentration of albumin at the dose level of 100 mg/kg bw/day, concentration of albumin and albumin to globulin ratio at the dose level of 300 mg/kg bw/day and concentration of total protein and albumin at the dose level of 1000 mg/kg bw/day. Changes of these values were not dose depended and all values were within historical control range. Therefore these changes were considered not to be test item-related.

3. TERMINAL FINDINGS

ORGAN WEIGHTS

No significant differences in absolute or relative organ weights were noted in females at any dose level.

MACROSCOPICAL FINDINGS

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

HISTOPATHOLOGY FINDINGS

Under the conditions of this experiment, test item did not induce histopathological lesions. All findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.

During examination of reproduction organs of infertile females no findings correlated to the infertility were noted in females.
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: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
For pup data see chapter: Any other information on results incl. tables
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: No effects observed
Key result
Abnormalities:
not specified
Key result
Developmental effects observed:
not specified

1. REPRODUCTION, BREEDING AND PUP DATA  

SUMMARY OF PERFORMANCE

 

P Animals Breeding for F1 Litters

 

Group
(mg/kg/day)

1
(0)

2
(100)

3
(300)

4
(1000)

Female numbers

45-55

56-66

67-77

78-88

Number of females paired

11

11

11

11

Number of females mated

11

11

11

11

Number of non pregnant females (A)

2

1

1

2

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

9

10

10

8

(A) Female nos. 46, 50, 59, 69, 83 and 85. (B) Female no. 87.

 

MATING PERFORMANCE AND FERTILITY

  

Mating performance and fertility were not affected by the treatment at any dose level.

  

Percentage of mating was 100% in all groups. Mating of all females was recorded during the first seven days of the pairing period.

 

Mean (median) precoital times were 2.9 (3), 2.6 (3), 2.9 (3) and 2.8 (3) days at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.

 

Six females were not pregnant: two in the control group, one at each dose level 100 and 300 mg/kg bw/day and two at the dose level of 1000 mg/kg bw/day. 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 81.8%, 90.9%, 90.9% and 81.8% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.

 

One female at the high dose level had two implantation sites but delivered no pups. Consequently, gestation index (number of females with living pups as percentages of females pregnant) was 88.9% at the high-dose level and 100% in remaining groups.

 

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 13.6, 15.5, 17.4 and 14.4 in order of ascending dose levels.

 

At the dose level of 300 mg/kg bw/day, mean number of corpora lutea per dam was statistically significantly higher when compared to the control group. In the absence of any dose dependency, this observation was considered not to be related to the treatment.

 

DURATION OF GESTATION

  

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

 

Mean duration of gestation was 21.6, 21.8, 21.8 and 21.6 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 was 13.0, 13.0, 14.2 and 14.4 per dam whereas mean incidence of post-implantation loss was 1.0, 1.0, 1.4 and 0.4 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 12.0, 12.2, 12.8 and 14.1 in order of ascending dose levels.

Birth index (number of pups born alive as a percentage of implantations) was 92.3%, 92.2%, 90.1% and 97.0% at the dose level of 0, 100, 300 and 1000 mg/kg bw/day.

 

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 eight pups (from 2 litters) were missing on day 4 and at the high dose level two pups (from two litters) were missing on day 2 of the lactation period. Mean postnatal loss during four days of lactation was 0.1, 0.1, 0.8 and 0.3% 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 99.1, 99.2, 93.8 and 98.2 in order of ascending dose levels.

 

At the dose level of 300 mg/kg bw/day, total number of pups lost was statistically significantly higher and viability index was statistically significantly lower when compared to the respective control values. These observations were due to a loss of seven pups in one litter. Because higher mortality of pups was noted only in one litter and in the absence of increased mortality of pups at the dose level of 1000 mg/kg bw/day, this observation was considered not to be related to the treatment.

 

EXTERNAL EXAMINATION OF 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.

 

In the control group, missing tail tip was noted at first litter check and during lactation in one pup. At the dose level of 300 mg/kg bw/day, all pups in one litter had reduced temperature on day 3 and several pups from this litter had reduced temperature on day 4 of the lactation period. At the dose level of 1000 mg/kg bw/day, one pup, which was dead at first litter check had no milk in the stomach and was partially cannibalized.

 

No further findings were noted in pups at any dose level.

 

SEX RATIOS OF PUPS

 

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 48%, 48%, 38% and 49% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day.

 

BODY WEIGHTS OF PUPS 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: 8.9 g, 9.2 g, 8.9 and 8.2 g and mean differences in body weights during lactation were +47.4%, +47.7%, 43.6% and +38.7%, at the dose levels of 0, 100, 300 and 1000 mg/kg/day, respectively.

 

At the dose level of 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 this dose level which was supported by observation that reduction of body weight gain was more pronounced in litters of higher size. Therefore this effect was not test item-related.

 

MACROSCOPICAL FINDINGS IN PUPS

 

No findings were found during necropsy of pups in any dose group.

 

2. IN-LIVE DATA OF PARENTAL MALES

 

VIABILITY / MORTALITY

 

All animals survived scheduled study period.

 

DAILY CLINICAL SIGNS OR OBSERVATIONS

 

Feces stained yellow were noted in all males in all dose groups starting from day 2 or 3 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.

 

Incidentally, scabs on the neck were noted in one male in the control group.

 

FINDINGS AT DETAILED WEEKLY CLINICAL OBSERVATIONS

 

No findings were noted during weekly detailed clinical observations in males.

 

FUNCTIONAL OBSERVATIONALBATTERY

 

None of the parameters under investigation during the functional observational battery gave an indication of a test item-related effect.

 

All findings recorded during the tests were considered not to be test item-related. Decreased or increased rearings, increased faeces balls and vocalization were noted in individual animals without dose dependency. Remaining parameters under investigation were similar in all groups.

 

LOCOMOTOR ACTIVITY

 

No effects on locomotor activity were noted in males 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: 1079, 1198, 1252 and 1207.

 

FOOD CONSUMPTION

 

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: ±0.0%, -1.2% and -4.6% during the pre-pairing period and -2.0%, -2.0% and -0.8% during the after pairing period (percentages refer to the respective values in the control group).

 

BODY WEIGHTS

 

At the dose levels of 1000 and 300 mg/kg bw/day, reduction in body weight gain was noted during the pre-pairing period; mean body weight gain within this period was +10% and +12% at the high- and mid-dose level respectively, compared to +14% in the control group. Reduction in body weight gain was statistically significant at the high-dose level starting from day 3 until the end of pre-pairing period and at the mid-dose level on days 7, 9, 11, 12 and 14 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.

Body weights of males in all dose groups were similar to the respective control values during the entire study period.

Because the reduction in body weight gain at the high- and mid-dose levels was reversible and did not result in any significant differences in body weights, this observation was considered not to be adverse.

 

Mean differences in body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day were respectively: +14%, +13%, +12% and +10% during the pre-pairing period, +3%, +2%, +3% and 3% during the pairing period and +1%, +1%, +1% and +2% during the after pairing period (percentages refer to the body weight change within the respective period).

 

2. CLINICAL LABORATORY INVESTIGATIONS IN PARENTAL MALES

 

HEMATOLOGY

 

No test item-related effects on hematology parameters were noted in males at any dose level.

 

At the dose level of 300 mg/kg bw/day, amount of large unstained cells (LUC) was statistically significantly lower when compared to the control value. No reduction of this value occurred at the dose level of 1000 mg/kg bw/day and therefore the difference at mid-dose level was not related to the treatment.

 

CLINICAL BIOCHEMISTRY

 

No test item-related effects on biochemistry parameters were noted in males at any dose level.

 

At the dose level of 300 mg/kg bw/day, potassium concentration was statistically significantly higher when compared to the control value. No increase of this value occurred at the dose level of 1000 mg/kg bw/day and therefore the difference at mid-dose level was not related to the treatment.

 

URINALYSIS

 

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

 

3. TERMINAL FINDINGS OF PARENTAL MALES

 

SEMINOLOGY AND SPERMATID COUNT

 

At the dose levels of 1000 and 300 mg/kg bw/day, statistically significant changes in motility of sperms were noted. Mean count of progressive sperms was reduced at the high dose level and it was 60.4% compared to 75.6% in the control group. This value is within the range of a limited pool of historical controls for OECD 422 (n=4) extended by OECD 416 data (n=5). Mean count of not motile sperms was increased and was 35.5% and 34.2% at the high- and mid-dose dose levels, respectively, compared to 20.2% in the control group. 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). In the absence of any findings during necropsy or histopathological examination of male reproductive organs as well as absence of any effects on reproduction, the differences in sperm motility were considered not to be adverse.

 

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 (testis) or slightly higher (epididymidis) when compared to the respective control values.

 

ORGAN WEIGHTS

 

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

 

At the dose level of 100 mg/kg bw/day, statistically significantly lower weight of testis was noted. This effect was due to reduced testis weights of one male (no. 15, malformation of testis in this male correlated with histopathological findings). No significant changes of testis weights were noted at the mid- and high dose levels and therefore these changes were not related to the treatment with the test item.

 

No further significant differences in absolute or relative organ weights were noted at any dose level.

 

MACROSCOPICAL FINDINGS

 

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

 

HISTOPATHOLOGY FINDINGS

 

Under the conditions of this experiment, the test item did not induce histopathological lesions. All findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.

There were no abnormal lesions encountered during sperm staging regarding completeness of stages and maturation of cell populations. Individual lesions recorded were within the range of background alterations that may be recorded in this type of study, in rats of this strain and age.

 

During examination of reproduction organs of infertile males, bilateral total tubular degeneration of testes was found in male no. 15 at the dose level of 100 mg/kg bw/day. This lesion was considered to be the reason for infertility of male no.15. No further findings correlated to the infertility were noted in reproduction organs of the remaining infertile males or females.

 

Conclusions:
This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item to rats. The test item was administered in vehicle (1.0% CMC / 0.05% Tween 80 in 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 32 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.

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 yellow were noted in all males and females receiving test item. This observation was due to staining properties of the test item.

At the dose levels of 1000 and 300 mg/kg bw/day in males, statistically significantly reduced body weight gain was noted during the pre-pairing period. During the remaining study period body weight gain at the high- and mid-dose levels was similar to the control values. Body weights at these dose levels were similar to the control values during the entire study period. Because reduction of body weight gain was reversible and no statistically significant differences in body weights were noted at the high- and mid-dose levels, this effect was considered not to be adverse.

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 at the dose levels of 1000 and 300 mg/kg bw/day. Statistically significant reduction in mean count of progressive sperms and increase in mean count of not motile sperms were noted at the high dose level and statistically significant increase in mean count of not motile sperms was noted at the mid-dose level. 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.

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 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 tets item 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 32 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 (1.0% CMC / 0.05% Tween 80 in highly purified water).

 

The following results were obtained:

 

MORTALITY AND GENERAL TOLERABILITY OF PARENTAL ANIMALS

 

All animals survived the scheduled study period.

 

During the treatment, yellow stained faeces was noted in all males and females in dose groups. This observation was due to staining properties of the test item.

 

No further test item-related observations were noted during clinical daily or detailed weekly observations in males or females at any dose level.

 

FUNCTIONAL OBSERVATIONAL BATTERY AND LOCOMOTOR ACTIVITY OF PARENTAL ANIMALS

 

None of the parameters under investigation during the functional observational battery gave an indication of a test item-related effect.

 

Locomotor activity was not affected by the treatment with test item 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 WEIGHT OF PARENTAL ANIMALS

 

In males at the dose levels of 1000 and 300 mg/kg bw/day, dose dependent and statistically significant reduction in 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- and mid-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 ANIMALS

 

At the dose level of 1000 mg/kg bw/day, reduction in mean count of progressive sperms and increase in mean count of not motile sperms were noted. At the dose level of 300 mg/kg bw/day, increase in mean count of not motile sperms was noted. 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 absence of any effects on reproduction, the differences in sperm motility were considered not to be adverse.

Sperm morphology and sperm count were not affected by the treatment at any dose level.

 

ORGAN WEIGHTS OF PARENTAL ANIMALS

 

No test item-related changes in organ weights 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.

All findings recorded during histopathological examination 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 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 IN PUPS

 

No findings were found during necropsy of pups in any dose group.

 

CONCLUSION

 

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

 

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 classiofication

There are no indications of adverse effects on reproduction in the available data base.

Additional information