4,4'-bis(dimethylamino)-4''-(methylamino)trityl alcohol

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Aug 2022 - Jan 2023

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Report date:

2023

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-bis(dimethylamino)-4''-(methylamino)trityl alcohol

Cas Number:

561-41-1

Molecular formula:

C24H29N3O

IUPAC Name:

4,4'-bis(dimethylamino)-4''-(methylamino)trityl alcohol

Test material form:

solid: particulate/powder

Remarks:

Violet powder

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Soruce: Hylasco Biotechnology (India) Private Limited.
CPCSEA Reg.No.1808/PO/RcBt/S/15/CPCSEA
Hyderabad – 500 078.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test animals:
Age upon receipt: 12 weeks
Body weight upon receipt: 441.12 - 503.10 g (males) and 251.41 - 288.53 g (females).

Environmental conditions:
a) Acclimatization: The animals were acclimatized for 11 days to experimental room conditions. The animals were examined for general health by a veterinarian upon receipt and prior to start of treatment.
b) Environmental conditions: Animals were housed in environmentally monitored air-conditioned room with adequate fresh air supply (air changes 12-14 per hour). The range of room temperature and relative humidity were 19.6 to 24.2°C and 45 to 70%, respectively, with a 12-hours light and a 12-hours dark cycle. The animal husbandry conditions such as temperature and relative humidity were recorded once a day.
c) Housing: Animals of the same sex and group were housed two or three per cage in standard polycarbonate cages (Size: L 421 mm, B 290 mm, and H 190 mm). During mating, one male and one female were housed together until conformation of mating. Pregnant females were separated from males and housed individually until sacrifice along with the litters. Enrichment and nesting materials were provided in the dam cages from the day of advanced gestation (i.e. from GD 18). The cages were fitted with stainless steel mesh top grill with facilities for holding pelleted feed and drinking water bottle.
d) Water: Reverse osmosis water was available ad libitum throughout the study period. The test results (analytical and bacteriological test) of collected water samples met acceptance criteria as per ISO 10500:2012 for drinking water.
e) Feed: The pellet feed (Purina Lab Diet 5L79 RAT AND MOUSE 18% (Lot No.25JUN20221) manufactured by PMI Nutrition International) was available ad libitum throughout acclimatization and experimental period. The analytical results of collected feed samples met acceptance criteria as per FSSC 22000.
f) Bedding: Autoclaved corn cob (Lot No.14) was used as bedding material and was changed along with the cage at least once a week. The material was manufactured by Rowan Agro Nature Private Limited. The corncob samples passed the requirement of Food Safety Standard Regulation (2011) specified with respect to tested parameters.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% CMC in Milli-Q-water

Details on exposure:

Oral administration was done at a fixed dose volume of 10 mL/kg body weight, based on the most recently recorded body weight of the animal.

Details on mating procedure:

After the premating period of two weeks, females were placed with a single male from the same group in a 1:1 ratio. Cohabitation was continued until there was evidence of vaginal plug and/or sperm in the vaginal smear or for a maximum period of 14 days. Thereafter, pregnant females were housed individually through gestation and lactation (until PND 13) along with litters. Two females (one each in G3 and G4), which were not confirmed pregnancy within 7 days even after re-mating with second proven male, were sacrificed. The day of confirmed mating was considered gestation day (GD) 0. Pre-coital time was calculated for each female.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test item formulation was determined prior to the start of treatment using a validated HPLC method. The samples were collected in duplicates (5 mL each) from the top, middle and bottom layers from all dose concentrations, covered with aluminium foil and stored at room temperature (bench top) for 6 hours. Further, the prepared formulation samples were covered with aluminium foil and stored at 2 to 8 °C for 24 hours. An aliquot was taken at post-storage for each dose concentration and analyzed for stability evaluation. The results indicated that formulations at all dose levels were stable for 6 hours at room temperature and for 24 hours at 2 to 8ºC. Formulation analysis for dose concentration verification and homogeneity was performed one time during the premating period and one time during the lactation period using a validated HPLC method. The samples were collected in duplicates (5 mL each) from the top, middle and bottom layers from all dose concentrations, and in duplicates (5 mL each) from the middle layer from vehicle control. The results showed that the mean concentrations of dose formulations were within ± 15% of the nominal concentrations (from 93.70% to 105.05%), and the RSD of the top, mid and bottom layers were ≤10% (from 0.33% to 2.22%) in all analysis. Therefore, all dose formulations prepared in this study were considered accurate, precise, and homogenous.

Duration of treatment / exposure:

The males were dosed before mating (2 weeks) and during mating (2 weeks) for a total of 28 days, with the exception of two males (one in G3 and the other in G4) that were used for re-mating and consequently dosed for a total of 35 days. Females were dosed two weeks prior to mating (with the objective of covering at least two complete oestrous cycles), during the variable time to conception (mating and re-mating period as applicable), the gestation period, and till post-natal day 13 (i.e. till the day before scheduled sacrifice).

Frequency of treatment:

Once daily

Details on study schedule:

Prior to treatment, i.e., on day 14 of the pre-exposure period, animals were grouped and allocated to their respective treatment groups using a body weight based stratified randomization procedure. At the commencement of the treatment, the weight variation of rats used did not exceed ±20% of the mean body weight in each sex and group. A minimum of 12 female animals per main study group were evaluated at the start of pre-treatment period for oestrous cyclicity. Females that failed to exhibit typical 4 - 5 days cycles were not included in the main study. After the pre-treatment period, 10 female rats were selected for each group.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

G4

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

G2

Doses / concentrations 3

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

G1

Doses / concentrations 4

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control (G1)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for dose selection: Dose levels were selected based on the results of a dose range finding (DRF) study performed at the test facility. In the DRF, 4 rats per sex per dose were treated at 0, 50 and 100 mg/kg bw/day. Male rats were treated before mating (2 weeks) and during mating (2 weeks). Female rats were treated before mating (2 weeks), during mating (2 weeks), during gestation, and on post-natal days 0, 1, 2 and 3. No mortality or morbidity was observed at 50 mg/kg bw/day, however 3 cases of morbidity (one female and two males) were observed at 100 mg/kg bw/day. Clinical signs were observed only at 100 mg/kg bw/day and included hypoactivity, piloerection and diarrhoea. Some changes in body weight gain and food intake were observed at 50 and 100 mg/kg bw/day. To avoid morbidity in the main study, a top dose of 75 mg/kg bw/day was selected. This top dose was mutually agreed upon by the Sponsor and the test facility.

Positive control:

None

Examinations

Parental animals: Observations and examinations:

The following in-life procedures, observation and measurements were made during the study for all animals.

MORBIDITY AND MORTALITY
The animals were observed for morbidity and mortality twice daily (morning and afternoon/evening) during the study period.

CLINICAL SIGNS OF TOXICITY
Animals were observed for clinical signs once per day during the acclimatization period. On the first three days of dosing, post dose observations were recorded twice at approximately 1-hour interval during the first four hours. On subsequent days, observations were recorded approximately at one-hour post-treatment. Special emphasis was given for pregnant female animals. Once before the first exposure and at least once a week thereafter, detailed clinical observations were made in all parental animals. These observations were made outside the home cage in a standard arena and preferably at the same time each day. Observations included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Observations were also made for the presence of changes in gait, posture, response to handling, clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling), difficult or prolonged parturition, and bizarre behaviour (e.g. self-mutilation or backwards walking).

BODY WEIGHTS
For both male and female rats, individual body weights were recorded on day 1 of treatment, weekly thereafter, and on the day of sacrifice. During gestation and lactation, female body weights were recorded on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (PND 0 or 1) and at least on PNDs 4 and 13.

FEED CONSUMPTION
Cage-wise food consumption was calculated by using the food consumed at weekly interval per cage and dividing by the number of rats per cage and the number of days in the intervening period to determine the food intake/rat/day. Food spilt was weighed and recorded at each food output recording session and during cage change. During gestation, food consumption was calculated on GD 0, 7, 14, and 20. During lactation, feed consumption was calculated on LD 0, 6, and 13. Food consumption was not measured during the cohabitation/mating period.

DAM OBSERVATIONS
Gestation duration was recorded and calculated from day 0 (GD 0) of pregnancy. Pregnant females were observed for difficulty and prolonged parturition as far as possible. The day of parturition was considered as PND 0.

THYROID HORMONE ANALYSIS
From adult females and males, blood samples were collected during terminal sacrifice. Aliquots of serum samples were stored at -70°C or colder until analysis. Serum samples from the adult males were measured for thyroid hormones (T4 and TSH). Serum T4 (Thyroxine) and TSH (Thyroid stimulating hormone) levels were measured using sandwich ELISA and competitive ELISA respectively, with commercially available reagents/kits from KRISHGEN Bio Systems, India.

Oestrous cyclicity (parental animals):

Vaginal smears were collected daily during the 2-week premating period to select females with normal cycle length (4 - 5 days)/pattern for study inclusion. Vaginal smears were also collected daily during the mating period until there was evidence of mating. When obtaining vaginal/cervical cells, care was taken to avoid damage of mucosa, which could induce pseudo pregnancy.

Litter observations:

Litters were examined as soon as possible after parturition to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than other pups) and the presence of gross abnormalities. Pups were weighed within 24 hours of parturition (PND 0), on PND 4, and on PND 13 at post-partum. Pups were also observed for any abnormal behaviour. The anogenital distance (AGD) of each pup was measured on PND 0 and normalized by dividing the AGD with the cube root of the respective pup's body weight. The number of nipples/areolae in male pups were counted on PND 13. Blood samples were collected from 2 pups (wherever number permits) from each litter on PND 4 and on PND 13. Aliquots of serum samples were stored at -70°C or colder until analysis. Serum samples from pups on PND 13 were measured for thyroid hormones (T4 and TSH). Serum T4 (Thyroxine) and TSH (Thyroid stimulating hormone) levels were measured using sandwich ELISA and competitive ELISA respectively, with commercially available reagents/kits from KRISHGEN Bio Systems, India.

Postmortem examinations (parental animals):

All adult animals were anaesthetized with isoflurane and euthanized by CO2 asphyxiation. All adult animals were subjected to a detailed necropsy by the study pathologists, and findings were recorded and graded. Special attention was paid to the organs of the reproductive system. The number of implantation sites were recorded for all the dams and vaginal smears were examined on the day of necropsy to determine the stage of oestrous cycle and allow correlation with histopathology of female reproductive organs. The testes and epididymides as well as prostate + seminal vesicles with coagulating glands of all adult male animals were collected, trimmed of any adherent tissue, as appropriate, and their wet weight was taken as soon as possible after dissection to avoid drying. Prostate + seminal vesicles with coagulating glands were preserved in the 10% neutral buffered formalin (NBF) and testes, epididymides were preserved in the modified Davidson’s fluid. The ovaries and the uterus (with cervix) of all adult female animals were collected, trimmed of any adherent tissue, as appropriate, and their wet weight was taken as soon as possible after dissection to avoid drying and preserved in the 10% neutral buffered formalin (NBF). From all adult males and females, thyroid glands were collected and preserved in the 10% neutral buffered formalin (NBF) for subsequent histopathological examination. The thyroid weight was determined after fixation. Trimming was done very carefully and only after fixation to avoid tissue damage. Any organs/tissues showing test item related gross changes, were preserved in the 10% neutral buffered formalin (NBF) for subsequent histopathological examination.

ABSOLUTE AND RELATIVE ORGAN WEIGHTS
Absolute organ weights and absolute organ weights relative to body weight (i.e. relative organ weights) were recorded for the organs as specified above.

HISTOPATHOLOGY
The detailed histopathological examinations were performed on the ovaries and uterus with cervix and vagina from all adult females and the testes, epididymides and prostate + seminal vesicles with coagulating glands from all adult males in G1 and G4. The thyroid along with parathyroid from all adult animals in G1 and G4 were also examined. The reproductive organs of animals failing to mate were also examined. The tissues were processed for routine paraffin embedding and 4 micron sections were stained with Mayer’s Hematoxylin Eosin stain. In addition, testes were sectioned at 4 micron and stained with PAS (Periodic acid–Schiff) reagent and Haematoxylin to aid in qualitative assessment of spermatogenesis. Unused tissues were archived.

Postmortem examinations (offspring):

All pups sacrificed at term were anaesthetized with isoflurane and euthanized by CO2 asphyxiation. All pups at necropsy were examined for internal and external gross abnormalities. From 1 male and 1 female pup from each litter (on PND 13), thyroid glands were collected and preserved in the 10% neutral buffered formalin (NBF) for subsequent histopathological examination. The thyroid weight was determined after fixation.

Statistics:

The following statistical methods were used to analyse the body weight, feed consumption, organ weights, thyroid hormone levels, as well as reproduction/developmental data.
• Data was summarized in tabular form. Statistical analysis was performed using Graphpad Software Inc., CA, USA (Version: 8.1.2).
• All the data was checked for normality and homogeneity before comparisons.
• All the normally distributed and homogenous data was subjected to One-Way ANOVA followed by Dunnett’s post hoc comparison.
• Data that fails the normality test and/or heterogeneous in nature was subjected to Krushkal Wallis ANOVA followed by Dunn’s post hoc comparisons.
• Unpaired T-test and Mann Whitney tests were applied for comparing two groups.
• Values were given as mean ± standard deviation (SD).
• All analyses and comparisons were evaluated at the ≤ 5% (P ≤ 0.05) level.
• All the statistically significant changes were denoted by an asterisk symbol (*) in the summary tables.

Reproductive indices:

a. Male Mating Index (%)

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

b. Male fertility index (%)

Number of males siring a litter
= ---------------------------------------- x 100
Number of males cohabited

c. Female mating index (%)

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

d. Fecundity index (%)

Number of pregnant females (confirmed at necropsy)
= ---------------------------------------------------------------------- x 100
Number of females with confirmed mating

e. Female fertility index (%)

Number of pregnant females (confirmed at necropsy)
= ---------------------------------------------------------------------- x 100
Number of females used for mating

f. Post implantation loss (%)

Number of implantations - Number of live foetuses/pups
= --------------------------------------------------------------------------- x 100
Number of implantations
g. Gestation index

Number of females with live pups born
= --------------------------------------------------------------- x 100
Number of females with evidence of pregnancy

Offspring viability indices:

h. Live birth index (%)

Number of viable pups born (at first observation)
= ----------------------------------------------------------------- x 100
Total Number of pups born (at first observation)

i. Day 4 survival index (%)

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

j. Day 13 survival index (%)

Number of viable pups on lactation day 13
= ------------------------------------------------------- x 100
Number of viable pups born

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All male and female rats in G2, G3 and G4 showed violet discoloration of faeces throughout the experimental period. All female rats in G4, except one non-pregnant animal, showed lethargy during the gestation and lactation. This lethargy was observed approximately 30 minutes after dosing and persisted up to 2 hours after dosing. No other clinical signs of toxicity were observed in the study.

Mortality:

no mortality observed

Description (incidence):

No morbidity or mortality was observed in the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In G4 males, statistically significant decrease in body weight was observed on day 8 of treatment (mean, 465.99 g) compared to G1 (mean, 493.05 g). Further, in G3 and G4, statistically significant decreases in body weights were observed on day 15 (mean, G3=483.94, G4=472.58 g), on day 22 (mean, G3=491.17, G4=479.16 g) and day 28 of treatment (mean, G3=495.73, G4=483.90 g) compared to G1 (mean, 504.05; 513.49; and 521.70 g on day 15, 22, and 28, respectively. Statistically significant decreases in the body weight gain were observed in G3 between day 1-8 (mean, 0.86 g) and day 1-28 (mean, 21.31 g) and in G4 between day 1-8 (mean, -9.13 g) and 1-28 (mean, 8.78 gram) compared to G1 (mean, 11.01 and 46.03 between day 1-8 and 1-28, respectively). The observed changes in body weight and body weight gain in G3 and G4 were considered to be adverse effects of treatment.
In female rats, no significant changes in body weight were observed in G2, G3 or G4, with the exception of a significant increase in average body weight in G2 on GD 20 (mean, 457.77 g) compared to G1 (mean, 433.23 g) that was considered to be incidental and not toxicologically relevant. Significant decreases in body weight gain were observed from treatment day 1-8 in G3 (mean, 3.89 g) and G4 (mean, 1.95 g) compared to G1 (mean, 9.76). This effect was considered to be treatment-related but transient as no significant changes in body weight gain were observed afterwards.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In males, statistically significant decreases in feed consumption were observed from day 1-8 in G3 (mean, 24.59 g) and G4 (mean, 22.64 g) compared to the G1 (mean, 32.27 g). In G4 males, a significant decrease in feed consumption was also observed from day 8-15 (mean, 28.79 gram) compared to G1 (mean, 31.69 gram). No significant changes in feed consumption were observed for the female rats. The significant changes in feed consumption in the male rats were associated with significant decreases in body weight and body weight gain and were therefore considered to be treatment-related and adverse.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

In adult male rats, no significant changes in T4 or TSH were observed at any dose level.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Microscopic findings in the adult animals were limited to an incidence of focal area of mononuclear cell infiltration in the epididymides in one G1 male with minimal severity. No microscopic findings were observed during the qualitative assessment of spermatogenesis stages or during the morphological examination of interstitial testicular cell structures in G4 compared to G1. The stage of estrous cycle in each female rat in G1 and G4 at the terminal sacrifice was in correlation with the histology of respective female reproductive organs.

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Oestrous cycle lengths in G2 (mean, 4.00 days), G3 (mean, 4.00 days) and G4 (mean, 4.50 days) were comparable with G1 (mean, 4.00 days) and there were no significant changes observed in oestrous cycle pattern at any dose level except in G4, in which one animal showed the irregularity during the premating period (on day 7 onwards). This isolated change in one animal in G4 was considered incidental and not toxicologically relevant.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Both male and female mating and fertility indexes were 100, 100, 90 and 90% in G1, G2, G3, and G4, respectively. Female fecundity indexes were 100, 100, 90, and 90% in G1, G2, G3, and G4, respectively. The number of female rats conceiving at 1-5 days after mating were 9/10, 8/10, 9/10, and 8/10 in G1, G2, G3, and G4, respectively. The number of female rats conceiving at 6-14 days after mating were 1/10, 2/10, 0/10, and 1/10 in G1, G2, G3, and G4, respectively. No re-mating was needed in G1 and G2. One animal each in G3 and G4 did not conceive during the initial mating period and were therefore subjected to re-mating with proven males from the same group. The failure of one animal each in G3 and G4 to achieve pregnancy was considered to be within normal biological variation for the species. For pregnant animals, no significant changes in gestation length were observed and gestation index was 100% in all groups. No significant changes in the number of implantations per dam were observed in G2 (mean, 15.00), G3 (mean, 14.22) or G4 (mean, 15.78) compared to G1 (mean, 12.90). No significant changes in percent post implantation losses were observed in G2 (3.27%), G3 (2.88%) or G4 (6.54%) compared to G1 (0.63%) and all of the values were consistent with the historical control range of the test facility (i.e.: 0.00 – 9.09%).

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

75 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive performance

Remarks on result:

other: NOAEL for reproductive toxicity

2

Key result

Dose descriptor:

LOAEL

Effect level:

75 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

Remarks on result:

other: LOAEL for general toxicity

3

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

Remarks on result:

other: NOAEL for general toxicity

4

Key result

Dose descriptor:

LOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: LOAEL for general toxicity

5

Key result

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

Remarks on result:

other: NOAEL for general toxicity

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No abnormal behavior was observed in any of the pups.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Live birth index was 100% in all groups and pup survival index was 100% on PND 4 in all groups. Pup survival indexes on PND 13 were 79.18, 86.63, 85.32 and 86.17% in G1, G2, G3 and G4, respectively.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In male pups on PND 13, a statistically significant decrease in TSH levels was observed in G4 (mean, 161.26 pg/ml) compared to G1 (mean, 168.06 pg/ml). In female pups on PND 13, T4 levels were statistically increased in G2 (mean, 40.10 ng/ml) and G3 (mean, 41.28 ng/ml) compared to G1 (mean, 33.40 ng/ml) whereas TSH levels were statistically increased in G2 (mean, 197.40 pg/ml) and G3 (mean, 198.82 pg/ml) compared to G1 (mean, 163.19 pg/ml). These significant changes in thyroid hormone levels in the male and female pups were not associated with any significant changes in thyroid weight. Furthermore, there were no gross lesions or histopathological findings in the thyroid from any of the examined pups in G1 and G4. The observed changes in thyroid hormone levels in the female pups also lacked any clear dose dependency. Therefore, the significant changes in thyroid hormone levels in the male and female pups were not considered to be adverse.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

No statistically significant changes in AGD or normalised AGD was observed at any dose level.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No nipples/areolae were retained in any of the male pups on PND 12.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No significant changes in thyroid weight were observed.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No gross pathological findings were observed in any of the pups.

Histopathological findings:

no effects observed

Description (incidence and severity):

No histopathological findings in the thyroid were observed in any of the examined pups.

Other effects:

no effects observed

Description (incidence and severity):

No statistically significant changes in litter size, litter weight, or sex ratio were observed in either G2, G3 or G4 when compared to G1.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

75 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

body weight and weight gain

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

other: litter size, litter weight, sex ratio.

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Table 1 - Details of Experimental Layout, Treatment and Sacrifice Schedule

Groups Dose (mg/kg bw/day)	Treatment group	No. of rats per Group			Treatment Schedule
		Male	Female	Total
G1 0	Vehicle control	10	10	20	Treatment commenced from the age of 14 weeks for males. The males were dosed for 28 days except two males were dosed up to day 35 (re- mated). This included two weeks prior mating, variable time of mating and post mating. Treatment commenced from the age of 14 weeks for females. This included two weeks of prior mating, variable time of mating and re-mating and gestation and up to lactation day 13. Two females (re-mated) were dosed up to the day 24 from the last day of mating period. The males were sacrificed on 29^th day except two males (re-mated), on day 36, and females sacrificed on lactation day 14 except two non-pregnant females, which was sacrificed on day 25 from the last day of mating period.
G2 25	Low dose - 4,4'-bis(dimethylamino)-4''-(methylamino)trityl alcohol	10	10	20
G3 50	Mid dose - 4,4'-bis(dimethylamino)-4''-(methylamino)trityl alcohol	10	10	20
G4 75	High dose - 4,4'-bis(dimethylamino)-4''-(methylamino)trityl alcohol	10	10	20

 

Table 2 - Summary of Mortality/Morbidity

Groups Dose (mg/kg bw/day)	Sex	Pre-terminal deaths
		Death during Treatment	Death during Gestation	Death during Lactation	Moribund Sacrifice	Total Mortality
							G1 0	M	0	NA	NA	0	0
F	0	0	0	0	0
G2 25	M	0	NA	NA	0	0
	F	0	0	0	0	0
G3 50	M	0	NA	NA	0	0
	F	0	0	0	0	0
G4 75	M	0	NA	NA	0	0
	F	0	0	0	0	0

             Key: N = 10 for G1, G2, G3 and G4; M= Male; F= Female;

Table 3 - Summary of Clinical Signs OF TOXICITY – MALES

Groups Dose (mg/kg bw/day)	Sex	No. of animals/Total no. treated animals	Clinical Signs
G1 0	M	10/10	Normal
G2 25	M	10/10	Violet discoloration of faeces
G3 50	M	10/10	Violet discoloration of faeces
G4 75	M	10/10	Violet discoloration of faeces

Key: N = 10 for G1, G2, G3 and G4; M=Male

 

Table 4 - Summary of Clinical Signs OF TOXICITY – FEMALES

Groups Dose (mg/kg bw/day)	Sex	No. of animals/Total no. treated animals	Clinical Signs
G1 0	F	10/10	Normal
G2 25	F	10/10	Violet discoloration of faeces
G3 50	F	10/10	Violet discoloration of faeces
G4 75	F	10/10	Violet discoloration of faeces
		9/10	Lethargy

Key: N = 10 for G1, G2, G3 and G4; F=Female

 

Home cage observation	Days	Groups / Dose (mg/kg bw/day)
		G1 / 0	G2 / 25	G3 / 50	G4 / 75
	Prior to Treatment	3R,6S,1A	3R,6S,1A	3R,5S,2A	4R,4S,2A
Animal body position/Posture   R - Rearing S - Sitting or Standing normally A - Asleep	8	3R,6S,1A	3R,6S,1A	3R,5S,2A	3R,5S,2A
	15	3R,7S,0A	3R,7S,0A	3R,7S,0A	3R,7S,0A
	22	3R,6S,1A	3R,7S,0A	2R,7S,1A	2R,7S,1A
	28	3R,6S,1A	2R,8S,0A	3R,5S,2A	3R,7S,0A
Respiration - Normal	Prior to Treatment , 8, 15, 22, 28	10/10	10/10	10/10	10/10
Vocalization - Normal		10/10	10/10	10/10	10/10
Hand-held observation
Ease of removal - Easy/Normal	Prior to Treatment , 8, 15, 22, 28	10/10	10/10	10/10	10/10
Handling reactivity- Easy to handle/ normal		10/10	10/10	10/10	10/10
Palpebral closure - Open		10/10	10/10	10/10	10/10
Lacrimation - No lacrimation		10/10	10/10	10/10	10/10
Nasal discharge - No discharge		10/10	10/10	10/10	10/10
Salivation - No salivation		10/10	10/10	10/10	10/10
Teeth - Normal		10/10	10/10	10/10	10/10
Fur coat/ Skin - Normal		10/10	10/10	10/10	10/10
Muscle tone/ Mass - Normal		10/10	10/10	10/10	10/10
Perineum wetness - None/normal		10/10	10/10	10/10	10/10
Tail - Normal		10/10	10/10	10/10	10/10
Open-field observation
Gait - Head is horizontal, abdomen rises slightly above floor, and body moves up and down slightly during walking	Prior to Treatment ,8, 15, 22, 28	10/10	10/10	10/10	10/10
Arousal - Normal		10/10	10/10	10/10	10/10
Clonic convulsion - None/ normal		10/10	10/10	10/10	10/10
Tonic Convulsion - None/ normal		10/10	10/10	10/10	10/10
Stereotype - None		10/10	10/10	10/10	10/10
Bizarre /Abnormal behaviour(s) - None		10/10	10/10	10/10	10/10
Fecal consistency - Formed/ normal		10/10	10/10	10/10	10/10

Table 5 - Summary of DETAILED Clinical Signs OF TOXICITY – MALES (PRE-MATING PERIOD)

                

                Key: N=10, G1- Vehicle control, G2-Low dose, G3-Mid dose, G4-High dose.

TABLE 5 (CONTD.) - SUMMARY OF DETAILED CLINICAL SIGNS OF TOXICITY – MALES (PRE-MATING PERIOD)

Open- field observation (contd.)
Rearing count
Group / Dose           (mg/kg bw/day)		Prior to Treatment	8	15	22	28
G1 / 0	Mean	16.90	12.60	14.30	14.60	14.30
	SD	2.59	2.73	2.69	3.58	2.49
G2 / 25	Mean	17.60	10.90	14.80	13.60	14.40
	SD	2.20	1.70	3.66	2.65	3.01
G3 / 50	Mean	18.80	10.80	14.60	14.60	14.60
	SD	1.99	1.83	3.01	3.01	3.20
G4 / 75	Mean	18.20	11.20	12.20	14.20	15.40
	SD	1.99	2.04	1.78	2.96	3.50
Urine pools (count)
G1 / 0	Mean	3.60	4.00	2.00	1.80	1.70
	SD	1.20	1.26	0.77	0.87	0.78
G2 / 25	Mean	4.00	2.60	1.70	1.90	1.90
	SD	1.41	1.36	0.90	0.83	1.04
G3 / 50	Mean	3.50	3.80	1.30	1.30	1.50
	SD	1.28	1.66	0.78	1.00	1.02
G4 / 75	Mean	3.20	4.00	1.60	1.20	1.70
	SD	0.98	1.48	0.80	1.08	0.78
Fecal (count)
G1 / 0	Mean	1.30	1.10	2.00	1.80	1.20
	SD	0.90	1.14	0.63	0.87	0.75
G2 / 25	Mean	1.80	0.70	1.40	1.40	1.20
	SD	0.87	0.78	0.80	0.66	1.08
G3 / 50	Mean	1.80	1.00	1.50	1.20	1.50
	SD	1.25	0.77	0.50	0.75	0.81
G4 / 75	Mean	1.60	0.50	1.40	1.20	1.30
	SD	1.02	0.67	0.49	0.75	0.78

 

                   Key: N=10, G1- Vehicle control, G2-Low dose, G3-Mid dose, G4-High dose.

 

TABLE 5 (CONTD.) - SUMMARY OF DETAILED CLINICAL SIGNS OF TOXICITY – FEMALES (PRE-MATING PERIOD)

Home cage observation	Days	Groups / Dose (mg/kg bw/day)
		G1 / 0	G2 / 25	G3 / 50	G4 / 75
	Prior to Treatment	3R,6S,1A	3R,6S,1A	4R,4S,2A	2R,6S,2A
Animal body position/Posture R - Rearing S - Sitting or Standing normally A - Asleep	8	3R,6S,1A	3R,5S,2A	3R,5S,2A	3R,7S,0A
	15	3R,6S,1A	2R,8S,0A	3R,7S,0A	3R,6S,1A
Respiration - Normal	Prior to Treatment, 8, 15,	10/10	10/10	10/10	10/10
Vocalization - Normal		10/10	10/10	10/10	10/10
Hand-held observation
Ease of removal - Easy/Normal	Prior to Treatment, 8, 15	10/10	10/10	10/10	10/10
Handling reactivity- Easy to handle/ normal		10/10	10/10	10/10	10/10
Palpebral closure - Open		10/10	10/10	10/10	10/10
Lacrimation - No lacrimation		10/10	10/10	10/10	10/10
Nasal discharge - No discharge		10/10	10/10	10/10	10/10
Salivation - No salivation		10/10	10/10	10/10	10/10
Teeth - Normal		10/10	10/10	10/10	10/10
Fur coat/ Skin - Normal		10/10	10/10	10/10	10/10
Muscle tone/ Mass - Normal		10/10	10/10	10/10	10/10
Perineum wetness - None/normal		10/10	10/10	10/10	10/10
Tail - Normal		10/10	10/10	10/10	10/10
Open-field observation
Gait - Head is horizontal, abdomen rises slightly above floor, and body moves up and down slightly during walking	Prior to Treatment, 8, 15	10/10	10/10	10/10	10/10
Arousal - Normal		10/10	10/10	10/10	10/10
Clonic convulsion - None/ normal		10/10	10/10	10/10	10/10
Tonic Convulsion - None/ normal		10/10	10/10	10/10	10/10
Stereotype - None		10/10	10/10	10/10	10/10
Bizarre /Abnormal behaviour(s) - None		10/10	10/10	10/10	10/10
Fecal consistency - Formed/ normal		10/10	10/10	10/10	10/10

 

                 Key: N=10, G1- Vehicle control, G2-Low dose, G3-Mid dose, G4-High dose.

 

TABLE 5 (CONTD.) - SUMMARY OF DETAILED CLINICAL SIGNS OF TOXICITY – FEMALES (PRE MATING PERIOD)

Open- field observation (contd.)
Rearing count
Group / Dose (mg/kg bw/day)		Prior to Treatment	8	15
G1 / 0	Mean	17.70	13.10	15.40
	SD	2.24	3.59	3.72
G2 / 25	Mean	17.70	13.10	15.40
	SD	2.24	3.59	3.72
G3 / 50	Mean	18.60	10.70	12.10
	SD	2.11	1.85	1.64
G4 / 75	Mean	19.20	10.60	12.30
	SD	1.83	2.06	1.68
Urine pools (count)
G1 / 0	Mean	3.70	3.70	2.00
	SD	1.27	1.10	0.77
G2 / 25	Mean	3.90	3.70	1.70
	SD	1.22	2.00	0.78
G3 / 50	Mean	3.30	4.00	1.80
	SD	1.00	1.55	0.87
G4 / 75	Mean	3.40	2.40	1.80
	SD	1.11	0.80	0.60
Fecal (count)
G1 / 0	Mean	1.50	1.00	1.70
	SD	0.81	0.89	0.78
G2 / 25	Mean	1.50	0.90	1.30
	SD	1.02	0.83	0.78
G3 / 50	Mean	1.70	0.60	1.30
	SD	0.00	0.80	0.78
G4 / 75	Mean	1.40	2.50	1.60
	SD	0.00	0.81	0.66

 

                  Key: n=10, G1- Vehicle control, G2-Low dose, G3-Mid dose, G4-High dose

 

TABLE 5 (CONTD.) - SUMMARY OF DETAILED CLINICAL SIGNS OF TOXICITY – MALES (RE MATING PERIOD)

Home cage observation	Days	Groups / Dose (mg/kg bw/day)
		G3 / 50	G4 / 75
Animal body position/Posture R - Rearing S - Sitting or Standing normally A - Asleep	29	S	S
	35	R	S
Respiration - Normal	35,43,50,57,59	1/1	1/1
Vocalization - Normal		1/1	1/1
Hand-held observation
Ease of removal - Easy/Normal	35,43,50,57,59	1/1	1/1
Handling reactivity- Easy to handle/ normal		1/1	1/1
Palpebral closure - Open		1/1	1/1
Lacrimation - No lacrimation		1/1	1/1
Nasal discharge - No discharge		1/1	1/1
Salivation - No salivation		1/1	1/1
Teeth - Normal		1/1	1/1
Fur coat/ Skin - Normal		1/1	1/1
Muscle tone/ Mass - Normal		1/1	1/1
Perineum wetness - None/normal		1/1	1/1
Tail - Normal		1/1	1/1
Open-field observation
Gait - Head is horizontal, abdomen rises slightly above floor, and body moves up and down slightly during walking	35,43,50,57,59	1/1	1/1
Arousal - Normal		1/1	1/1
Clonic convulsion - None/ normal		1/1	1/1
Tonic Convulsion - None/ normal		1/1	1/1
Stereotype - None		1/1	1/1
Bizarre /Abnormal behaviour(s) - None		1/1	1/1
Fecal consistency - Formed/ normal		1/1	1/1

 

Key: n=1 for G3 and n=1 for G3; G3-Mid dose, G4-High dose

 

TABLE 5 (CONTD.) - SUMMARY OF DETAILED CLINICAL SIGNS OF TOXICITY – MALES (RE MATING PERIOD)

Open- field observation (contd.)
Rearing count
Group / Dose (mg/kg bw/day)	29	35
G3 / 50	14	15
G4 / 75	10	11
Urine pools (count)
G3 / 50	2	3
G4 / 75	2	3
Fecal (count)
G3 / 50	1	2
G4 / 75	4	3

 

Key: n=1 for G3 and n=1 for G3; G3-Mid dose, G4-High dose

 

TABLE 5 (CONTD.) - SUMMARY OF DETAILED CLINICAL SIGNS OF TOXICITY – FEMALES (RE MATING PERIOD)

Home cage observation	Days	Groups / Dose (mg/kg bw/day)
		G3 / 50	G4 / 75
	35	1R,0S,0A	1R,0S,0A
Animal body position/Posture   R - Rearing S - Sitting or Standing normally A - Asleep	43	0R,1S,0A	0R,1S,0A
	50	1R,0S,0A	0R,1S,0A
	57	0R,1S,0A	1R,0S,0A
	59	1R,0S,0A	1R,0S,0A
Respiration - Normal	35,43,50,57,59	1/1	1/1
Vocalization - Normal		1/1	1/1
Hand-held observation
Ease of removal - Easy/Normal	35,43,50,57,59	1/1	1/1
Handling reactivity- Easy to handle/ normal		1/1	1/1
Palpebral closure - Open		1/1	1/1
Lacrimation - No lacrimation		1/1	1/1
Nasal discharge - No discharge		1/1	1/1
Salivation - No salivation		1/1	1/1
Teeth - Normal		1/1	1/1
Fur coat/ Skin - Normal		1/1	1/1
Muscle tone/ Mass - Normal		1/1	1/1
Perineum wetness - None/normal		1/1	1/1
Tail - Normal		1/1	1/1
Open-field observation
Gait - Head is horizontal, abdomen rises slightly above floor, and body moves up and down slightly during walking	35,43,50,57,59	1/1	1/1
Arousal - Normal		1/1	1/1
Clonic convulsion - None/ normal		1/1	1/1
Tonic Convulsion - None/ normal		1/1	1/1
Stereotype - None		1/1	1/1
Bizarre /Abnormal behaviour(s) - None		1/1	1/1
Fecal consistency - Formed/ normal		1/1	1/1

 

Key: n=1 for G3 and n=1 for G3; G3-Mid dose, G4-High dose.

TABLE 5 (CONTD.) - SUMMARY OF DETAILED CLINICAL SIGNS OF TOXICITY – FEMALES (RE MATING PERIOD)

 

Open- field observation (contd.)
Rearing count
Group / Dose (mg/kg bw/day)	35	43	50	57	59
G3 / 50	9	10	9	12	10
G4 / 75	14	13	13	14	13
Urine pools (count)
G3 / 50	2	3	2	2	2
G4 / 75	3	2	3	3	3
Fecal (count)
G3 / 50	1	1	1	2	1
G4 / 75	0	2	2	2	1

 

Key: n=1 for G3 and n=1 for G3; G3-Mid dose, G4-High dose.

 

Table 6 - Summary of DETAILED Clinical Signs OF TOXICITY – FEMALES (GESTATION PERIOD)

Home cage observation   Animal body position/Posture R - Rearing S - Sitting or Standing normally A - Asleep	Days	Groups / Dose (mg/kg bw/day)
		G1 / 0	G2 / 25	G3 / 50	G4 / 75
	GD 0	2R,8S,0A	1R,9S,0A	2R,8S,0A	2R,8S,0A
	GD 7	4R,6S,0A	3R,7S,0A	4R,6S,0A	4R,6S,0A
	GD 14	3R,7S,0A	7R,3S,0A	4R,6S,0A	2R,8S,0A
	GD 20	6R,4S,0A	6R,4S,0A	4R,5S,1A	5R.5S,0A
Respiration - Normal	GD 0,7,14,20	10/10	10/10	10/10	10/10
Vocalization - Normal		10/10	10/10	10/10	10/10
Hand-held observation
Ease of removal - Easy/Normal	GD 0,7,14,20	10/10	10/10	10/10	10/10
Handling reactivity- Easy to handle/ normal		10/10	10/10	10/10	10/10
Palpebral closure - Open		10/10	10/10	10/10	10/10
Lacrimation - No lacrimation		10/10	10/10	10/10	10/10
Nasal discharge - No discharge		10/10	10/10	10/10	10/10
Salivation - No salivation		10/10	10/10	10/10	10/10
Teeth - Normal		10/10	10/10	10/10	10/10
Fur coat/ Skin - Normal		10/10	10/10	10/10	10/10
Muscle tone/ Mass - Normal		10/10	10/10	10/10	10/10
Perineum wetness - None/normal		10/10	10/10	10/10	10/10
Tail - Normal		10/10	10/10	10/10	10/10
Open-field observation
Gait - Head is horizontal, abdomen rises slightly above floor, and body moves up and down slightly during walking	GD 0,7,14,20	10/10	10/10	10/10	10/10
Arousal - Normal		10/10	10/10	10/10	10/10
Clonic convulsion - None/ normal		10/10	10/10	10/10	10/10
Tonic Convulsion - None/ normal		10/10	10/10	10/10	10/10
Stereotype - None		10/10	10/10	10/10	10/10
Bizarre /Abnormal behaviour(s) - None		10/10	10/10	10/10	10/10
Fecal consistency - Formed/ normal		10/10	10/10	10/10	10/10

 

Key: n=10 for G1, n=10 for G2, n=9 for G3, n=9 for G4 and G1- Vehicle control, G2-Low dose, G3-Mid dose,

G4-High dose

TABLE 6 (CONTD.) - SUMMARY OF DETAILED CLINICAL SIGNS OF TOXICITY – FEMALES (GESTATION PERIOD)

Open- field observation (contd.)
Rearing count
Group / Dose (mg/kg bw/day)		GD 0	GD 7	GD 14	GD 20
G1 / 0	Mean	10.40	10.50	10.10	10.00
	SD	1.11	1.36	0.83	1.10
G2 / 25	Mean	10.80	10.80	10.00	10.60
	SD	1.33	1.08	1.00	1.28
G3 / 50	Mean	10.50	10.50	10.50	10.00
	SD	1.20	1.20	1.20	1.00
G4 / 75	Mean	10.10	10.60	10.40	9.90
	SD	1.22	0.92	1.28	0.83
Urine pools (count)
G1 / 0	Mean	2.00	3.40	2.20	2.20
	SD	0.63	1.02	0.60	0.60
G2 / 25	Mean	2.30	2.20	2.10	2.30
	SD	0.46	0.60	0.70	0.64
G3 / 50	Mean	2.90	2.30	2.40	2.50
	SD	0.70	0.64	0.80	0.67
G4 / 75	Mean	2.80	2.20	2.60	2.60
	SD	0.75	0.60	0.80	0.80
Fecal (count)
G1 / 0	Mean	0.90	1.50	0.90	0.90
	SD	0.70	0.81	0.54	0.83
G2 / 25	Mean	0.50	0.70	0.90	0.90
	SD	0.50	0.46	0.83	0.83
G3 / 50	Mean	0.80	0.80	0.90	1.30
	SD	0.60	0.60	0.54	1.00
G4 / 75	Mean	0.70	1.10	0.80	0.70
	SD	0.64	0.54	0.60	0.64

 

Key: n=10 for G1, n=10 for G2, n=9 for G3, n=9 for G4 and G1- Vehicle control, G2-Low dose, G3-Mid dose,

G4-High dose

 

 

Table 7 - Summary of DETAILED Clinical Signs OF TOXICITY – FEMALES (LACTATION PERIOD)

Home cage observation	Days	Groups / Dose (mg/kg bw/day)
		G1 / 0	G2 / 25	G3 / 50	G4 / 75
	LD 6	3R,7S,0A	4R,6S,0A	3R,6S,0A	3R,6S,0A
Animal body position/Posture   R - Rearing S - Sitting or Standing normally A – Asleep	LD 13	5R,5S,0A	4R,6S,0A	4R,5S,0A	4R,5S,0A
Respiration – Normal	LD 6,13	10/10	10/10	10/10	10/10
Vocalization – Normal		10/10	10/10	10/10	10/10
Hand-held observation
Ease of removal - Easy/Normal	LD 6,13	10/10	10/10	9/9	9/9
Handling reactivity- Easy to handle/ normal		10/10	10/10	9/9	9/9
Palpebral closure - Open		10/10	10/10	9/9	9/9
Lacrimation - No lacrimation		10/10	10/10	9/9	9/9
Nasal discharge - No discharge		10/10	10/10	9/9	9/9
Salivation - No salivation		10/10	10/10	9/9	9/9
Teeth - Normal		10/10	10/10	9/9	9/9
Fur coat/ Skin - Normal		10/10	10/10	9/9	9/9
Muscle tone/ Mass - Normal		10/10	10/10	9/9	9/9
Perineum wetness - None/normal		10/10	10/10	9/9	9/9
Tail - Normal		10/10	10/10	9/9	9/9
Open-field observation
Gait - Head is horizontal, abdomen rises slightly above floor, and body moves up and down slightly during walking	LD 6,13	10/10	10/10	9/9	9/9
Arousal - Normal		10/10	10/10	9/9	9/9
Clonic convulsion - None/ normal		10/10	10/10	9/9	9/9
Tonic Convulsion - None/ normal		10/10	10/10	9/9	9/9
Stereotype - None		10/10	10/10	9/9	9/9
Bizarre /Abnormal behaviour(s) - None		10/10	10/10	9/9	9/9
Fecal consistency - Formed/ normal		10/10	10/10	9/9	9/9

 

Key: n=10 for G1, n=10 for G2, n=9 for G3, n=9 for G4 and G1- Vehicle control, G2-Low dose, G3-Mid dose,

G4-High dose

TABLE 7 (CONTD.) - SUMMARY OF DETAILED CLINICAL SIGNS OF TOXICITY – FEMALES (LACTATION PERIOD)

Open- field observation (contd.)
Rearing count
Group / Dose (mg/kg bw/day)		LD 6	LD 13
G1 / 0	Mean	19.80	20.67
	SD	1.08	1.63
G2 / 25	Mean	19.80	20.20
	SD	0.87	1.40
G3 / 50	Mean	19.44	20.22
	SD	0.96	1.47
G4 / 75	Mean	19.89	20.33
	SD	0.99	1.76
Urine pools (count)
G1 / 0	Mean	2.00	2.50
	SD	0.77	0.81
G2 / 25	Mean	2.10	3.10
	SD	0.54	0.54
G3 / 50	Mean	1.89	2.67
	SD	0.74	0.67
G4 / 75	Mean	2.33	2.67
	SD	0.67	0.67
Fecal (count)
G1 / 0	Mean	1.40	1.50
	SD	0.49	0.67
G2 / 25	Mean	1.50	1.90
	SD	0.50	0.70
G3 / 50	Mean	1.56	1.56
	SD	0.68	0.83
G4 / 75	Mean	1.67	1.00
	SD	0.47	0.67

 

Key: n=10 for G1, n=10 for G2, n=9 for G3, n=9 for G4 and G1- Vehicle control, G2-Low dose, G3-Mid dose,

G4-High dose

 

Table 8 - Summary of Body Weights– Males

Average Body Weights (g) - Males
Days
Groups Dose (mg/kg bw/day)		1	8	15	22	28
G1 0	Mean	475.67	493.05	504.05	513.49	521.70
	SD	17.26	18.32	17.36	14.31	14.87
	N	10	10	10	10	10
G2 25	Mean	474.76	486.92	495.83	504.84	511.02
	SD	17.48	20.74	22.05	19.74	20.00
	N	10	10	10	10	10
G3 50	Mean	474.43	475.29	483.94*	491.17*	495.73*
	SD	19.08	17.84	14.24	14.94	17.26
	N	10	10	10	10	10
G4 75	Mean	475.12	465.99*	472.58*	479.16*	483.90*
	SD	15.83	16.97	15.30	15.22	13.18
	N	10	10	10	10	10

          Key: N = 10 for G1, G2; N= 09 for G3 and G4; Values are in Mean ± SD ; *= Statistically significant from vehicle control (P ≤ 0.05).

 

Table 9 - SUMMARY OF NET BODY WEIGHT GAIN – Males

Groups Dose (mg/kg bw/day)	From To	1 8	8 15	15 22	22 28	Abs Gain 1 28	Total Gain % 1 28
G1 0	Mean	17.37	11.01	9.44	8.21	46.03	9.71
	SD	5.75	3.65	3.81	2.38	6.35	1.56
	N	10	10	10	10	10	10
G2 25	Mean	12.16	8.91	9.01	6.19	36.27	7.65
	SD	4.84	5.03	5.32	2.09	9.64	2.05
	N	10	10	10	10	10	10
G3 50	Mean	0.86*	8.65	7.23	4.10	21.31*	4.54*
	SD	6.34	8.70	4.25	6.35	12.88	2.85
	N	10	10	10	10	10	10
G4 75	Mean	-9.13*	6.59	6.59	3.49	8.78*	1.88*
	SD	6.52	5.05	3.33	4.26	7.99	1.73
	N	10	10	10	10	10	10

 

          Key: N = 10 for G1, G2; N= 09 for G3 and G4; Values are in Mean ± SD; *= Statistically significant from vehicle control (P ≤ 0.05).

 

TABLE 10 - SUMMARY OF BODY WEIGHTS DURING PRE MATING PERIOD – FEMALES

Body Weights During Pre-Mating Period – Females
Days
Groups Dose (mg/kg bw/day)		1	8	15
G1 0	Mean	272.20	281.96	286.99
	SD	9.90	11.34	8.47
	N	10	10	10
G2 25	Mean	271.07	285.39	291.82
	SD	11.55	11.73	11.38
	N	10	10	10
G3 50	Mean	270.33	274.22	280.48
	SD	9.30	11.35	12.92
	N	10	10	10
G4 75	Mean	272.03	273.98	279.23
	SD	7.85	10.81	11.99
	N	10	10	10

          Key: N = 10 for G1, G2; N= 09 for G3 and G4; Values are in Mean ± SD.

 

TABLE 11 - SUMMARY OF NET BODY WEIGHT GAIN DURING PRE-MATING PERIOD – FEMALES

Groups Dose (mg/kg bw/day)		From To	1 8	8 15	Abs. Gain 1 15	Total % Gain 1 15
G1 0	Mean		9.76	5.03	14.79	5.46
	SD		3.70	4.20	3.34	1.35
	N		10	10	10	10
G2 25	Mean		14.32	6.42	20.74	7.68
	SD		4.40	1.30	3.55	1.41
	N		10	10	10	10
G3 50	Mean		3.89*	6.26	10.15	3.73
	SD		3.53	4.16	6.04	2.18
	N		10	10	10	10
G4 75	Mean		1.95*	5.25	7.20	2.65
	SD		6.72	6.61	9.21	3.41
	N		10	10	10	10

 

          Key: N = 10 for G1, G2; N= 09 for G3 and G4; Values are in Mean ± SD; *= Statistically significant from vehicle control (P ≤ 0.05).

 

TABLE 12 - SUMMARY OF CAGE WISE AVERAGE FOOD INTAKE – MALES (PRE-MATING Period)

Groups Dose (mg/kg bw/day)	Weeks
	1		2
G1 0	Mean	32.27	31.69
	SD	0.79	0.45
	N	10	10
G2 25	Mean	29.71	31.06
	SD	1.55	1.51
	N	10	10
G3 50	Mean	24.59*	30.34
	SD	1.17	0.65
	N	10	10
G4 75	Mean	22.64*	28.79*
	SD	0.95	0.65
	N	10	10

 

               Key: N = 10 for G1, G2; N= 09 for G3 and G4; Values are in Mean ± SD; *= Statistically significant from vehicle control (P ≤ 0.05).

 

TABLE 13- SUMMARY OF CAGE WISE AVERAGE FOOD INTAKE (PRE-MATING PERIOD) – FEMALES

Groups Dose (mg/kg bw/day)	Weeks
	1		2
G1 0	Mean	21.13	22.11
	SD	1.56	1.12
	N	10	10
G2 25	Mean	19.98	22.02
	SD	1.45	1.31
	N	10	10
G3 50	Mean	18.65	23.13
	SD	1.25	1.59
	N	10	10
G4 75	Mean	18.87	23.16
	SD	2.18	0.76
	N	10	10

 

     Key: N = 10 for G1, G2; N= 09 for G3 and G4; Values are in Mean ± SD

 

 

TABLE 14 - SUMMARY OF MATERNAL BODY WEIGHTS (g) and BOdy weight changes DURING GESTATION PERIOD

MATERNAL BODY WEIGHTS DURING GESTATION PERIOD (g)
Groups Dose (mg/kg bw/day)		Body weight on Gestation Day				Body weight change during Gestation (Day)
		0	7	14	20	0-7	7-14	14-20	0-20
G1 0	Mean	285.85	315.76	346.24	433.23	26.54	32.43	85.01	143.97
	SD	11.37	11.05	14.19	26.10	5.99	6.45	14.01	19.07
	N	10	10	10	10	10	10	10	10
G2 25	Mean	289.91	325.33	361.33	457.77*	35.27	38.19	96.25	169.71
	SD	12.87	23.66	27.63	35.34	15.73	6.48	16.67	29.81
	N	10	10	10	10	10	10	10	10
G3 50	Mean	284.63	310.11	338.04	421.97	22.15	32.86	80.13	135.14
	SD	13.38	14.94	15.21	17.47	5.61	5.08	3.91	7.17
	N	09	09	09	09	09	09	09	09
G4 75	Mean	281.92	308.18	341.33	438.33	24.63	32.49	86.49	143.62
	SD	12.54	14.24	12.89	24.94	8.29	4.42	14.18	18.27
	N	09	09	09	09	09	09	09	09

 

         Key: N = 10 for G1, G2; N= 09 for G3 and G4; Values are in Mean ± SD; *= Statistically significant from vehicle control (P ≤ 0.05).

 

Table 15 - Summary of MATERNAL FOOD INTAKE DURING GESTATION PERIOD

Maternal food intake (g) during Gestation period
Gestation Period (Day)
Groups Dose (mg/kg bw/day)	From To	0 7	7 14	14 20	0 20
G1 0	Mean	26.10	28.12	27.38	27.20
	SD	2.28	1.71	2.16	1.02
	N	10	10	10	10
G2 25	Mean	25.72	29.69	27.90	27.77
	SD	3.22	3.05	3.21	1.99
	N	10	10	10	10
G3 50	Mean	24.75	27.79	26.12	26.22
	SD	2.40	1.67	2.51	1.52
	N	09	09	09	09
G4 75	Mean	24.26	26.92	25.93	25.70
	SD	2.47	2.03	2.51	1.35
	N	09	09	09	09

                   

          Key: N = 10 for G1, G2; N= 09 for G3 and G4; Values are in Mean ± SD

 

Table 16 -Summary of Maternal Body Weights (g) During THE LACTATION Period

Groups Dose (mg/kg bw/day)		Body weight (g) during Lactation period (Day)			Body weight change during Lactation period (Day)
		0	4	13	0 - 4	4 - 13
G1 0	Mean	334.80	341.24	347.16	6.44	5.92
	SD	23.97	15.46	20.27	14.76	8.61
	N	10	10	10	10	10
G2 25	Mean	346.40	350.12	351.41	3.72	1.28
	SD	21.65	25.32	25.03	16.32	14.44
	N	10	10	10	10	10
G3 50	Mean	321.87	329.59	331.67	7.72	2.08
	SD	19.61	18.30	22.59	13.17	8.43
	N	10	10	10	10	10
G4 75	Mean	318.42	325.03	329.50	6.62	4.47
	SD	18.54	17.46	17.27	12.64	7.88
	N	10	10	10	10	10

          Key: N = 10 for G1, G2, G3 and G4; Values are in Mean ± SD

 

Table 17 - Summary of Maternal Food Intake (g) During the LACTATION period

Groups Dose (mg/kg bw/day)	Maternal food intake (g) during Lactation period
		Per day during Lactation (Day)
		0 - 6	6 - 13
G1 0	Mean	38.25	35.32
	SD	2.10	4.42
	N	10	10
G2 25	Mean	37.95	35.73
	SD	3.26	2.97
	N	10	10
G3 50	Mean	35.85	34.15
	SD	2.27	1.84
	N	09	09
G4 75	Mean	35.39	33.78
	SD	4.08	2.57
	N	09	09

      Key: N = 10 for G1, G2, G3 and G4; Values are in Mean ± SD

 

Table 18 - Summary of OESTOUS CYCLICITY – PRE-MATING

Groups Dose (mg/kg. bw/day)	Mean cycle length (days)	No. of animals with normal cycle	No. of animals with abnormal cycle
G1 0	4.00	10	0
G2 25	4.00	10	0
G3 50	4.50	10	0
G4 75	4.00	09	1

 

                              Key: N = 10 for G1, G2, G3 and G4; Values are in Mean ± SD

 

Table 19 –Summary REPORT OF REPRODUCTIVE / developmental effect

OBSERVATIONS	VALUES
Groups	G1	G2	G3	G4
Dose (mg/kg bw/day)	0	25	50	75
Pairs started (N)	10	10	10	10
Females showing evidence of copulation (N)	10	10	9	9
Females achieving pregnancy (N)	10	10	9	9
Conceiving days 1 - 5 (N)	9	8	9	8
Conceiving days 6 - 14 (N)	1	2	-	1
Conceiving during remating (N)	-	-	-	-
Pregnancy ≤ 21 days (N)	2	3	-	4
Pregnancy = 22 days (N)	8	6	9	5
Pregnancy ≥ 23 days (N)	-	1	-	-
Dams with live young born (N)	10	10	9	9
Dams with live young at day 4 pp (N)	10	10	9	9
Implants/dam (mean)	12.90	15.00	14.22	15.78
Live pups per dam on PND 0 (mean)	12.50	14.50	13.78	14.67
Live pups per dam on PND 4 (mean)	12.50	14.50	13.78	14.67
Live pups per dam on PND 13 (mean)	10.00	12.60	11.78	12.67
Sex ratio (m/f) on PND 0 (mean)	1.67	1.24	1.41	1.86
Sex ratio (m/f) on PND 4 (mean)	1.67	1.24	1.41	1.86
Pup weight (gm)  on PND 0 – Male	7.45	7.28	7.50	7.19
Pup weight (gm) on PND 0 – Female	6.99	6.93	7.21	6.60
Pup body weight (gm) on PND 0 (both sexes combined)	7.22	7.10	7.36	6.89
Pup weight (gm)  on PND 4 – Male	13.01	11.45	11.57	11.31
Pup weight (gm)  on PND 4 – Female	12.27	11.04	11.38	10.87
Pup body weight (gm) on PND 4 (both sexes combined)	12.64	11.24	11.48	11.09
Pup weight (gm)  on PND 13 – Male	34.13	31.77	31.60	30.61
Pup weight (gm)  on PND 13 – Female	32.99	31.17	31.02	29.40
Pup body weight (gm) on PND 13 (both sexes combined)	33.56	31.47	31.31	30.01
Litter weight (gm)  on PND 0 (mean)	91.22	98.10	101.27	91.57
Litter weight (gm) on PND 4 (mean)	156.82	161.28	140.94	161.06
Litter weight on PND 13 (mean)	360.40	389.25	333.32	343.00
AGD pups (mm)- Male	3.07	3.00	2.97	2.99
AGD pups (mm)- Female	1.46	1.13	1.13	1.16
Normalized AGD pups (mm) – Male	1.45	1.44	1.44	1.44
Normalized AGD pups (mm) – Female	1.17	1.13	1.13	1.16
No of nipples/ Areolae - male pups on PND 12 (mean)	0.0	0.0	0.0	0.0
No. of abnormal pups
Dams with 0	0	0	0	0
Dams with 1	0	0	0	0
Dams with ≥ 2	0	0	0	0

 

TABLE 19 (CONTD.) – STUDY SUMMARY REPORT OF REPRODUCTIVE / DEVELOPMENTAL EFFECT

OBSERVATIONS	VALUES
Groups	G1	G2	G3	G4
Dose (mg/kg bw/day)	0	25	50	75
LOSS OF OFFSPRING
Pre -natal (implantations minus live births)
Females with 0	10	10	09	09
Females with 1	0	0	0	0
Females with 2	0	0	0	0
Females with ≥ 3	0	0	0	0
Post-natal (live births minus alive at post-natal day 4)
Females with 0	0	0	0	0
Females with 1	0	0	0	0
Females with 2	0	0	0	0
Females with ≥ 3	0	0	0	0
Post-natal (live births minus alive at post-natal day 13)
Females with 0	0	0	0	0
Females with 1	0	0	0	0
Females with 2	0	0	0	0
Females with ≥ 3	0	0	0	0

 

Table 20 – Reproduction Indices

Indices	Groups Dose (mg/kg bw/day)	G1	G2	G3	G4
		0	25	50	75
Male mating index (%)		100	100	90	90
Male fertility index (%)		100	100	90	90
Female mating index (%)		100	100	90	90
Female fecundity index (%)		100	100	90	90
Female fertility index (%)		100	100	90	90
Post-implantation loss (%) – Mean		0.63	3.27	2.88	6.54
Gestation index (%)		100	100	100	100
Live birth index (%)		100	100	100	100
Day 4 survival index (%)		100	100	100	100
Day 13 survival index (%)		79.18	86.63	85.32	86.17

 

Table 21 – SUMMARY OF Implantation in dam

Groups Dose (mg/kg bw/day)	Implantations
G1 0	Mean	12.90
	SD	3.25
	N	10
G2 25	Mean	15.00
	SD	1.76
	N	10
G3 50	Mean	14.22
	SD	1.86
	N	09
G4 75	Mean	15.78
	SD	2.05
	N	09

                                                Key: N = 10 for G1, G2 and N = 09 for G3 and G4; Values are in Mean ± SD.

 

              TABLE 22 - SUMMARY OF STAGE OF OESTROUS CYCLE (VAGINAL SMEAR) AT TERMINAL SACRIFICE) – FEMALES             

Stage of Estrous Cycle	Groups Dose (mg/kg bw/day)
	G1 0	G2 25	G3 50	G4 75
P	2	1	-	1
PE	1	1	1	2
E	4	2	1	3
EM	-	-	1	-
M	3	3	4	2
MD	-	2	-	1
D	-	1	2	-
DP	-	-	1	1

 

    Key: N= 10; P=Proestrus; E=Estrus; M=Metestrus=Diestrus; Transitional stages: P/E, E/M, M/D and D/P.        

 

TABLE 23- Summary of MEAN LITTER WEIGHT (g) during the Lactation Period

Groups Dose (mg/kg bw/day)		Mean Litter Weight (g) During the Lactation Period
		PND 0	PND 4
G1 0	Mean	91.22	156.82
	SD	16.66	25.87
	N	128	128
G2 25	Mean	98.10	161.28
	SD	9.53	21.95
	N	145	145
G3 50	Mean	101.27	140.94
	SD	9.10	51.67
	N	124	124
G4 75	Mean	91.57	161.06
	SD	33.90	18.01
	N	132	132

 

                                       Key: N = 128 for G1, 128 for G2, 124 for G3,132 for G4 and values are G2, G3 and; Values are in Mean ± SD

 

 

TABLE 24 - Summary of MEAN BODY WEIGHT (g) of MALE Pups during the Lactation Period

Groups Dose (mg/kg bw/day)		Mean Body Weight (g) of Male Pups During the Lactation Period
		PND 0	PND 4	PND 13
G1 0	Mean	7.45	13.01	34.13
	SD	0.95	1.95	3.54
	N	72	72	71
G2 25	Mean	7.26	11.45	31.77
	SD	0.51	1.56	3.70
	N	75	75	75
G3 50	Mean	7.50	11.57	31.60
	SD	0.41	0.83	2.23
	N	68	68	68
G4 75	Mean	7.19	11.31	30.61
	SD	0.49	1.18	2.93
	N	80	80	80

 

Key: N= 10; Values are in Mean ± SD

 

TABLE 25 - Summary of MEAN BODY WEIGHT (g) of FEMALE Pups during the Lactation Period

Groups Dose (mg/kg bw/day)		Mean Body Weight (g) of Female Pups During the Lactation Period
		PND 0	PND 4	PND 13
G1 0	Mean	6.99	12.27	32.99
	SD	0.91	1.95	2.98
	N	56	56	37
G2 25	Mean	6.93	11.04	31.17
	SD	0.64	1.91	3.86
	N	70	70	50
G3 50	Mean	7.21	11.38	31.02
	SD	0.49	1.10	1.75
	N	56	56	38
G4 75	Mean	6.60	10.87	29.40
	SD	0.66	1.30	2.61
	N	52	52	34

 

                                       Key: N= 10; Values are in Mean ± SD; * = significantly higher than vehicle control (P ≤ 0.05).

 

TABLE 26 - Summary of Anogenoital distance – MALE PUPS

Groups Dose (mg/kg bw/day)		Absolute AGD (mm)	Normalised AGD
G1 0	Mean	3.07	1.45
	SD	0.11	0.02
	N	72	72
G2 25	Mean	3.00	1.44
	SD	0.15	0.03
	N	75	75
G3 50	Mean	2.97	1.44
	SD	0.09	0.01
	N	68	68
G4 75	Mean	2.99	1.44
	SD	0.07	0.01
	N	80	80

 

                       Key: N = 49 for G1, N= 62 for G2, N= 50 for G3 and N=52 for G4; Values are in Mean ± SD; Normalized AGD = AGD (mm)/ 3√pups body weight (gm).

 

TABLE 27- Summary of Anogenoital distance – FEMALE PUPS

Groups Dose (mg/kg bw/day)		Absolute AGD (mm)	Normalised AGD
G1 0	Mean	1.46	1.17
	SD	0.06	0.10
	N	49	49
G2 25	Mean	1.47	1.13
	SD	0.12	0.03
	N	62	62
G3 50	Mean	1.43	1.13
	SD	0.15	0.04
	N	50	50
G4 75	Mean	1.57	1.16
	SD	0.13	0.03
	N	52	52

 

         Key: N = 49 for G1, N= 62 for G2, N= 50 for G3 and N=52 for G4; Values are in Mean ± SD; Normalized AGD = AGD (mm)/ 3√pups body weight (gm).

 

TABLE 28 - Summary of SEX RATIO ON PND 0

Groups Dose (mg/kg bw/day)		Sex ratio (m/f)
G1 0	Mean	1.67
	SD	1.37
	N	128
G2 25	Mean	1.24
	SD	0.78
	N	145
G3 50	Mean	1.41
	SD	0.88
	N	124
G4 75	Mean	1.86
	SD	1.34
	N	132

Key: N (Total no. pups) = 128 for G1, N= 62 for G2, N= 50 for G3 and N=52 for G4; Values are in Mean ± SD; Values are in Mean ± SD; m/f =male pups /female pups.

 

TABLE 28 (CONTD.) - SUMMARY OF SEX RATIO ON PND 4

Groups Dose (mg/kg bw/day)		Sex ratio (m/f)
G1 0	Mean	1.67
	SD	1.37
	N	128
G2 25	Mean	1.24
	SD	0.78
	N	145
G3 50	Mean	1.41
	SD	0.88
	N	124
G4 75	Mean	1.86
	SD	1.34
	N	132

Key: N (Total no. pups) = 128 for G1, N= 62 for G2, N= 50 for G3 and N=52 for G4; Values are in Mean ± SD; Values are in Mean ± SD; m/f =male pups /female pups.

 

TABLE 29 -Summary of NIPPLE RETENSION/ areolae- MALE PUPS

Groups Dose (mg/kg bw/day)		Total no. of nipple retention/ areolae
G1 0	Mean	0.00
	SD	0.00
	N	72
G2 25	Mean	0.00
	SD	0.00
	N	75
G3 50	Mean	0.00
	SD	0.00
	N	68
G4 75	Mean	0.00
	SD	0.00
	N	80

                                      Key: N= 72 for G1, N=75 for G2, N=68 for G3 and N=80 for G4; Values are in Mean ± SD; Values are in Mean ± SD.

 

Table 30 - Summary of THYROID HORMONE ANALYSIS – MALES/SIREs

Parameters
Groups Dose (mg/kg bw/day)		T4	TSH
		ng/mL	pg/mL
G1 0	Mean	30.45	182.67
	SD	9.44	19.20
	N	10	10
G2 25	Mean	29.92	184.72
	SD	8.58	9.56
	N	10	10
G3 50	Mean	35.67	183.99
	SD	11.03	7.65
	N	10	10
G4 75	Mean	39.71	176.70
	SD	7.97	7.30
	N	10	10

 

                          Key: N = 10 for G1, G2; N = 9 for G3, G4; Values are in Mean ± SD; Values are in Mean ± SD

 

Table 31 - Summary of THYROID HORMONE ANALYSIS – DAY 13 PROGENY-MALE PUPS

Parameters
Groups Dose (mg/kg bw/day)		T4	TSH
		ng/mL	pg/mL
G1 0	Mean	31.83	168.06
	SD	6.47	4.97
	N	10	10
G2 25	Mean	31.77	172.05
	SD	6.36	5.73
	N	10	10
G3 50	Mean	26.81	167.28
	SD	5.54	6.03
	N	10	10
G4 75	Mean	32.71	161.26*
	SD	10.05	4.87
	N	10	10

Key: N = 10 for G1, G2; N = 9 for G3, G4; Values are in Mean ± SD; Values are in Mean ± SD; *= Statistically significant from vehicle control (P ≤ 0.05).

 

Table 32 - Summary of THYROID HORMONE ANALYSIS – DAY 13 PROGENY-FEMALE PUPS

Parameters
Groups Dose (mg/kg bw/day)		T4	TSH
		ng/mL	pg/mL
G1 0	Mean	33.40	163.19
	SD	4.31	17.50
	N	10	10
G2 25	Mean	40.10*	197.40*
	SD	5.35	7.41
	N	10	10
G3 50	Mean	41.28*	198.82*
	SD	4.59	5.34
	N	10	10
G4 75	Mean	36.24	193.05
	SD	8.68	4.70
	N	10	10

Key: N = 10 for G1, G2, G3 and G4; Values are in Mean ± SD; Values are in Mean ± SD; *= Statistically significant from vehicle control (P ≤ 0.05).

 

Table 33– SUMMARY OF TERMINAL FASTING BODY WEIGHT AND ABSOLUTE ORGAN WEIGHTS (g) – MALES

Groups Dose (mg/kg/day)		Fasting Body Weight and Absolute Organ Weights (g) – Males
		FBW	Testes	epididymis	Prostate+seminal vesicles with Coagulating glands	Thyroid with Parathyroid
		(g)	(g)	(g)	(g)	(g)
G1 0	Mean	507.69	4.096	1.889	3.851	0.320
	SD	12.09	0.130	0.268	0.842	0.141
	N	10	10	10	10	10
G2 25	Mean	497.16	4.128	1.896	3.857	0.229
	SD	24.42	0.466	0.214	0.494	0.050
	N	10	10	10	10	10
G3 50	Mean	485.81*	4.590	1.850	3.668	0.266
	SD	17.47	1.415	0.126	0.394	0.059
	N	10	10	10	10	10
G4 75	Mean	472.65*	4.149	1.738	3.519	0.292
	SD	14.68	0.373	0.153	0.327	0.072
	N	10	10	10	10	10

        Key: N= number of animals; FBW=Fasting Body Weight Values are in Mean ± SD; * = significantly lesser than vehicle control (P ≤ 0.05).

 

Table 34– SUMMARY OF TERMINAL FASTING BODY WEIGHT AND ABSOLUTE ORGAN WEIGHTS (g) – FEMALES

Groups Dose (mg/kg/day)		Fasting Body Weight and Absolute Organ Weights (g) – Females
		FBW	Ovaries	Uterus	Thyroid with parathyroid
		(g)	(g)	(g)	(g)
G1 0	Mean	336.01	0.235	0.649	0.165
	SD	19.78	0.029	0.038	0.061
	N	10	10	10	10
G2 25	Mean	337.65	0.257	0.651	0.165
	SD	22.80	0.040	0.074	0.057
	N	10	10	10	10
G3 50	Mean	321.27	0.233	0.680	0.204
	SD	22.02	0.022	0.041	0.085
	N	9#	9#	9#	9#
G4 75	Mean	318.92	0.237	0.669	0.191
	SD	22.30	0.028	0.047	0.060
	N	9#	9#	9#	9#

      Key: N= number of animals; FBW=Fasting Body Weight Values are in Mean ± SD; # - One animal in G3 and G4 was non-pregnant

 

TABLE 35- SUMMARY OF RELATIVE ORGAN WEIGHTS (%) – MALES

Groups Dose (mg/kg/day)		Relative Organ Weights (%) – Males
		Testes	epididymis	Prostate+seminal vesicles with Coagulating glands	Thyroid with Parathyroid
		(%)	(%)	(%)	(%)
G1 0	Mean	0.807	0.373	0.761	0.063
	SD	0.028	0.056	0.183	0.028
	N	10	10	10	10
G2 25	Mean	0.832	0.381	0.778	0.046
	SD	0.100	0.036	0.107	0.011
	N	10	10	10	10
G3 50	Mean	0.948	0.381	0.755	0.055
	SD	0.305	0.029	0.073	0.013
	N	10	10	10	10
G4 75	Mean	0.878	0.368	0.746	0.062
	SD	0.080	0.033	0.081	0.015
	N	10	10	10	10

Key: N= number of animals; Values are in Mean ± SD

 

TABLE 36 – SUMMARY OF RELATIVE ORGAN WEIGHTS (%) – FEMALES

Groups Dose (mg/kg/day)		Relative Organ Weights (%)  – Females
		Ovaries	Uterus	Thyroid with parathyroid
		(%)	(%)	(%)
G1 0	Mean	0.070	0.194	0.049
	SD	0.012	0.011	0.017
	N	10	10	10
G2 25	Mean	0.077	0.194	0.049
	SD	0.015	0.026	0.018
	N	10	10	10
G3 50	Mean	0.075	0.212*	0.065
	SD	0.012	0.013	0.025
	N	9#	9#	9#
G4 75	Mean	0.078	0.218	0.058
	SD	0.015	0.029	0.019
	N	9#	9#	9#

          Key: N= number of animals; Values are in Mean ± SD; * = significantly lesser than vehicle control (P ≤ 0.05); # - One animal in G3 and G4 was non-pregnant

 

Table 37 – Summary of THYROID WEIGHT – DAY 13 PROGENY- MALE PUPS

Parameters
Groups Dose (mg/kg bw/day)	Thyroid with parathyroid weight (g)
G1 0	Mean	0.031
	SD	0.006
	N	10
G2 25	Mean	0.027
	SD	0.007
	N	10
G3 50	Mean	0.028
	SD	0.004
	N	9#
G4 75	Mean	0.027
	SD	0.005
	N	9#

               Key: N= number of animals; # - One animal in G3 and G4 was non-pregnant.

 

Table 38 – Summary of THYROID WEIGHT – DAY 13 PROGENY- feMALE PUPS

Parameters
Groups Dose (mg/kg bw/day)	Thyroid with parathyroid weight (g)
G1 0	Mean	0.028
	SD	0.008
	N	10
G2 25	Mean	0.026
	SD	0.007
	N	10
G3 50	Mean	0.027
	SD	0.008
	N	9#
G4 75	Mean	0.029
	SD	0.005
	N	9#

            Key: N= number of animals; # - One animal in G3 and G4 was non-pregnant.

 

Table 39- SUMMARY OF EXTERNAL AND GROSS PATHOLOGICAL OBSERVATIONS – MALES

Group	G1	G2	G3	G4
No. of male Animals Observed	10	10	10	10
External Observations^
NAD	10/10	10/10	10/10	10/10
Internal Observations^
NAD	10/10	0/10	0/10	0/10
Intestine – Bluish discoloration	0/10	10/10	10/10	10/10

 

                       Key: N= number of animals; NAD= No Abnormality Detected, ^= Incidence

 

Table 40 – SUMMARY OF EXTERNAL AND GROSS PATHOLOGICAL OBSERVATIONS – FEMALES

Group	G1	G2	G3	G4
No. of Females/Dams	10	10	10	10
External Observations^
NAD	10/10	10/10	10/10	10/10
Internal Observations^
NAD	10/10	0/10	0/10	0/10
Intestine – Bluish discoloration	0/10	10/10	10/10	10/10

 

                    Key: N= number of animals; NAD= No Abnormality Detected;

 

Table 41 – SUMMARY OF EXTERNAL AND GROSS PATHOLOGICAL OBSERVATIONS OF PUPS

Group	G1	G2	G3	G4
No. of Pup Observed	128	145	124	132
External Observations^
NAD	128/128	145/145	124/124	132/132

 

      Key: NAD= No Abnormality Detected.

 

Table 42-SUMMARY: HISTOPATHOLOGY OBSERVATIONS – MALE AND FEMALE

Tissue/ Findings/Sex	Males		Females
Dose Group	G1	G4	G1	G4
Number Examined	10	10	10	09
NAD	9/10	10/10	10/10	09/09
Epididymides
Infiltration, Mononuclear cells, focal, Minimal	1/10	0/10	0/10	0/09

 

Key: NAD= No Abnormality Detected.

 

Table 43 -SUMMARY: HISTOPATHOLOGY OBSERVATIONS – FEMALES, NON-PREGNANT

Tissue/ Findings/Sex	Females
Dose Group	G3	G4
Number Examined	1	1
NAD	1/1	1/1

 

Key: NAD= No Abnormality Detected.

 

APPENDIX 16: INDIVIDUAL ANIMAL HISTOPATHOLOGY PUPS (PND 13) - MALE & FEMALE (G1)

 

Organ/Microscopic Finding

Dam Number

11

12

13

14

15

16

17

18

19

20

Thyroid

NAD

NAD

NAD

NAD

NAD

NAD

NAD

NAD

NAD

NAD

Parathyroid

NAD

NAD

NAD

NAD

NAD

NAD

NAD

NAD

NAD

NAD

 

Note: Severity: - 1: Minimal; 2: Mild; 3: Moderate,4: Severe; Distribution: - F: Focal; M: Multifocal; D: Diffused; NAD: No Abnormality Detected

 

APPENDIX 17: INDIVIDUAL ANIMAL HISTOPATHOLOGY – PUPS (PND 13) – MALE & FEMALE – (G4)

Organ/Microscopic Finding

Dam Number

71

72

73

74

76

77

78

79

80

Thyroid

NAD

NAD

NAD

NAD

NAD

NAD

NAD

NAD

NAD

Parathyroid

NAD

NAD

NAD

NAD

NAD

NAD

NAD

NAD

NAD

 

Note: Severity: - 1: Minimal; 2: Mild; 3: Moderate,4: Severe; Distribution: - F: Focal; M: Multifocal; D: Diffused; NAD: No Abnormality Detected

Applicant's summary and conclusion

Conclusions:

Based on the above data and under the experimental conditions used in this study, the following effect levels were derived:

- NOAEL for general toxicity in male rats was considered to be 25 mg/kg bw/day
- LOAEL for general toxicity in male rats was considered to be 50 mg/kg bw/day based on significant decreases in average body weight, body weight gain, food intake, and terminal fasting body weight at ≥50 mg/kg bw/day.
- NOAEL for general toxicity in female rats was considered to be 50 mg/kg bw/day.
- LOAEL for general toxicity in female rats was considered to be 75 mg/kg bw/day based on clinical signs of toxicity (lethargy) which was observed during gestation and lactation in all of the adult female rats, except in one that was found to be non-pregnant.
- NOAEL for reproductive and developmental toxicity was considered to be 75 mg/kg bw/day as no adverse effects on reproduction or development were observed. 

Executive summary:

The purpose of this reproduction/developmental screening test in Wistar rats was to generate toxicological information concerning the effects of test item, 4,4'-bis(dimethylamino)-4''-(methylamino) trityl alcohol (CAS: 561-41-1) on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition. One vehicle control (G1) and three graded dose levels of 25 (G2), 50 (G3) and 75 (G4) mg/kg bw/day, with each group consisting of 10 male and 10 female rats with normal estrous cyclicity, were employed in the study. The test item, 4,4'-bis(dimethylamino)-4''-methylamino) trityl alcohol in 0.5% CMC (in Milli-Q water), was administered by oral gavage to animals of both sexes at the doses of 25 (G2), 50 (G3) and 75 (G4) mg/kg bw/day throughout the study period. Similarly, the vehicle (i.e. 0.5% CMC in Milli-Q water) was administered to the male and female rats in the vehicle control group (G1). Oral administration was done at a fixed dose volume of 10 mL/kg bw. The males were dosed before mating (2 weeks) and during mating (2 weeks) for a total of 28 days, with the exception of two males (one in G3 and the other in G4) that were used for re-mating and consequently dosed for a total of 35 days. Females were dosed two weeks prior to mating (with the objective of covering at least two complete oestrous cycles), during the variable time to conception (mating and re-mating period as applicable), the gestation period and till post-natal day 13 (i.e. till the day before scheduled sacrifice). The stability of the test item formulation was determined prior to the start of treatment and the results indicated that formulations at all dose levels were stable for 6 hours at room temperature and for 24 hours at 2 to 8ºC. Formulation analysis for dose concentration verification and homogeneity was performed one time during the premating period and one time during the lactation period. The results of the dose formulation analyses showed that the mean concentrations of dose formulations were within ± 15% of the nominal concentration (from 93.70% to 105.05%), and the RSD of the top, mid and bottom layers were ≤10% (from 0.33% to 2.22%) in all analysis. Therefore, all dose formulations prepared in this study were considered accurate, precise, and homogenous. The following parameters were evaluated in this study: Mortality/morbidity, clinical signs, body weight, feed consumption, oestrous cyclicity, clinical pathology, thyroid hormone analysis, pre-coital interval, gestation length, litter size/litter weights on post-natal day (PND) 0, 4 and 13, sex ratio, anogenital distance (AGD), nipple retention of male pups on PND 12, pups survival index on PND 4 and 13, external observations, male/female mating index, male/female fertility index, fecundity index, gestation index, live birth index, implantation sites count, post implantation loss, organ weights and macro and micro examination of tissues/organs. Results: Mortality/Morbidity: No morbidity or mortality was observed in the study. Clinical Signs of Toxicity: All male and female rats in G2, G3 and G4 showed violet discoloration of faeces throughout the experimental period. All female rats in G4, except one non-pregnant animal, showed lethargy during the gestation and lactation. This lethargy was observed approximately 30 minutes after dosing and persisted up to 2 hours after dosing. No other clinical signs of toxicity were observed in the study. Body Weights / Body Weight Changes of Adult Animals: In G4 males, statistically significant decrease in body weight was observed on day 8 of treatment (mean, 465.99 g) compared to G1 (mean, 493.05 g). Further, in G3 and G4, statistically significant decreases in body weights were observed on day 15 (mean, G3=483.94, G4=472.58 g), on day 22 (mean, G3=491.17, G4=479.16 g) and day 28 of treatment (mean, G3=495.73, G4=483.90 g) compared to G1 (mean, 504.05; 513.49; and 521.70 g on day 15, 22, and 28, respectively. Statistically significant decreases in the body weight gain were observed in G3 between day 1-8 (mean, 0.86 g) and day 1-28 (mean, 21.31 g) and in G4 between day 1-8 (mean, -9.13 g) and 1-28 (mean, 8.78 gram) compared to G1 (mean, 11.01 and 46.03 between day 1-8 and 1-28, respectively). The observed changes in body weight and body weight gain in G3 and G4 males were considered to be adverse effects of treatment. In females, no significant changes in body weight were observed in G2, G3 or G4, with the exception of a significant increase in average body weight in G2 on GD 20 (mean, 457.77 g) compared to G1 (mean, 433.23 g) that was considered to be incidental and not toxicologically relevant. Significant decreases in body weight gain were observed from treatment day 1-8 in G3 (mean, 3.89 g) and G4 (mean, 1.95 g) compared to G1 (mean, 9.76). This effect was considered to be treatment-related but transient as no significant changes in body weight gain were observed afterwards. Feed Consumption: In males, statistically significant decreases in feed consumption were observed from day 1-8 in G3 (mean, 24.59 g) and G4 (mean, 22.64 g) compared to the G1 (mean, 32.27 g). In G4 males, a significant decrease in feed consumption was also observed from day 8-15 (mean, 28.79 gram) compared to G1 (mean,31.69 gram). No significant changes in feed consumption were observed for the female rats. The significant changes in feed consumption in the male rats were associated with significant decreases in body weight and body weight gain and were therefore considered to be treatment-related and adverse. Oestrous Cyclicity: Oestrous cycle lengths in G2 (mean, 4.00 days), G3 (mean, 4.00 days) and G4 (mean, 4.50 days) were comparable with G1 (mean, 4.00 days) and there were no significant changes observed in oestrous cycle pattern at any dose level except in G4, in which one animal showed the irregularity during the premating period (on day 7 onwards). This isolated change in one animal in G4 was considered incidental and not toxicologically relevant. Thyroid Hormone Analysis: No significant changes in thyroid hormone levels were observed in the adult male rats at any dose level. In male pups on PND 13, a statistically significant decrease in TSH levels was observed in G4 (mean, 161.26 pg/ml) compared to G1 (mean, 168.06 pg/ml). In female pups on PND 13, T4 levels were statistically increased in G2 (mean, 40.10 ng/ml) and G3 (mean, 41.28 ng/ml) compared to G1 (mean, 33.40 ng/ml) whereas TSH levels were statistically increased in G2 (mean, 197.40 pg/ml) and G3 (mean, 198.82 pg/ml) compared to G1 (mean, 163.19 pg/ml). These significant changes in thyroid hormone levels in the male and female pups were not associated with any significant changes in thyroid weight. Furthermore, there were no gross lesions or histopathological findings in the thyroid from any of the examined pups in G1 and G4. The observed changes in thyroid hormone levels in the female pups also lacked clear dose dependency. Therefore, the significant changes in thyroid hormone levels in the male and female pups were not considered to be adverse. Reproductive Performance and Development Indices: Both male and female mating and fertility indexes were 100, 100, 90 and 90% in G1, G2, G3, and G4, respectively. Female fecundity indexes were 100, 100, 90, and 90% in G1, G2, G3, and G4, respectively. The number of female rats conceiving at 1-5 days after mating were 9/10, 8/10, 9/10, and 8/10 in G1, G2, G3, and G4, respectively. The number of female rats conceiving at 6-14 days after mating were 1/10, 2/10, 0/10, and 1/10 in G1, G2, G3, and G4, respectively. No re-mating was needed in G1 and G2. One animal each in G3 and G4 not conceived during the initial mating period were subjected to re-mating with proven males from the same group. The failure of one animal each in G3 and G4 to achieve pregnancy was considered to be within normal biological variation for the species. For pregnant animals, no significant changes in gestation length were observed and gestation index was 100% in all groups. No significant changes in the number of implantations per dam were observed in G2 (mean, 15.00), G3 (mean, 14.22) or G4 (mean, 15.78) compared to G1 (mean, 12.90). No significant changes in percent post implantation losses were observed in G2 (3.27%), G3 (2.88%) or G4 (6.54%) compared to G1 (0.63%) and all of the values were consistent with the historical control range of the test facility (i.e.: 0.00 – 9.09%). Offspring Data: No statistically significant changes in litter size, litter weight, mean male pup body weight, mean female pup body weight, sex-combined pup weight, or sex ratio were observed in either G2, G3 or G4 when compared to G1. No statistically significant changes in AGD or normalised AGD was observed at any dose level. No nipples/areolae were retained in any of the male pups on PND 12. Live birth index was 100% in all groups and pup survival index was 100% on PND 4 in all groups. Pup survival indexes on PND 13 were 79.18, 86.63, 85.32 and 86.17% in G1, G2, G3 and G4, respectively. All pups were normal upon external examination. There were no significant changes observed in pup thyroid weight at any dose level. Terminal Fasting Body Weight, Absolute Organ Weight, and Relative Organ Weight: In male rats, significant decreases in terminal fasting body weights were observed in G3 (mean, 485.81 g) and G4 (mean 472.65 g) compared to G1 (mean, 507.69 g). This effect on terminal fasting body weight in G3 and G4 were considered to be adverse. In female rats, no significant changes in terminal fasting body weights were observed. Significant changes in relative organ weight were limited to a significant increase in relative uterus weight in G3 females (mean, 0.212%) compared to G1 females (mean, 0.194%) that was considered incidental and not toxicologically relevant. Necropsy and Gross Pathology: No gross findings were observed except for bluish discoloration of the gastrointestinal tract that was observed in all animals in G2, G3 and G4. This discoloration was attributed to the test item but was not considered to be adverse. No gross findings were observed in any of the pups. Histopathological Observations: Microscopic findings in the adult animals were limited to an incidence of focal area of mononuclear cell infiltration in the epididymides of one control group male animal (Animal No. 03) with minimal severity. No microscopic findings were observed during the qualitative assessment of spermatogenesis stages or during the morphological examination of interstitial testicular cell structures in G4 compared to G1. The stage of estrous cycle in each female rat in G1 and G4 at the terminal sacrifice was in correlation with the histology of respective female reproductive organs. In the pups that were subjected to microscopic examinations of the thyroids in G1 and G4, no histopathological findings were observed. Conclusion: Based on the above data and under the experimental conditions used in this study, the following effect levels were derived:

• NOAEL for general toxicity in male rats was considered to be 25 mg/kg bw/day.


• LOAEL for general toxicity in male rats was considered to be 50 mg/kg bw/day based on significant decreases in average body weight, body weight gain, food intake, and terminal fasting body weight at ≥50 mg/kg bw/day.


• NOAEL for general toxicity in female rats was considered to be 50 mg/kg bw/day.


• LOAEL for general toxicity in female rats was considered to be 75 mg/kg bw/day based on clinical signs of toxicity (lethargy) which was observed during gestation and lactation in all of the adult female rats, except in one that was found to be non-pregnant.


• NOAEL for reproductive and developmental toxicity was considered to be 75 mg/kg bw/day as no adverse effects on reproduction or development were observed.