Dodecan-1-ol, ethoxylated

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Reproductive toxicity study

In the OECD 422 study performed with Wistar rats, NOAEL for reproductive and developmental toxicity was found at 700 mg/kg bw/day. Adverse effects in general health included significant decreases in locomotor activity in male rats treated at 300 and 700 mg/kg bw/day and dullness in all rats (both male and females) treated at 700 mg/kg bw/day.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jan 2022 to Aug 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat is a common species used for conducting preclinical toxicological studies and recommended by various regulatory guidelines such as OECD Test Guideline 422.

Sex:

male/female

Details on test animals or test system and environmental conditions:

SOURCE OF ANIMALS:
Hylasco Biotechnology (India) Pvt. Ltd., CPCSEA Reg.No.1808/PO/RcBt/S/15/CPCSEA, Hyderabad – 500 078.

ANIMAL HUSBANDRY:
Acclimatization: The animals were acclimatized for 9 days to experimental room conditions. A veterinarian at the receipt and prior to start of treatment examined the animals for general health.

Environmental conditions: Animals were housed in environmentally monitored air-conditioned room with conditions such as adequate fresh air supply (air changes 13-14 per hour), the range of room temperature and relative humidity were 20.6 to 22.4 0C, 51 to 62 % respectively, with 12 hours’ light and 12 hours’ dark cycle. The anial husbandry conditions such as temperature and relative humidity were recorded once in a day.

Housing: Animals of the same sex and group were housed in two and three per cage in standard polycarbonate cages (Size: L 421 X B 290 X 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/nesting materials were provided in the dam cages from the day of advanced gestation (GD18). The cages were fitted with stainless steel mesh top grill with facilities for holding pelleted feed and drinking water bottle.

Water: Reverse osmosis water was provided ad libitum throughout the study period. The results of analysis of water samples are appended to the report.

Feed: The pellet feed was provided ad libitum throughout acclimatization and experimental period. SDS RM 5 (Batch 4939) manufactured by SDS, UK was provided.

Bedding: Autoclaved corncob were used as bedding material and changed along with the cage once a week. Contaminant and microbial analysis report was appended to the report.

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Milli-Q

Details on exposure:

Dose administration was performed at approximately the same time each day for all animals (within ± 2 hours) throughout the treatment period. The dose volume administered to each rat was at an equivolume of 10 mL/kg body weight throughout the study. The dose volumes were adjusted based on the most recently recorded body weight of each individual rat.

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. Mating was performed for only main study animals. Cohabitation has been continued until there was evidence of vaginal plug and/or sperm in the vaginal smear or for a maximum period of 14 days. Subsequently, pregnant females were housed individually through gestation and lactation (until PND 13) along with litters. Four females (one from each G2, G3 and two from G4), which was not mated within 14 days of pairing with the first male, have been re-mated with proven male of the same group for 7 days. Two females (one in each G2 and G4), which were not confirmed pregnancy within 7 days even after re-mating with second proven male, have been sacrificed by 26th day from last day of re-mating. The day of confirmed mating was designated as Gestation day 0 (GD 0). The pre-coital time was calculated for each female.
Animals in the recovery groups have not been allowed for mating and consequently not been used for the assessment of reproductive/developmental toxicity.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation analysis was performed using a Waters LC-MS/MS system.
Homogeneity and dose concentration analysis was performed by assessing replicate samples from the top, middle and bottom strata of all three dosages from preparation vessels. Results obtained before treatment (first week of pre-mating period) and during last week of lactation were within the acceptable range of ±15% to the nominal concentration with less than 10% RSD (relative standard deviation).
Stability analysis was performed during method validation. Samples were collected from the top, middle and bottom strata of all three dosage concentrations and were stored at room temperature for 6 hours. Further the prepared formulation samples were stored at 2 to 8 ºC for 24 hours. An aliquot was taken at post storage for each dose concentration and analysed for stability evaluation. Results obtained were within the acceptable range of ±15% to the nominal concentration with less than 10% RSD (relative standard deviation).

Duration of treatment / exposure:

The males were dosed two weeks before mating and during the mating period for a total of 28 days, with the exception one animal form G2, one animal from G3 and two animals from G4 which were dosed for a total of 35 days for the provision of re-mating. Females were dosed two weeks before 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 up to post-natal day 12 (i.e. up to the day before scheduled sacrifice), for a total of approx. 71 days. For recovery group animals (i.e. G1R and G4R), treatment was withdrawn after completion of total 28 days of treatment period and the animals were observed for 14 days after the last day of treatment.

Frequency of treatment:

Once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

G1 / G1R (vehicle control)

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

G2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

G3

Dose / conc.:

700 mg/kg bw/day (nominal)

Remarks:

G4 / G4R

No. of animals per sex per dose:

Main study (G1, G2, G3 and G4): 10 rats per sex per dose
Recovery groups (G1R and G4R): 5 rats per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose levels were selected based on Dose range finding study (Study number: VBPL- 014/2021) with the same test item and in consultation with the sponsor. Prior to treatment, animals were grouped and allocated to their respective treatment groups using a body weight based stratified randomization. 12 female animals per each main study group were examined during 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 exclusion/randomization, 10 female rats were selected for each main study group.

Positive control:

None

Parental animals: Observations and examinations:

IN-LIFE OBSERVATIONS AND MEASUREMENTS

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/mortality twice daily (morning and afternoon/ evening) during the experimental period.
For unscheduled death, necropsy was conducted at the earliest.

CLINICAL SIGNS OF TOXICITY
Animals were observed for clinical signs at least 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 in the first four hours. Subsequent post dose observations were recorded at one hour. 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). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards) shall also be recorded.

BODY WEIGHTS
For both male and female rats, individual body weights were recorded at the beginning (on day 1) of the treatment, weekly thereafter and on the day of sacrifice.
During pregnancy, female’s 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 12, and on the day of sacrifice. These observations were reported individually for each adult animal.

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.
Food consumption has not been measured during the cohabitation/mating period.

DAMS OBSERVATIONS
The duration of gestation period 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
Blood samples were collected from 2 pups (wherever number permits) from each litter on PND 4 (as per the criteria mentioned in section 26) and PND 13. 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 pups on PND 13 and the adult males were measured for thyroid hormones (T4 and TSH).
Serum T4 (Thyroxine) and TSH (Thyroid stimulating hormone) levels were measured using a commercially available kit, KINESISDx (USA) by competitive ELISA method. The method of analysis was attached as Annexure 1.

FUNCTIONAL OBSERVATION BATTERY (FOB)
The following neurological examinations were conducted for randomly selected 5 males and 5 females in each main group towards the end of the dosing period (last week). For the recovery groups, neurological examinations were carried out towards the end of recovery period (last week).
a) Home Cage Observations
Each rat was observed in the home cage for activity level and scored.
b) Handheld Observations
These observations were made while holding the subject animal. Animals were observed for lacrimation and salivation, hair coat characteristics (color/staining, alopecia, and piloerection), presence and degree of eyelid closure, ocular abnormalities and muscle tone or mass. Each observation was recorded using scoring/ranking system as mentioned in test facility SOP.
c) Open Field Observation
Rats were placed (one at a time) in an open arena, on a flat surface with a clean absorbent paper and observed for at least 2 minutes. Absorbent paper was replaced for each rat. During this observation period, each rat was evaluated as it moves freely/unperturbed and the below parameters were recorded using a scoring/ranking system as mentioned in test facility SOP:
• Activity (locomotion)
• Rearings
• Observation of reactivity and arousal
• Observation of gait and postural characteristics
• Observation of involuntary/abnormal motor movements (tremors, twitches, clonic convulsions, tonic convulsions, stereotypic and bizarre behaviour)
d) Sensory Reactivity Measurements
After approximately two minutes of observation, while the rat was in the open field arena, the following sensory reactivity measurements were performed and recorded using a scoring/ranking system as mentioned in test facility SOP:
• Approach response
• Touch response
• Click response
• Tail-pinch response
• Pupil response
• Aerial righting reflex
e) Landing Hind limbs Foot splay
The landing hind limbs foot splay was performed by dropping the rat on a horizontal surface from a short height and measuring the distance between the hind feet upon landing. The hind feet of the rat were gently pressed to an inkpad just prior to testing. The rat was suspended in prone position and then dropped from a height of approximately 30 cm on to a recording sheet, which had the details of study no., animal no. and group. A clean recording sheet was used for each individual rat. Three readings were recorded for each rat and the average of three individual foot splay values were calculated and presented in the report.

f) Grip Performance
Forelimbs and hindlimbs grip performance was tested using Grip strength meter (Orchid Scientific & Innovative India Pvt. Ltd; Model: GSM 02RS). Three trials were conducted for each rat. The average value for trials (forelimb and hindlimb, respectively) were calculated and presented in the report.
g) Motor Activity
The locomotor activity was tested using actimeter (Orchid Scientific & Innovative India Pvt. Ltd; Model: ACT01). The value for each animal were recorded and presented in the report.

Oestrous cyclicity (parental animals):

OESTROUS CYCLE
Oestrous cyclicity was monitored for two weeks (pre-exposure) prior to the start of treatment to select for the females for the study with normal cycle length (4 - 5 days)/pattern. Further, vaginal smears were monitored daily from the beginning of the treatment. Then vaginal smears were collected daily to monitor oestrous cycle for a minimum of two weeks during the pre-mating period and continued monitoring into 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 or 1), on PND 4 and 13 of post-partum. Pups were also observed for any abnormal behaviour.
The AGD (Anogenital distance) of each pup was measured on the same day on PND 0. Pup body weight was collected on the day, the AGD is measured and the AGD was normalized by dividing with the cube root of respective pup body weight. The number of nipples/areolae in male pups were counted on PND 12.

Postmortem examinations (parental animals):

PATHOLOGY

Blood Collection
At the end of the treatment period, randomly selected, 5 males and 5 females in each main group and all animals of G1R and G4R at the end of recovery period were fasted overnight (water allowed), anaesthetized with isoflurane anaesthesia. Blood samples were collected from the retro-orbital sinus plexus with a micro-hematocrit non-heparinized glass capillary tube.
Samples were collected for hematology, coagulation parameters and clinical chemistry analysis. For hematology and coagulation parameters, blood samples were collected in tubes containing 10% Dipotassium Ethylene Diamine Tetra Acetic acid (K2-EDTA) and Sodium citrate, respectively. For clinical chemistry, blood samples were collected in a plain tube, allowed to clot, and the serum was collected after centrifugation for analysis.

HEMATOLOGY
The following haematological examination was estimated using a hematology analyzer (Horiba Medical: ABX SCIL Vet ABC).
Sl. No. Parameters Units
1 Total Leukocyte Count (WBC) 103 cells/mm3
2 Erythrocyte Count (RBC) 106 cells/mm3
3 Reticulocytes %
4 Hemoglobin (HGB) g/dL
5 Hematocrit (HCT) %
6 Platelet Count 103 cells/mm3
7 Mean corpuscular volume (MCV) µm3
8 Mean Corpuscular Hemoglobin (MCH) pg
9 Mean Corpuscular Hemoglobin Concentration (MCHC) g/dL
10 Differential Leucocytes Count (DLC) %
Blood smears were prepared and stained with Giemsa stain for enumerating differential count. Differential cell count was done manually under microscope. The cell count was expressed in percentage (%). A minimum of 100 cells were counted per blood smear namely Lymphocytes (LYM), Monocytes (MON), Neutrophils (NEU), Eosinophils (EOS) and Basophils (BAS).
Blood samples collected for coagulation analysis were centrifuged at 2500 rpm for 10 minutes for separation of plasma. Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT) and Fibrinogen (FN) analysis was carried out with plasma using coagulation analyser. Coagulation parameters analysis was performed using Start Max (Stago) Coagulation analyzer.

CLINICAL CHEMISTRY
The following clinical chemistry parameters were estimated by using Randox Daytona Plus analyzer (Randox Laboratories Ltd, United Kingdom; Model: RX Daytona plus). Serum was separated by centrifugation of whole blood samples in a refrigerated centrifuge at 2500 rpm for 10 minutes.
S. No. Parameters Units
1 Albumin (ALB) g/L
2 Albumin/Globulin ratio (A/G) %
3 Alanine Aminotransferase (ALT) U/L
4 Aspartate Aminotransferase (AST) U/L
5 Alkaline Phosphatase (ALP) U/L
6 Blood Urea Nitrogen (BUN) mg/dL
7 Creatinine (CRE) mg/dL
8 Glucose (Glu) mg/dL
9 Globulin (Glob) mg/dL
10 Serum Urea (URE) mg/dL
11 Total Protein (TP) g/L
12 Total Cholesterol (TCHO) mg/dL
13 Total bile acid µmol/L
Electrolytes
14 Sodium (Na+) mmol/L
15 Potassium (K+) mmol/L

GROSS NECROPSY
The adult animals sacrificed at term were euthanized by CO2 asphyxiation. The adult animals were subjected to detailed necropsy by pathologist and findings were recorded and scored. Special attention was paid to the organs of the reproductive system. Dead animals were examined as soon as possible for any defects and/or cause of death.
The number of implantation sites were recorded for all the dams. Vaginal smear was examined on the day of necropsy to determine the stage of oestrous cycle.

TISSUE COLLECTION AND PRESERVATION
All tissues/organs specified in Table A were collected from all of the animals in each group to specifically examine endocrine and reproductive effects. In addition, all tissues/organs specified in Table B were collected from the randomly selected five male and five female rats (as mentioned in section 29.1) to examine systemic effects (including endocrine and reproductive effects). Wet weights of the organs specified in below tables were recorded by trimming of any adherent tissue immediately after dissection to avoid drying. Collected tissues were preserved in 10 % neutral buffered formalin and Davidson’s fluid and modified Davidson’s fluid as applicable.

A. Tissues Collected from All Animals for Endocrine and Reproductive Effects
Tissue/Organ Organ Weight Collection Preservation Microscopic Examination
All gross lesions - X X
Adrenals - X X
Epididymides X X X
Skin with mammary gland (male and female) - X -
Ovaries X X X
Uterus (along with cervix) X X -
Vagina - X -
Prostate and Seminal vesicles with coagulating glands X X X
Testes* X X X
Thyroid with Parathyroid# X X X
Key: * = collected and fixed in modified Davidson’s fluid; X = activity performed; # = collected from one male and female pups on PND 13 in addition to the parent animals.

B. Additional Tissues Collected from the Randomly Five Male and Five Female Animals for Systemic Effects
Tissue/Organ Organ Weight Collection
Preservation Microscopic Examination
Adrenal glands X X X
Aorta - X X
Axillary/neck lymph nodes - X X
Bone marrow smear (femur) - X X
Brain (cerebrum, cerebellum, and medulla/pons) X X X
Cecum - X X
Colon - X X
Duodenum - X X
Oesophagus - X X
Eyes (with optic nerve) a - X X
Femur bone with distal joint - X X
Gross lesions - X X
Heart X X X
Ileum with Peyer’s Patch - X X
Jejunum - X X
Kidneys X X X
Larynx - X X
Liver X X X
Lungs c - X X
Mesenteric lymph nodes - X X
Mandibular lymph node - X X
Nerve, sciatic - X X
Oviducts - X X
Pancreas - X X
Pituitary gland - X X
Rectum - X X
Salivary gland - X X
Skeletal muscle - X X
Spinal cord (cervical, thoracic and lumbar) - X X
Spleen X X X
Stomach - X X
Thymus X X X
Trachea - X X
Ureters - X X
Urinary bladder c - X X
Key: a = collected and fixed in Davidson’s fluid, b= collected and fixed in modified Davidson’s fluid, c= infused with formalin at necropsy, X= activity performed.


ABSOLUTE AND RELATIVE ORGAN WEIGHTS
Absolute and relative organ weights were recorded for the organs as specified above.

HISTOPATHOLOGY
Tissues collected from all animals in the control and high dose groups were examined microscopically for histopathological changes. Histopathological examination of testes also included a qualitative assessment of stages of spermatogenesis. All gross lesions were examined in all the groups. The reproductive organs of animals failing to mate were examined in all the dose groups.
The tissues were processed for routine paraffin embedding and 4 micron sections were stained with Mayer’s Haematoxylin Eosin. In addition, testes were stained with PAS (Periodic acid–Schiff) reagent and Haematoxylin to aid in qualitative assessment of spermatogenesis. Unused tissues were archived.

Postmortem examinations (offspring):

All the pups on PND 13 were examined for external gross abnormalities. Microscopic examinations of the thryoids and parathyroids of the pups were also performed.

Statistics:

The following statistical methods were used to analyse the body weight, feed consumption, organ weights, and clinical pathology parameters:
• 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 heterogenous 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:

FOLLOWING REPRODUCTIVE INDICES WERE CALCULATED:
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:

Live birth index (%)

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

Day 4 survival index (%)

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

Day 13 survival index (%)

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

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No clinical signs were observed in either G2 or G3 during the study period. All animals in G4 and G4R showed dullness throughout the treatment period, which started on the second day of treatment. All animals in the G4R group showed recovery from the dullness after the withdrawal of treatment. No other clinical signs of toxicity were observed in G4 or G4R.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There was no morbidity/mortality at any dose level, except for one dam at 700 mg/kg bw/day that died on GD 21. The exact cause of death could not be determined, but it was possibly due to dystocia. The gross pathological examination of the animal that died on GD 21 showed autolysis of all organs which was considered to be due to natural post-mortem changes.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No significant changes in body weight or body weight gain were observed in G2, G3 or G4 during the treatment period.
In G4R males, significant decreases in absolute body weight were observed on day 15 of treatment (mean, 367.58 gram) compared to G1R males (mean, 389.13 gram), on the 8th day of recovery (mean 393.20 gram) compared to G1R males (mean, 429.49 gram), and on the 14th day of recovery (mean, 393.79 gram) compared to G1R males (mean 430.36 gram). Further in G4R males, statistically significant decreases in absolute body weight gain were observed between treatment days 1-8 (average loss of 0.31 gram) and between day 1 to 42 of the study (average gain of 28.44 gram) compared to G1R (average body weight gain between treatment days 1 to 8: 14.93g; and between day 1 to 42 of study: 64.28g). A significant decrease in % body weight gain from day 1 to 42 of study was also observed in G4R males (mean, 7.8% gain) compared to G1R males (mean, 17.7% gain). No significant changes in absolute body weight or body weight gain were observed in G4R females. The observed significant changes in absolute body weight and body weight gain in G4R males during recovery were considered to be treatment-related but most likely non-adverse since the differences compared to the G1R males were rather small, for example the differences in absolute body weight in G4R males compared to G1R males were less than 8.5% on both occasions.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No significant changes in food intake were observed in any of the groups during the study period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No significant changes in haematology were observed, with the exception of a significant increase in MCH in G2 females compared to G1 females. This effect was not attributed to the test item due to lack of dose dependency. No significant changes in coagulation parameters were observed in any of the main study groups. A significant decrease in fibrinogen was observed in G4R females (mean, 210.9 mg/dl) compared to G1R females (mean, 266.27 mg/dl). This significant decrease in fibrinogen was considered incidental as it could not be corroborated with any gross or histopathological findings.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In male rats the following significant changes were observed: significant increases in urea in G2 (mean, 42.44 mg/dl) and G3 (mean, 42.89 mg/dl) compared to G1 (mean, 35.49 md/dl); significant decreases in creatinine in G3 (mean, 0.58 md/dl) and G4 (mean, 0.57 md/dl) compared to G1 (mean, 0.65 mg/dl); a significant increase in ALT in G2 (mean, 108.10 U/l) compared to G1 (mean, 79.56 U/l); a significant decrease in albumin in G2 (mean, 34.15 mg/dl) compared to G1 (mean, 36.75 mg/dl); and a significant decrease in total bile acids in G4 (mean, 18.45 mmol/l) compared to G1 (mean, 37.81 mmol/l). The observed changes in urea, ALT and albumin in G2/G3 were considered incidental due to lack of dose-dependency. The observed changes in creatinine and total bile acids in G3/G4 were not considered to be adverse effects of treatment due to lack of associated gross or histopathological findings and since no significant changes in creatinine or total bile acids were observed after the recovery period.
In female rats the following significant changes were observed: a significant decrease in urea in G3 (mean, 39.63 mg/dl) compared to G1 (mean, 77.21 mg/dl); a significant increase in total protein levels in G3 (mean, 75.10 g/l) compared to G1 (mean, 66.48 g/l); and a significant increase in ALT in G4R (mean, 81.40 U/l) compared to G1R (mean, 58.72 U/l). The observed changes in urea and total protein levels in G3 were considered incidental due to lack of dose-dependency. The observed increase in ALT in G4R was attributed to an unusually low mean value in the G1R, i.e. the mean value of ALT in G1R was only 58.72 U/l compared to the mean value of 137.28 U/l in the G1 control group in the main study. The increase in ALT in G4R was therefore considered incidental.

Endocrine findings:

no effects observed

Description (incidence and severity):

In adult males, there were no significant changes observed in T4 or TSH.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Dose-dependent and statistically significant decreases in fore limb and hind limb grip strengths were observed in G3 males (mean fore limb, 677.3 g/f; mean hind limb, 421.9 g/f) and G4 males (mean fore limb, 582.7 g/f; mean hind limb, 408.1 g/f) compared to G1 (mean fore limb, 769.7 g/f; mean hind limb, 462.7 g/f). In addition to this, significant decreases in locomotor activity were observed in G3 males in terms of total movements (mean, 1328.4 counts) compared to G1 (mean, 1455.2 counts) and in G4 in terms of Y-movements, Z-movements, and total movements (mean total movements 1227.0 counts). The above effects in G3 and G4 males were considered to be treatment-related and adverse, however reversible as no significant changes in any FOB parameter were observed in the G4R males. For female rats, significant changes in FOB parameters were limited to a significant decrease in Z-movements in G3 (mean 445.8 counts) compared to G1 (mean 488.4 counts). This effect lacked dose-dependency and was therefore not considered to be treatment-related. No other significant changes in the functional observatory battery (which included sensory reactivity measurement) were observed.

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):

The microscopic examinations of the animals in G1 and G4 did not show any lesions of pathological significance that could be attributed to the test item.
The histopathological findings in adult male rats included: minimal, multifocal dilatation of gastric glands in one G1 rat; minimal involution in the thymus in one G1 rat and in one G4 rat; minimal, multifocal mononuclear cellular infiltration in the alveolar tissue in one G1 rat; mild, multifocal mononuclear cellular infiltration in the alveolar tissue in one G4 rat; minimal mononuclear cellular infiltration in the heart in one G4 rat; mild, multifocal cortical vacuolization in adrenal gland in one G1 rat; unilateral accessory cortical tissue in adrenal gland in two G4 rats; diffused bilateral cell debris in epididymis in one G1 rat; diffused unilateral cell debris in epididymis in one G4 rat; bilateral degeneration/atrophy in seminiferous tubules in one G1 rat; unilateral degeneration/atrophy in seminiferous tubules in one G4 rat; and minimal multifocal presence of multinucleated giant cell in one G4 rat.
The histopathological findings in adult female rats included: minimal, multifocal dilatation of gastric glands in one G1 rat and in one G4 rat; minimal involution in the thymus in one G1 rat and in one G4 rat; and unilateral accessory cortical tissue in adrenal gland in one G1 rat.
The above microscopic findings observed in the male and female rats were considered incidental as they occurred randomly across G1 and G4, were mild/minimal severity, and/or can be expected for Wistar rats of this age (Elizabeth McInnes, 2012).

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Dose formulation analysis results of performed before treatment (second week of pre-exposure period) and during last week of lactation were within the acceptable range of ± 15 % to the nominal concentration with less than 10% RSD (relative standard deviation).

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No significant change in oestrous cycle length was observed in G2 (mean, 4.18 days), G3 (mean, 4.00 days) or G4 (mean, 4.08 days) compared to G1 (mean, 4.20 days) and no significant changes in oestrous cyclicity were observed.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Male and female mating indexes were 100, 90, 100, and 90% in G1, G2, G3, and G4 respectively and female fecundity and fertility indexes were 100, 90, 100, and 90% in G1, G2, G3, and G4 respectively. Higher pre-coital intervals were observed in G3 and G4 compared to G1. The number of female rats conceiving at 1-5 days after mating were 6/10, 5/10, 3/10, and 2/10 in G1, G2, G3, and G4 respectively. The number of female rats conceiving at 6-14 days after mating were 4/10, 4/10, 6/10, and 6/10 in G1, G2, G3, and G4, respectively. No remating was needed in G1. One animal each in G2, G3 and G4 conceived during the remating period. The higher pre-coital intervals in G3 and G4 were not considered as adverse since there were no corroborating effects on any other reproductive or developmental parameters such as mating index and fertility index. No significant changes in gestation length were observed in any of the groups. Gestation indexes were 100, 100, 100 and 88.9% in G1, G2, G3, and G4, respectively, i.e. one out of 9 conceived dams in G4 did not deliver any live offspring. The lower gestation index in G4 was not statistically secured and was considered to be within normal biological range. No significant difference in the number of implantations per dam were observed in G2 (mean, 9.7), G3 (mean, 10.7) or G4 (mean, 9.4) compared to G1 (mean, 9.0). A dose-dependent trend of increased % post-implantation loss was observed in G2 (mean, 6.6%), G3 (mean, 8.5%) and G4 (mean, 9.8%) compared to G1 (mean, 0%). Since the % post-implantation losses in G2, G3 and G4 were less than 10% and could not be corroborated with any other relevant reproductive or developmental parameter in the study, the higher pre-implantation losses in G2, G3 and G4 were not considered to be adverse in nature.

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive performance

Effect level:

700 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Key result

Dose descriptor:

LOAEL

Remarks:

General toxicity

Effect level:

700 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

Key result

Dose descriptor:

NOAEL

Remarks:

General toxicity

Effect level:

300 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

haematology

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

other:

Remarks on result:

other: NOAEL was considered to be 300 mg/kg bw/day

Key result

Dose descriptor:

LOAEL

Remarks:

General toxicity

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other:

Remarks on result:

other: NOAEL was considered to be 300 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Remarks:

General toxicity

Effect level:

100 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

haematology

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

other:

Remarks on result:

other: NOAEL was considered to be 100 mg/kg bw/day

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

No significant changes in the number of live pups on PND 0 or PND 4 were observed. Pup survival indexes on PND 4 were 99.9, 92.7, 90.0, and 99.17% in G1, G2, G3 and G4 respectively, with no statistically significant effect observed. Pup survival indexes on PND 13 were 88.9, 78.4, 71.5 and 83.0% in G1, G2, G3 and G4 respectively, with no statistically significant effect observed.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant changes in litter weight were observed on PND 0 or 4 and no significant changes in male or female pup weight were observed on PND 0, 4 or 13.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In female pups, a dose-dependent and statistically significant increase in TSH levels was observed in G3 (mean, 6015.50 pg/ml) and G4 (mean, 6236.18 pg/ml) compared to G1 (mean, 5233.88 pg/ml). Similarly in female pups, a dose-dependent and statistically significant increase in T4 levels was observed in G3 (mean, 119.05 ng/ml) and G4 (mean, 121.83 ng/ml) compared to G1 (mean, 103.94 ng/ml). In male pups, no significant changes were observed except for a statistically significant decrease in T4 in G3 (mean 102.60 ng/ml) compared to G1 (mean, 109.73 ng/ml) which was not attributed to the test item due to lack of dose-dependency. The observed changes in TSH and T4 levels in the female pups were considered to be potential treatment-related effects but non-adverse in nature as there were no gross or histopathological findings in any of the thyroid glands from the female pups.

Urinalysis findings:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

No significant changes in AGD or normalised AGD was observed in either male or female pups at any dose level.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No nipple retention was observed in any of the male pups in G1, G2, G3 or G4.

Gross pathological findings:

no effects observed

Description (incidence and severity):

All pups in G1, G2, G3 and G4 were normal upon external examination.

Histopathological findings:

no effects observed

Description (incidence and severity):

No findings were made in any of the thyroids or parathyroids from the pups.

Other effects:

no effects observed

Description (incidence and severity):

No significant difference in sex ratio were observed between the groups on PND 0 or PND 4.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

700 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

body weight and weight gain

clinical biochemistry

gross pathology

histopathology: non-neoplastic

other:

Remarks on result:

other: NOAEL was considered to be 700 mg/kg bw/day

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Table 1 - Summary of Clinical Signs – MALES

GroupsDose(mg/kg bw/day)	Sex	No. of animals/Total no. treated animals	Clinical Signs
G10	M	10/10	Normal
G2100	M	10/10	Normal
G3300	M	10/10	Normal
G4700	M	10/10	Dullness
G1R0	M	5/5	Normal
G4R700	M	5/5	Dullness/normal*

Note: *- Dullness was observed from day 1 of treatment to the end of the treatment, whereas after treatment cessation, recovery group (G4R) animals were recovered and found normal.

 

Table 2 - Summary of Clinical Signs – FEMALES

GroupsDose(mg/kg bw/day)	Sex	No. of animals/Total no. treated animals	Clinical Signs
G10	F	10/10	Normal
G2100	F	10/10	Normal
G3300	F	10/10	Normal
G4700	F	10/10	Dullness
G1R0	F	5/5	Normal
G4R700	F	5/5	Dullness/normal*

Note: *- Dullness was observed from day - 1 to the end of the treatment, whereas after treatment cessation, recovery group (G4R) animals were recovered and found normal.

 

Table 3 - Summary of Body Weights – Males

Average Body Weights (g) - Males
Days
GroupsDose(mg/kg bw/day)		1	8	15	22	28	35*
G10	Mean	381.21	391.04	409.80	431.82	434.71	-
	SD	22.02	27.18	29.86	28.22	32.70	-
	N	10	10	10	10	10	-
G2100	Mean	386.22	401.05	415.16	431.97	430.50	440.83
	SD	19.49	21.06	27.00	26.87	26.14	0
	N	10	10	10	10	10	1
G3300	Mean	381.54	392.34	401.19	413.95	420.33	477.88
	SD	18.84	23.03	31.71	40.71	31.85	0
	N	10	10	10	10	10	1
G4700	Mean	380.02	383.45	399.12	416.72	420.52	434.88
	SD	20.58	24.44	32.84	34.63	40.37	65.70
	N	10	10	10	10	10	2

          Key: Values are in Mean ± SD; * - Extended mating

 

TABLE 3 (CONTD.) - SUMMARY OF BODY WEIGHTS – RECOVERY MALES                                                                                                                                                                       

Average Body Weights (g) – Recovery Males
Days
GroupsDose(mg/kg bw/day)		1	8	15	22	29	36	42
G1R0	Mean	366.08	381.01	389.13	409.96	418.02	429.49	430.36
	SD	13.78	16.49	15.00	15.06	18.24	17.90	19.66
	N	5	5	5	5	5	5	5
G4R700	Mean	365.35	365.04	367.58*	380.50	381.58	393.20*	393.79*
	SD	15.03	21.34	13.94	26.93	32.77	24.99	21.97
	N	5	5	5	5	5	5	5

 Key: Values are in Mean ± SD; *= Statistically significant from vehicle control (P ≤ 0.05)

 

Table 4 - SUMMARY OF NET BODY WEIGHT GAIN –Males                                                                                                                                                                                                                        

GroupsDose(mg/kg bw/day)	FromTo	18	815	1522	2228	Abs Gain128	TotalGain %128
G10	Mean	9.83	18.77	22.02	2.87	53.49	14.03
	SD	9.20	6.15	7.39	11.01	20.69	5.47
	n	10	10	10	10	10	10
G2100	Mean	14.82	14.11	16.81	-1.47	44.28	11.53
	SD	11.11	8.88	5.33	8.51	20.53	5.49
	n	10	10	10	10	10	10
G3300	Mean	10.80	8.84	12.77	6.38	38.79	10.14
	SD	12.40	9.39	12.39	13.34	22.36	5.82
	n	10	10	10	10	10	10
G4700	Mean	3.43	15.67	17.61	3.80	40.50	10.48
	SD	9.21	10.90	8.61	10.20	23.25	5.74
	n	10	10	10	10	10	10

Key: Values are in Mean ± SD

 

TABLE 4 (CONTD.) - SUMMARY OF NET BODY WEIGHT GAIN – RECOVERY MALES

	Average Body Weights (g) – Recovery Males
	Days
GroupsDose(mg/kg bw/day)		FromTo	18	815	1522	2229	2936	3642	Abs Gain142	Total% Gain142
G1R0		Mean	14.93	8.12	20.83	8.07	11.46	0.87	64.28	17.69
		SD	8.88	8.77	2.84	10.81	4.99	28.79	23.76	6.85
		N	5	5	5	5	5	5	5	5
G4R700		Mean	-0.31*	2.54	12.92	1.08	11.62	0.59	28.44*	7.77*
		SD	9.21	7.90	13.85	7.33	17.23	5.82	12.32	3.31
		N	5	5	5	5	5	5	5	5

           Key: Values are in Mean ± SD; *= Statistically significant from vehicle control (P ≤ 0.05)

 

TABLE 5 - SUMMARY OF BODY WEIGHTS DURING PRE MATING PERIOD – FEMALES                                                                                                                          

Body Weights During Pre Mating Period – Females
Days
GroupsDose(mg/kg bw/day)		1	8	15
G10	Mean	261.80	266.32	269.88
	SD	14.53	10.35	12.36
	n	10	10	10
G2100	Mean	263.45	264.30	268.54
	SD	15.11	13.83	14.03
	n	10	10	10
G3300	Mean	261.96	263.63	270.25
	SD	15.90	11.18	8.60
	n	10	10	10
G4700	Mean	261.39	261.98	270.04
	SD	15.11	15.58	17.55
	n	10	10	10

Key: Values are in Mean ± SD

 

TABLE 5 (CONTD.) - SUMMARY OF BODY WEIGHTS – RECOVERY FEMALES

Average Body Weights (g) – Recovery Females
Days
GroupsDose(mg/kg bw/day)		1	8	15	22	29	36	42
G1R0	Mean	259.92	258.53	263.06	267.27	272.31	280.37	279.72
	SD	17.89	24.58	17.09	17.10	19.73	18.12	21.01
	N	5	5	5	5	5	5	5
G4R700	Mean	260.65	260.07	267.26	272.77	274.60	279.54	276.16
	SD	16.45	14.25	9.64	8.85	12.56	10.08	11.63
	N	5	5	5	5	5	5	5

Key: Values are in Mean ± SD

 

TABLE 6 - SUMMARY OF NET BODY WEIGHT GAIN DURING PRE MATING PERIOD – FEMALES                                                                                                                   

GroupsDose(mg/kg bw/day)		FromTo	18	815	Abs. Gain115	Total% Gain115
G10	Mean		4.52	3.56	8.08	3.22
	SD		7.95	8.00	11.49	4.46
	N		10	10	10	10
G2100	Mean		0.85	4.24	5.09	2.06
	SD		7.25	7.83	12.95	4.96
	N		10	10	10	10
G3300	Mean		1.67	6.62	8.29	3.34
	SD		5.37	4.62	8.68	3.44
	N		10	10	10	10
G4700	Mean		0.59	8.06	8.65	3.32
	SD		5.90	7.07	9.10	3.36
	N		10	10	10	10

Key: Values are in Mean ± SD

 

TABLE 6 (CONTD.) - SUMMARY OF NET BODY WEIGHT GAIN – RECOVERY FEMALES                                                                                                                                                                

	Average Body Weights (g) – Recovery Female
	Days
GroupsDose(mg/kg bw/day)		FromTo	18	815	1522	2229	2936	3642	Abs Gain142	Total% Gain142
G1R0		Mean	-1.38	4.53	4.20	5.05	8.06	-0.65	19.81	7.74
		SD	11.87	10.85	4.68	9.14	2.08	3.14	16.95	6.58
		N	5	5	5	5	5	5	5	5
G4R700		Mean	-0.58	7.19	5.51	1.83	4.94	-3.38	15.51	6.11
		SD	2.84	5.83	4.23	5.25	4.08	3.80	10.19	4.29
		N	5	5	5	5	5	5	5	5

           Key: Values are in Mean ± SD

 

TABLE 7 - SUMMARY OF CAGE WISE AVERAGE FOOD INTAKE – MALES (PRE MATING Period)

GroupsDose(mg/kg bw/day)		Weeks
		1	2
G10	Mean	26.98	28.97
	SD	1.57	1.60
	N	10	10
G2100	Mean	27.69	28.52
	SD	1.63	1.15
	N	10	10
G3300	Mean	27.37	27.00
	SD	1.42	1.02
	N	10	10
G4700	Mean	25.40	28.00
	SD	1.04	2.11
	N	10	10

Key: Values are in Mean ± SD

 

TABLE 7 (CONTD.) - SUMMARY OF CAGE WISE AVERAGE FOOD INTAKE – RECOVERY MALES                                                                                                                                                              

Average Cage Wise Food Intake (g) – Recovery Male
Weeks
GroupsDose(mg/kg bw/day)		1	2	3	4	5	6
G1R0	Mean	28.26	28.61	28.34	27.68	28.96	24.37
	SD	1.60	1.56	1.56	2.06	0.97	2.12
	N	5	5	5	5	5	5
G4R700	Mean	25.62	25.36	28.32	26.68	28.70	26.70
	SD	1.50	2.04	2.48	1.51	2.05	1.45
	N	5	5	5	5	5	5

Key: Values are in Mean ± SD

 

TABLE 8 - SUMMARY OF CAGE WISE AVERAGE FOOD INTAKE (PRE MATING PERIOD) – FEMALES

GroupsDose(mg/kg bw/day)		Weeks
		1	2
G10	Mean	17.80	18.20
	SD	1.30	1.82
	N	10	10
G2100	Mean	17.80	16.39
	SD	1.46	0.55
	N	10	10
G3300	Mean	18.80	17.02
	SD	1.30	0.72
	N	10	10
G4700	Mean	19.61	16.82
	SD	1.16	0.85
	N	10	10

Key: Values are in Mean ± SD

 

TABLE 8 (CONTD.) - SUMMARY OF CAGE WISE AVERAGE FOOD INTAKE – RECOVERY FEMALES

Average Cage Wise Food Intake (g) – Recovery Female
Treatment period						Recovery period
GroupsDose(mg/kg bw/day)		1-8	8-15	15-22	22-29	29-36	36-42
G1R0	Mean	18.30	16.67	17.36	18.02	18.94	17.35
	SD	1.52	1.55	2.02	2.08	1.00	1.56
	N	5	5	5	5	5	5
G4R700	Mean	19.33	16.00	19.02	17.67	18.69	17.35
	SD	1.50	0.99	1.00	1.20	0.55	0.64
	N	5	5	5	5	5	5

Key: Values are in Mean ± SD

 

TABLE 9 - SUMMARY OF MATERNAL BODY WEIGHTS (g) and BOdy weight changes DURING GESTATION PERIOD

MATERNAL BODY WEIGHTS DURING GESTATION PERIOD (g)
GroupsDose(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
G10	Mean	271.74	295.37	324.17	385.35	22.13	28.80	61.18	112.10
	SD	15.09	18.53	24.61	38.00	8.91	9.33	18.06	25.34
	N	10	10	10	10	10	10	10	10
G2100	Mean	278.27	292.79	322.91	389.33	13.07	27.11	59.78	99.96
	SD	19.10	18.85	18.26	25.49	6.17	9.93	26.48	39.90
	N	9	9	9	9	9	9	9	9
G3300	Mean	277.05	296.53	323.93	393.33	19.48	27.40	69.40	116.28
	SD	10.40	7.37	13.98	27.59	7.50	10.58	18.97	30.31
	N	10	10	10	10	10	10	10	10
G4700	Mean	270.09	286.76	321.97	390.75	15.00	31.69	61.90	108.59
	SD	19.35	21.75	24.69	28.72	9.54	14.48	25.26	40.79
	N	9	9	9	9	9	9	9	9

Key: Values are in Mean ± SD

 

Table 9 (CONTD.) - Summary of MATERNAL FOOD INTAKE DURING GESTATION PERIOD

Maternal food intake (g) during Gestation period
Gestation Period  (Day)
GroupsDose(mg/kg bw/day)	FromTo	07	714	1420	020
G10	Mean	22.70	23.80	22.95	23.15
	SD	2.93	2.05	1.36	2.11
	N	10	10	10	10
G2100	Mean	20.59	20.82	20.36	20.59
	SD	7.48	7.50	7.28	7.42
	N	9	9	9	9
G3300	Mean	22.66	24.56	23.30	23.51
	SD	1.14	1.87	1.94	1.65
	N	10	10	10	10
G4700	Mean	20.44	21.08	20.73	20.75
	SD	7.45	7.62	7.55	7.54
	N	9	9	9	9

Key: Values are in Mean ± SD

 

Table 10 -Summary of Maternal Body Weights (g) During THE LACTATION Period

GroupsDose(mg/kg bw/day)		Body  weight (g) during Lactation period (Day)			Body weight change during Lactation period (Day)
		0	4	12	0 - 4	4 - 12
G10	Mean	318.24	325.95	338.84	7.70	12.89
	SD	24.65	20.15	19.73	15.42	8.67
	N	10	10	10	10	10
G2100	Mean	300.91	310.54	329.58	9.63	19.04
	SD	32.81	33.36	30.59	4.21	35.44
	N	9	9	9	9	9
G3300	Mean	309.26	315.19	326.63	5.93	11.44
	SD	17.18	15.66	18.37	10.38	5.68
	N	10	10	10	10	10
G4700	Mean	298.67	304.03	312.72	5.36	8.68
	SD	30.90	31.08	29.35	7.21	5.31
	N	8	8	8	8	8

Key: Values are in Mean ± SD

 

Table 10 (CONTD.) - Summary of Maternal Food Intake (g) During the LACTATION period

GroupsDose(mg/kg bw/day)	Maternal food intake (g) during Lactation period
		Per day during Lactation (Day)
		0 - 4	4 - 12
G10	Mean	26.40	22.51
	SD	2.25	1.13
	N	10	10
G2100	Mean	27.07	22.81
	SD	8.66	1.03
	N	9	9
G3300	Mean	27.38	23.06
	SD	0.83	1.11
	N	10	10
G4700	Mean	20.89	18.75
	SD	11.28	9.95
	N	8	8

Key: Values are in Mean ± SD

 

Table 11 - Summary of THYROID HORMONE ANALYSIS – MALES/SIREs

Sex: Male		Parameters
GroupsDose(mg/kg bw)		T4	TSH
		ng/mL	pg/mL
G10	Mean	113.41	5216.43
	SD	10.58	844.31
	N	10	10
G2100	Mean	106.79	5477.67
	SD	12.50	760.63
	N	10	10
G3300	Mean	118.36	5899.04
	SD	3.57	691.23
	N	10	10
G4700	Mean	119.33	5865.42
	SD	5.36	431.13
	N	10	10

Key: Values are in mean ± SD

 

Table 11 (CONTD.) - Summary of THYROID HORMONE ANALYSIS – DAY 13 PROGENY

Sex: Male		Parameters
GroupsDose(mg/kg bw)		T4	TSH
		ng/mL	pg/mL
G10	Mean	109.73	5566.75
	SD	3.58	679.53
	N	10	10
G2100	Mean	109.52	5496.24
	SD	4.77	262.92
	N	9	9
G3300	Mean	102.60*	5555.52
	SD	3.96	580.24
	N	9	9
G4700	Mean	111.09	5511.55
	SD	6.98	544.40
	N	8	8

Key: Values are in mean ± SD; * = Statistically significant from vehicle control (P ≤ 0.05)

 

Table 11 (contd.) - Summary of THYROID HORMONE ANALYSIS – DAY 13 PROGENY

Sex: Female		Parameters
GroupsDose(mg/kg bw)		T4	TSH
		ng/mL	pg/mL
G10	Mean	103.94	5233.88
	SD	13.53	677.14
	N	10	10
G2100	Mean	109.02	5577.88
	SD	4.46	335.05
	N	9	9
G3300	Mean	119.05*	6015.50*
	SD	10.27	366.68
	N	9	9
G4700	Mean	121.83*	6236.18*
	SD	5.31	363.91
	N	8	8

Key: Values are in mean ± SD; * = Statistically significant from vehicle control (P ≤ 0.05)

 

Table 12 - Summary of OESTOUS CYCLICITY – PRE MATING

GroupsDose(mg/kg. bw/day)	Mean cycle length (days)	No. of animals with normal cycle	No. of animals with abnormal cycle
G1 0	4.20	10	0
G2100	4.18	10	0
G3300	4.00	10	0
G4700	4.08	10	0

 

 

 

 

 

 

TABLE 13 - SUMMARY OF FUNCTIONAL OBSERVATION BATTERY (MALES)

Parameter	Groups (n)
	G1	G2	G3	G4	G1R	G4R
Home cage observations
Posture
Sitting or standing normally	3	4	3	3	3	2
Standing on hind limbs	2	1	2	1	2	3
Asleep (lying on side)	0	0	0	1	0	0
Respiration
Normal	5	5	5	5	5	5
Clonic involuntary movement
None/normal	5	5	5	5	5	5
Tonic involuntary movement
None/normal	5	5	5	5	5	5

 

 

TABLE 13 (CONTD) - SUMMARY OF FUNCTIONAL OBSERVATION BATTERY (MALES)

Parameter	Groups (n)
	G1	G2	G3	G4	G1R	G4R
Vocalization
None/normal	5	5	5	5	5	5
Convulsions
Absent – N	5	5	5	5	5	5
Hand-held observations
Reactivity
Easy/ normal (animal does not resist)	5	5	5	5	5	5
Handling
Squeaks or does not squeak but exhibit mild resistance; easy to handle/ normal	5	5	5	5	5	5
Palpebral closure
Open	5	5	5	5	5	5
Lacrimation
No lacrimation/ normal	5	5	5	5	5	5

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TABLE 13 (CONTD) - SUMMARY OF FUNCTIONAL OBSERVATION BATTERY (MALES) 

Parameter	Groups (n)
	G1	G2	G3	G4	G1R	G4R
Salivation
No salivation/ normal	5	5	5	5	5	5
Piloerection
None/ normal	5	5	5	5	5	5
Perineum Wetness
N: Absence of wetness	5	5	5	5	5	5
Body Temperature Range (ºC)	35.9 - 36.7	35.8 – 36.6	35.8 – 36.8	35.8 – 36.7	35.4-36.7	35.2-36.8
Open Field Activity
Clonic involuntary movement
None/ Normal	5	5	5	5	5	5
Tonic involuntary movement
None/ Normal	5	5	5	5	5	5

 

 

TABLE 13 (CONTD) - SUMMARY OF FUNCTIONAL OBSERVATION BATTERY (MALES)

Parameter	Groups (n)
	G1	G2	G3	G4	G1R	G4R
Gait
N:Head is horizontal, abdomen rises slightly above floor, and body moves up and down slightly during walking	5	5	5	5	5	5
Movements
Normal	5	5	5	5	5	5
Arousal
Normal (keeps guard up and engages in exploratory activity)	5	5	5	5	5	5
Tremors
None	5	5	5	5	5	5
Occurrence of stereotype	0	0	0	0	0	0
Abnormal behaviour	0	0	0	0	0	0
Number of Defecations (Mean)	0.8	0.4	0.8	0.6	1.2	1.0
Number of Urinations (Mean)	2.0	2.4	2.6	2.8	1.8	1.6
Number of Rearings (Mean)	7.4	6.6	4.8	4.8	5.6	7.8
Sensory Reactivity Measurements
Approach Response
Slowly approaches, sniffs and pulls back/ normal	5	5	5	5	5	5
Touch Response
Slowly approaches, sniffs and pulls back/ normal	5	5	5	5	5	5

 

TABLE 13 (CONTD) - SUMMARY OF FUNCTIONAL OBSERVATION BATTERY (MALES)

Parameter	Groups (n)
	G1		G2		G3		G4		G1R		G4R
Eyelid reflex
Blink/ Normal	5	5		5		5		5		5
Pinna reflex
Auricle twitches/ normal	5	5		5		5		5		5
Sound response
Mild reaction, hears sound/normal	5	5		5		5		5		5
Tail pinch response
Looks back, moves forward and lightly squeaks/normal	5	5		5		5		5		5
Pupillary reflex
Contracts	5	5		5		5		5		5
Aerial righting reflex
Lands on four limbs/ normal	5	5		5		5		5		5
Tail limb reflex
Present (Y)	5	5		5		5		5		5
Abdominal tone
Normal (proper hardness)	5	5		5		5		5		5

 

TABLE 13 (CONTD) - SUMMARY OF FUNCTIONAL OBSERVATION BATTERY (MALES)

Parameter	Groups (n)
	G1	G2	G3	G4	G1R	G4R
Limb tone
Normal (proper hardness)	5	5	5	5	5	5
Landing Foot Splay (Mean ± SD (mm))	64.13 ± 5.29	69.07 ± 10.23	65.60 ± 2.78	75.00 ± 7.77	58.13 ±10.96	51.47 ±10.45
Grip strength fore limb (Mean ± SD (g/f))	769.67 ±21.54	737.73 ±30.34	677.33 * ±22.56	582.73 * ±15.50	652.80 ±84.30	657.53 ±103.95
Grip strength hind limb (Mean ± SD (g/f))	462.73 ±17.39	458.40 ±11.39	421.93 * ±10.79	408.07 * ±5.56	470.07 ±58.58	458.13 ±48.69
Locomotor activity (Mean ± SD)
X - Movements	541.40 ±51.59	529.80 ±58.32	510.60 ±42.58	527.60 ±37.96	686.60 ±74.52	687.40   ±14.24
Y - Movements	520.00 ±39.14	519.00 ±28.11	483.20 ±49.48	409.60 *±11.84	680.60 ±13.24	707.40 ±44.09
Z - Movements	393.80 ±54.12	420.80 ±37.51	334.60 ±44.38	289.80 * ±40.60	740.20 ±45.39	750.40 ±104.65
Total Movements (X+Y+Z)	1455.20 ±48.24	1469.60 ±48.46	1328.40 * ±67.93	1227.00* ±60.76	2107.40 ±67.93	2145.20 ±126.23

Note: * = Statistically significant from vehicle control (P ≤ 0.05)

 

TABLE 14 - SUMMARY OF FUNCTIONAL OBSERVATION BATTERY (FEMALES)

Parameter	Groups (n)
	G1	G2	G3	G4	G1R	G4R
Home cage observations
Posture
Sitting or standing normally	3	3	2	3	4	3
Standing on hind limbs	2	2	3	1	1	2
Respiration
Normal	5	5	5	4	5	5
Clonic involuntary movement
None/normal	5	5	5	4	5	5
Tonic involuntary movement
None/normal	5	5	5	4	5	5

 

TABLE 14 (CONTD) - SUMMARY OF FUNCTIONAL OBSERVATION BATTERY (FEMALES)

Parameter	Groups (n)
	G1	G2	G3	G4	G1R	G4R
Vocalization
None/normal	5	5	5	4	5	5
Convulsions
Absent – N	5	5	5	4	5	5
Hand-held observations
Reactivity
Easy/ normal (animal does not resist)	5	5	5	4	5	5
Handling
Squeaks or does not squeak but exhibit mild resistance; easy to handle/ normal	5	5	5	4	5	5
Palpebral closure
Open	5	5	5	4	5	5
Lacrimation
No lacrimation/ normal	5	5	5	4	5	5

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TABLE 14 (CONTD) - SUMMARY OF FUNCTIONAL OBSERVATION BATTERY (FEMALES) 

Parameter	Groups (n)
	G1	G2	G3	G4	G1R	G4R
Salivation
No salivation/ normal	5	5	5	4	5	5
Piloerection
None/ normal	5	5	5	4	5	5
Perineum Wetness
N: Absence of wetness	5	5	5	4	5	5
Body Temperature Range (ºC)	35.70-37.10	35.80-37.00	35.80-38.00	35.90-37.00	35.80-36.70	35.00-36.70
Open Field Activity
Clonic involuntary movement
None/ Normal	5	5	5	4	5	5
Tonic involuntary movement
None/ Normal	5	5	5	4	5	5

 

TABLE 14 (CONTD) - SUMMARY OF FUNCTIONAL OBSERVATION BATTERY (FEMALES)

Parameter	Groups (n)
	G1	G2	G3	G4	G1R	G4R
Gait
N:Head is horizontal, abdomen rises slightly above floor, and body moves up and down slightly during walking	5	5	5	4	5	5
Movements
Normal	5	5	5	4	5	5
Arousal
Normal (keeps guard up and engages in exploratory activity)	5	5	5	4	5	5
Tremors
None	5	5	5	4	5	5
Occurrence of stereotype	0	0	0	0	0	0
Abnormal behaviour	0	0	0	0	0	0
Number of Defecations (Mean)	1.4	2.2	1.8	2.3	1.2	1.0
Number of Urinations (Mean)	1.8	1.6	2.2	1.8	2.0	1.8
Number of Rearings (Mean)	7.2	7.4	6.8	7.0	9.2	7.6
Sensory Reactivity Measurements
Approach Response
Slowly approaches, sniffs and pulls back/ normal	5	5	5	4	5	5
Touch Response
Slowly approaches, sniffs and pulls back/ normal	5	5	5	4	5	5

 

TABLE 14 (CONTD) - SUMMARY OF FUNCTIONAL OBSERVATION BATTERY (FEMALES)

Parameter	Groups (n)
	G1	G2	G3	G4	G1R	G4R
Eyelid reflex
Blink/ Normal	5	5	5	4	5	5
Pinna reflex
Auricle twitches/ normal	5	5	5	4	5	5
Sound response
Mild reaction, hears sound/normal	5	5	5	4	5	5
Tail pinch response
Looks back, moves forward and lightly squeaks/normal	5	5	5	4	5	5
Pupillary reflex
Contracts	5	5	5	4	5	5
Aerial righting reflex
Lands on four limbs/ normal	5	5	5	4	5	5
Tail limb reflex
Present (Y)	5	5	5	4	5	5
Abdominal tone
Normal (proper hardness)	5	5	5	4	5	5

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TABLE 14 (CONTD) - SUMMARY OF FUNCTIONAL OBSERVATION BATTERY (FEMALES)

Parameter	Groups (n)
	G1	G2	G3	G4	G1R	G4R
Limb tone
Normal (proper hardness)	5	5	5	5	5	5
Landing Foot Splay (Mean ± SD (mm))	63.87 ±13.63	66.27 ±7.38	71.40 ±7.83	62.25 ±10.39	67.6716.91	50.00*↓ ±3.57
Grip strength fore limb (Mean ± SD (g/f))	675.00  ±16.37	684.53  ±63.09	651.20 ±12.30	635.17 ±79.98	504.20 ±46.28	610.40 ±104.35
Grip strength hind limb (Mean ± SD (g/f))	410.67 ±2.12	427.27 ±24.23	394.93 ±12.79	396.50 ±49.29	582.20 ±121.91	536.60 ±46.64
Locomotor activity (Mean ± SD)
X - Movements	518.40 ±43.48	522.80 ±49.34	505.40 ±21.37	467.00 ±42.17	616.80 ±61.90	650.60 ±44.09
Y - Movements	486.60 ±18.43	478.40 ±41.58	457.80 ±26.66	510.25 ±45.04	659.00 ±41.25	677.80 ±63.50
Z - Movements	488.40 ±20.17	450.00 ±13.56	445.80 * ±25.90	444.00 ±37.35	705.00 ±91.36	765.20 ±87.85
Total Movements (X+Y+Z)	1493.40 ±43.43	1451.20 ±37.38	1409.00 ±45.46	1421.25 ±123.35	1980.80 ±170.34	2093.60 ±172.15

Note: * = Statistically significant from vehicle control (P ≤ 0.05)

 

Table 15 –Summary REPORT OF REPRODUCTIVE / developmental effect

OBSERVATIONS	VALUES
Groups	G1	G2	G3	G4
Dose (mg/kg bw/day)	0	100	300	700
Pairs started (N)	10	10	10	10
Females showing evidence of copulation (N)	10	9	10	9
Females achieving pregnancy (N)	10	9	10	9
Conceiving days 1 - 5 (N)	6	5	3	2
Conceiving days 6 - 14 (N)	4	4	6	6
Conceiving during remating (N)	0	0	1	1
Pregnancy ≤ 21 days (N)	4	3	4	6
Pregnancy = 22 days (N)	5	6	6	3
Pregnancy ≥ 23 days (N)	1	0	0	0
Dams with live young born (N)	10	9	10	8
Dams with live young at day 4 pp (N)	10	9	9	8
Implants/ dam (mean)	9.00	9.67	10.70	9.38
Live pups/ dam at day 0 (mean)	9.00	9.11	10.30	8.63
Live pups/ dam at day 4 (mean)	7.70	7.56	8.40	7.00
Live pups/ dam at day 13 (mean)	7.70	7.56	8.20	7.00
Sex ratio (m/f) at birth (mean)	1.72	1.70	1.35	1.21
Sex ratio (m/f) at day 4 (mean)	1.68	1.70	1.20	1.24
Litter weight at birth (mean)	63.41	56.36	67.36	59.22
Litter weight at day 4 (mean)	107.08	99.02	112.27	96.79
Pup weight at birth (mean) - Male	7.16	7.06	6.80	7.40
Pup weight at birth (mean) - Female	6.86	5.85	6.27	5.60
Pup weight at day 4 (mean) - Male	12.37	12.05	11.03	11.83
Pup weight at day 4 (mean) - Female	11.90	10.49	10.99	9.57
Pup weight at day 13 (mean) - Male	28.60	26.90	27.85	27.40
Pup weight at day 13 (mean) - Female	28.56	26.83	26.83	23.50
AGD pups (mm) – Male	2.43	2.54	2.43	2.45
AGD pups (mm) - Female	1.35	1.28	1.37	1.15
Normalized AGD pups  - Male	1.27	1.33	1.28	1.26
Normalized AGD pups - Female	0.71	0.77	0.75	0.71
No of nipples/ Areolae - male pups at day 12 (mean)	0	0	0	0
ABNORMAL PUPS
Dams with 0	10	9	9	8
Dams with 1	0	0	0	0
Dams with  ≥  2	0	0	0	0

 

TABLE 15 (CONTD.) – STUDY SUMMARY REPORT OF REPRODUCTIVE / DEVELOPMENTAL EFFECT (CONTD.)

OBSERVATIONS	VALUES
Groups	G1	G2	G3	G4
Dose (mg/kg. bw/day)	0	100	300	700
LOSS OF OFFSPRING
Pre -natal (implantations minus live births)
Females with 0	10	6	8	5
Females with 1	0	2	2	1
Females with 2	0	0	0	1
Females with ≥  3	0	1	0	1
Post-natal (live births minus alive at post-natal day 4)
Females with 0	9	9	9	7
Females with 1	1	0	1	1
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	10	9	9	8
Females with 1	0	0	0	0
Females with 2	0	0	1	0
Females with ≥  3	0	0	0	0

 

Table 16 – Reproduction/Developmental Indices

Indices	Groups Dose (mg/kg. bw/day)	G1	G2	G3	G4
		0	100	300	700
Male mating index (%)		100	90	100	90
Male fertility index (%)		100	90	100	90
Female mating index (%)		100	90	100	90
Female fecundity index (%)		100	90	100	90
Female fertility index (%)		100	90	100	90
Post-implantation loss (%) - Mean		0.0	6.6	8.5	9.8
Gestation index (%)		100	100	100	88.88
Live birth index (%)		100	100	100	100
Day 4 survival index (%)		98.75	100	98.8	98.18
Day 13 survival index (%)		98.75	100	97.6	98.18

 

 

 

TABLE 17 – SUMMARY OF TERMINAL FASTING BODY WEIGHT AND ABSOLUTE ORGAN WEIGHTS (g) – MALES

GroupsDose(mg/kg/day)	Fasting Body Weight And Absolute Organ Weights (g)
		FBW	Liver	Kidneys	Spleen	Adrenals	Heart	Thymus	Brain
		(g)	(g)	(g)	(g)	(g)	(g)	(g)	(g)
G1 0	Mean	409.37	13.09	3.04	1.24	0.10	1.33	0.37	2.07
	SD	29.79	0.42	0.19	0.23	0.00	0.10	0.06	0.20
	N	5	5	5	5	5	5	5	5
G2100	Mean	411.92	12.96	2.84	1.28	0.10	1.37	0.35	2.03
	SD	18.20	0.27	0.29	0.25	0.01	0.13	0.07	0.07
	N	5	5	5	5	5	5	5	5
G3300	Mean	45.30	13.19	2.86	1.31	0.09	1.28	0.31	1.98
	SD	18.44	0.06	0.21	0.27	0.01	0.07	0.09	0.16
	N	5	5	5	5	5	5	5	5
G4700	Mean	390.65	14.25*	3.50	1.25	0.08*	1.36	0.40	2.03
	SD	37.08	1.23	0.18	0.13	0.01	0.24	0.07	0.13
	N	5	5	5	5	5	5	5	5

Key: Values are in Mean ± SD; *= Statistically significant from vehicle control (P ≤ 0.05)                                                                                         

 (P ≤ 0.05)

ORGAN WEIGHTS (g) – MALES

GroupsDose(mg/kg/day)	Fasting Body Weight And Absolute Organ Weights (g)
		FBW	Testes	Epididymis	Prostrate + seminal vesicles with coagulation glands	Thyroid with parathyroid
		(g)	(g)	(g)	(g)	(g)
G10	Mean	421.44	4.06	1.61	3.53	0.43
	SD	32.43	0.19	0.14	0.21	0.07
	N	10	10	10	10	10
G2100	Mean	420.52	3.89	1.64	3.55	0.48
	SD	25.53	0.39	0.16	0.26	0.06
	N	10	10	10	10	10
G3300	Mean	407.11	4.02	1.59	3.44	0.47
	SD	35.24	0.31	0.23	0.38	0.04
	N	10	10	10	10	10
G4700	Mean	409.80	3.87	1.55	3.46	0.49*
	SD	39.21	0.38	0.14	0.25	0.05
	N	10	10	10	10	10

Key: Values are in Mean ± SD; * = Statistically significant from vehicle control (P ≤ 0.05)                                                                                         

 

TABLE 18 – SUMMARY OF TERMINAL FASTING BODY WEIGHT AND ABSOLUTE ORGAN WEIGHTS (g) – FEMALES

GroupsDose(mg/kg/day)	Fasting Body Weight And Absolute Organ Weights (g)
		FBW	Liver	Kidneys	Spleen	Adrenals	Heart	Thymus	Brain
		(g)	(g)	(g)	(g)	(g)	(g)	(g)	(g)
G1 0	Mean	323.96	13.42	1.65	0.88	0.09	1.09	0.27	1.82
	SD	7.44	0.43	0.32	0.20	0.01	0.08	0.05	0.05
	N	5	5	5	5	5	5	5	5
G2100	Mean	321.31	12.98	1.95	1.04	0.08	1.12	0.25	1.91
	SD	31.37	0.67	0.20	0.18	0.01	0.07	0.03	0.11
	N	5	5	5	5	5	5	5	5
G3300	Mean	321.91	13.53	2.12	0.86	0.25	1.13	0.34	1.92
	SD	8.76	1.04	0.30	0.45	0.38	0.10	0.02	0.10
	N	5	5	5	5	5	5	5	5
G4700	Mean	305.48	13.17	2.01	1.08	0.09	1.07	0.27	1.85
	SD	33.92	0.47	0.18	0.11	0.01	0.04	0.11	0.10
	N	4	4	4	4	4	4	4	4

Key: Values are in Mean ± SD

GroupsDose(mg/kg/day)	Fasting Body Weight And Absolute Organ Weights (g)
		FBW	Ovaries	Uterus	Thyroid with parathyroid
		(g)	(g)	(g)	(g)
G1 0	Mean	323.95	0.23	0.75	0.47
	SD	19.43	0.02	0.07	0.06
	N	10	10	10	10
G2100	Mean	299.46	0.24	0.69	0.47
	SD	38.36	0.02	0.05	0.04
	N	10	10	10	10
G3300	Mean	312.09	0.22	0.72	0.47
	SD	17.94	0.02	0.05	0.06
	N	10	10	10	10
G4700	Mean	301.14	0.24	0.74	0.50
	SD	24.91	0.03	0.08	0.03
	N	8	8	8	8

Key: Values are in Mean ± SD

 

TABLE 19 – SUMMARY OF TERMINAL FASTING BODY WEIGHT AND ABSOLUTE ORGAN WEIGHTS (g) – RECOVERY MALES

GroupsDose(mg/kg/day)	Fasting Body Weight and Absolute Organ Weights (g) – Recovery Males
		FBW	Liver	Kidneys	Spleen	Adrenals	Heart	Thymus	Brain	Testes	Epididymes	Prostate+seminal vesicles with coagularting glands	Thyroid with parathyroid
		(g)	(g)	(g)	(g)	(g)	(g)	(g)	(g)	(g)	(g)	(g)	(g)
G1R0	Mean	421.88	13.02	2.89	1.08	0.10	1.52	0.36	2.03	3.33	1.46	3.26	0.47
	SD	17.36	0.85	0.21	0.24	0.01	0.15	0.08	0.05	0.40	0.27	0.62	0.06
	N	5	5	5	5	5	5	5	5	5	5	5	5
G4R700	Mean	383.62	12.98	2.47	1.08	0.09	1.32	0.34	2.03	3.75	1.52	2.93	0.49
	SD	22.85	1.21	0.16	0.07	0.01	0.12	0.07	0.15	0.41	0.12	0.46	0.06
	N	5	5	5	5	5	5	5	5	5	5	5	5

Key: Values are in Mean ± SD

 

TABLE 20 – SUMMARY OF TERMINAL FASTING BODY WEIGHT AND ABSOLUTE ORGAN WEIGHTS (g) – RECOVERY FEMALES

GroupsDose(mg/kg/day)					Fasting Body Weight and Absolute Organ Weights (g) – Recovery Females
		FBW		Liver		Kidneys	Spleen	Adrenals	Heart	Thymus	Brain	Ovaries	Uterus	Thyroid with parathyroid
		(g)		(g)		(g)	(g)	(g)	(g)	(g)	(g)	(g)	(g)	(g)
G1R0	Mean	268.32		8.62		1.98	0.74	0.09	1.09	0.42	1.93	0.20	0.69	0.48
	SD	21.29		0.72		0.23	0.08	0.01	0.04	0.06	0.11	0.05	0.13	0.02
	N	5		5		5	5	5	5	5	5	5	5	5
G4R700	Mean	267.37		8.97		1.93*	0.71	0.10	1.17	0.42	1.98	0.21	0.72	0.50
	SD	11.96		0.94		0.16	0.10	0.01	0.27	0.12	0.17	0.02	0.07	0.05
	N	5		5		5	5	5	5	5	5	5	5	5

Key: Values are in Mean ± SD; * = Statistically significant from vehicle control recovery (P ≤ 0.05)

TABLE 21 – SUMMARY OF RELATIVE ORGAN WEIGHTS – MALES

GroupsDose(mg/kg/day)	Relative Organ Weights (%)
		Liver	Kidneys	Spleen	Adrenals	Heart	Thymus	Brain
		(%)	(%)	(%)	(%)	(%)	(%)	(%)
G1 0	Mean	3.21	0.75	0.31	0.02	0.33	0.09	0.51
	SD	0.22	0.06	0.07	0.00	0.02	0.01	0.04
	N	5	5	5	5	5	5	5
G2100	Mean	3.15	0.69	0.31	0.02	0.33	0.08	0.49
	SD	0.17	0.05	0.05	0.00	0.03	0.01	0.01
	N	5	5	5	5	5	5	5
G3300	Mean	3.26	0.71	0.32	0.02	0.32	0.08	0.49
	SD	0.15	0.05	0.06	0.00	0.01	0.02	0.03
	N	5	5	5	5	5	5	5
G4700	Mean	3.65*	0.78	0.32	0.02	0.35	0.10	0.52
	SD	0.18	0.04	0.04	0.00	0.08	0.01	0.04
	N	5	5	5	5	5	5	5

Key: Values are in Mean ± SD; * = Statistically significant from vehicle control (P ≤ 0.05)

                                                                                          

GroupsDose(mg/kg/day)	Relative Organ Weights (%) Males
		Testes	Epididymis	Prostrate + seminal vesicles with coagulation glands	Thyroid with parathyroid
		(%)	(%)	(%)	(%)
G1 0	Mean	0.97	0.38	0.84	0.10
	SD	0.07	0.04	0.07	0.02
	N	10	10	10	10
G2100	Mean	0.93	0.39	0.85	0.11
	SD	0.08	0.02	0.07	0.02
	N	10	10	10	10
G3300	Mean	0.99	0.39	0.84	0.12
	SD	0.12	0.04	0.06	0.02
	N	10	10	10	10
G4700	Mean	0.95	0.38	0.85	0.12
	SD	0.11	0.04	0.08	0.01
	N	10	10	10	10

Key: Values are in Mean ± SD

 

TABLE 22 – SUMMARY OF RELATIVE ORGAN WEIGHTS – FEMALES

GroupsDose(mg/kg/day)	Relative Organ Weights (%)
		Liver	Kidneys	Spleen	Adrenals	Heart	Thymus	Brain
		(%)	(%)	(%)	(%)	(%)	(%)	(%)
G1 0	Mean	4.14	0.51	0.27	0.03	0.34	0.08	0.56
	SD	0.14	0.10	0.06	0.00	0.02	0.01	0.02
	N	5	5	5	5	5	5	5
G2100	Mean	4.08	0.61	0.33	0.03	0.35	0.08	0.60
	SD	0.52	0.10	0.06	0.00	0.04	0.02	0.05
	N	5	5	5	5	5	5	5
G3300	Mean	4.20	0.63	0.31	0.03	0.34	0.11	0.58
	SD	0.30	0.04	0.06	0.00	0.02	0.01	0.03
	N	5	5	5	5	5	5	5
G4700	Mean	4.35	0.66*	0.35	0.03	0.35	0.09	0.62
	SD	0.50	0.07	0.04	0.00	0.03	0.05	0.11
	N	5	5	5	5	5	5	5

Key: Values are in Mean ± SD; * = Statistically significant from vehicle control (P ≤ 0.05)                                                                                                                                                       

GroupsDose(mg/kg/day)	Relative Organ Weights (%) Females
		Ovaries	Uterus	Thyroid with parathyroid
		(%)	(%)	(%)
G1 0	Mean	0.07	0.23	0.15
	SD	0.01	0.04	0.01
	N	10	10	10
G2100	Mean	0.08	0.23	0.16
	SD	0.01	0.02	0.03
	N	10	10	10
G3300	Mean	0.07	0.23	0.15
	SD	0.01	0.02	0.03
	N	10	10	10
G4700	Mean	0.08	0.25	0.17
	SD	0.01	0.02	0.02
	N	8	8	8

Key: Values are in Mean ± SD

 

TABLE 23 - SUMMARY OF RELATIVE ORGAN WEIGHTS – RECOVERY MALES

GroupsDose(mg/kg/day)	Relative Organ Weights (%) – Recovery Males
		FBW	Liver	Kidneys	Spleen	Adrenals	Heart	Thymus	Brain	Testes	Epididymis	Prostrate + seminal vesicles with coagulation glands	Thyroid with parathyroid
		(%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)
G1R0	Mean	421.88	3.09	0.68	0.26	0.02	0.36	0.09	0.48	0.79	0.34	0.77	0.11
	SD	17.36	0.27	0.05	0.07	0.00	0.04	0.02	0.02	0.07	0.06	0.12	0.01
	N	5	5	5	5	5	5	5	5	5	5	5	5
G4R700	Mean	383.62	3.40	0.65	0.28	0.02	0.34	0.09	0.53	0.98*	0.40	0.77	0.13
	SD	22.85	0.46	0.06	0.01	0.00	0.03	0.01	0.06	0.13	0.04	0.16	0.02
	N	5	5	5	5	5	5	5	5	5	5	5	5

Key: Values are in Mean ± SD; * - Statistically significant from vehicle control recovery (P ≤ 0.05)

 

TABLE 24 - SUMMARY OF RELATIVE ORGAN WEIGHTS –RECOVERY FEMALES

GroupsDose(mg/kg/day)					Relative Organ Weights (%) – Recovery Females
		FBW		Liver		Kidneys	Spleen	Adrenals	Heart	Thymus	Brain	Ovaries	Uterus	Thyroid with parathyroid
		(%)		(%)		(%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)	(%)
G1R0	Mean	268.32		3.22		0.74	0.28	0.03	0.41	0.16	0.72	0.08	0.25	0.18
	SD	21.29		0.21		0.10	0.05	0.00	0.03	0.03	0.09	0.02	0.03	0.01
	N	5		5		5	5	5	5	5	5	5	5	5
G4R700	Mean	267.37		3.35		0.72	0.26	0.04	0.44	0.16	0.74	0.08	0.27	0.19
	SD	11.96		0.21		0.05	0.03	0.00	0.11	0.04	0.05	0.01	0.03	0.01
	N	5		5		5	5	5	5	5	5	5	5	5

Key: Values are in Mean ± SD

 

TABLE 25 - 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	9/10	10/10	10/10	10/10
Reddish discoloration of Liver	1/10	0/10	0/10	0/10

         Key: n=10; NAD= No Abnormality Detected, ^= Incidence

 

TABLE 26 - SUMMARY OF EXTERNAL AND GROSS PATHOLOGICAL OBSERVATIONS – (FEMALES AND PROGENY)

Group	G1	G2	G3	G4
No. of Females/Dams	10	10	10	10
External Observations^
NAD	10/10	10/10	10/10	9/10
Dark secretion at genetalia	0/10	0/10	0/10	1/10
Internal Observations^
NAD	7/10	9/10	10/10	8/10
Reddish discoloration of Liver	3/10	0/10	0/10	0/10
Cysts on both the ovaries	0/10	1/10	0/10	1/10
Autolysis	0/10	0/10	0/10	1/10
No. of progeny observed	78	68	82	56
External Observations (Progeny )^
NAD	78/78	68/68	82/82	56/56

         Key: n=10; NAD= No Abnormality Detected, ^= Incidence

 

TABLE 27 - SUMMARY OF EXTERNAL AND GROSS PATHOLOGICAL OBSERVATIONS – MALES RECOVERY

Group	G1R	G4R
No. of male Animals Observed	5	5
External Observations^
NAD	5/5	5/5
Internal Observations^
NAD	5/5	5/5

 

 

 

 

 

 

TABLE 28 - SUMMARY OF EXTERNAL AND GROSS PATHOLOGICAL OBSERVATIONS – FEMALES RECOVERY

Group	G1R	G4R
No. of Female Animals Observed	5	5
External Observations^
NAD	5/5	5/5
Internal Observations^
NAD	5/5	5/5

 

 

 

 

 

 

TABLE 29: SUMMARY: HISTOPATHOLOGY OBSERVATIONS – MALE AND FEMALE

Tissue/ Findings/Sex	Males		Females
Dose Group	G1	G4	G1	G4
Adrenals Glands Corticular vacuolization increased-Bilateral Multifocal mild	1/10	0/10	0/10	0/9
Accessory cortical tissue unilateral	0/10	2/10	1/10	0/9
Epididymis  Cell debris-  Diffused Bilateral	1/10	0/10	0/10	0/9
Cell debris Diffused unilateral	0/10	1/10	0/10	0/9
Testes Degeneration/atrophy of seminiferous tubules - Bilateral	1/10	0/10	0/10	0/9
Degeneration/atrophy’s of seminiferous tubules - Unilateral	0/10	1/10	0/10	0/9
Multinucleated giant cell – minimal multifocal	0/10	1/10	0/10	0/9
Stomach glandular Dilation-gastric glands multifocal minimal	1/5	0/5	1/5	1/4
Thymus Involution-age related Minimal	1/5	1/5	1/5	1/4
Lungs Alveolar Mono Nuclear Cell infiltration multifocal minimal	1/5	0/5	0/5	0/4
Alveolar Mono Nuclear Cell infiltration Multifocal mild	0/5	1/5	0/5	0/4
Heart Infiltration of mononuclear cells multifocal minimal	0/5	1/5	0/5	0/4

                                                                                                                          Note: - Organs/tissues of parent animals and progeny with no abnormality detected (NAD) are not included in this table.

Conclusions:

Based on the experimental conditions in this study and toxicological data generated, the following effects levels were established for Dodecan-1-ol, ethoxylated (CAS No.: 9002-92-0) in Wistar rats:

- The No Observed Adverse Effect Level (NOAEL) for general toxicity in the male rats was considered to be 100 mg/kg bw/day.
- The Lowest Observed Adverse Effect Level (LOAEL) for general toxicity in male rats was considered to be 300 mg/kg bw/day based on dose-dependent decreases in locomotor activity at ≥300 mg/kg bw/day.
- The NOAEL for general toxicity in the female rats was considered to be 300 mg/kg bw/day.
- The LOAEL for general toxicity in female rats was considered to be 700 mg/kg bw/day based on dullness observed at 700 mg/kg bw/day in all of the female rats throughout the dosing period.
- The NOAEL for reproductive and developmental endpoints was considered to be 700 mg/kg bw/day.

Executive summary:

The purpose of this Combined oral (gavage) repeated dose toxicity with the reproduction/developmental screening test in Wistar rats with 2 weeks recovery period was to generate toxicological information concerning the effects of Dodecan-1-ol, ethoxylated (CAS No: 9002-92-0) on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of the conceptus and parturition. The aim of this study was also to provide information on systemic toxicity, target organ toxicity, the No Observed Adverse Effect Levels (NOAELs), the Lowest Observed Adverse Effect Levels (LOAELs), and the reversibility, persistence, or delayed occurrence of toxic effects (if any) after cessation of treatment. One vehicle control (G1) and three graded dose levels of 100 (G2), 300 (G3) and 700 (G4) mg/kg bw/day with each group consisting of 10 male and 10 female (with normal estrous cyclicity) rats were employed in the study. In addition to the main groups, two recovery groups G1R and G4R each consisted of 5 male and 5 female rats were included to observe reversibility, persistence, or delayed occurrence of toxic effects after cessation of treatment. The test item, i.e. Dodecan-1-ol, ethoxylated (CAS No: 9002-92-0) was suspended in Milli-Q water. Freshly prepared formulations of test item were administered orally to animals of both sex at the doses of 100 (G2), 300 (G3) and 700 (G4 and G4R) mg/kg bw/day throughout the treatment period. Similarly, the vehicle (Milli-Q water) was administered to the male and female rats from the vehicle control group and vehicle control recovery (G1 and G1R). The males were dosed two weeks before mating and during the mating period for a total of 28 days, with the exception one animal form G2, one animal from G3 and two animals from G4 which were dosed for a total of 35 days for the provision of re-mating. Females were dosed two weeks before 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 up to post-natal day 12 (i.e. up to the day before scheduled sacrifice), for a total of approx. 71 days. For recovery group animals (i.e. G1R and G4R), treatment was withdrawn after completion of total 28 days of treatment period and the animals were observed for 14 days after the last day of treatment. Oral administration by gavage carried at a fixed dose volume of 10 mL/kg bw. Formulation analyses for dose concentration verification and homogeneity were performed twice during the period treatment. The following parameters were evaluated in this study: Mortality/morbidity, clinical signs of adults and progeny, body weight, feed consumption, oestrous cycle, clinical pathology, thyroid hormone analysis, functional observation battery, pre-coital interval, gestation length, litter size/litter weights on Post-natal day (PND) 0, 4 and 13, pups sex ratio, Anogenital distance (AGD) of pups, nipple retention of male pups on PND 12, pups survival index on PND 4 and 13, external observations of pups, 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. Morbidity and Mortality: There was no morbidity/mortality at any dose level, except for one dam at 700 mg/kg bw/day that died on GD 21. The exact cause of death could not be determined, but it was possibly due to dystocia. The gross pathological examination of the animal that died on GD 21 showed autolysis of all organs which was considered to be due to natural post-mortem changes. Clinical Signs of Toxicity: No clinical signs were observed in either G2 or G3 during the study period. All animals in G4 and G4R showed dullness throughout the treatment period, which started on the second day of treatment. All animals in the G4R group showed recovery from the dullness after the withdrawal of treatment. No other clinical signs of toxicity were observed in G4 or G4R. Body Weights / Body Weight Changes of Adult Animal: No significant changes in body weight or body weight gain were observed in G2, G3 or G4 during the treatment period. In G4R males, significant decreases in absolute body weight were observed on day 15 of treatment (mean, 367.58 gram) compared to G1R males (mean, 389.13 gram), on the 8^th day of recovery (mean 393.20 gram) compared to G1R males (mean, 429.49 gram), and on the 14^th day of recovery (mean, 393.79 gram) compared to G1R males (mean 430.36 gram). Further in G4R males, statistically significant decreases in absolute body weight gain were observed between treatment days 1-8 (average loss of 0.31 gram) and between day 1 to 42 of the study (average gain of 28.44 gram) compared to G1R (average body weight gain between treatment days 1 to 8: 14.93g; and between day 1 to 42 of study: 64.28g). A significant decrease in % body weight gain from day 1 to 42 of study was also observed in G4R males (mean, 7.8% gain) compared to G1R males (mean, 17.7% gain). No significant changes in absolute body weight or body weight gain were observed in G4R females. The observed significant changes in absolute body weight and body weight gain in G4R males during recovery were considered to be treatment-related but most likely non-adverse since the differences compared to the G1R males were rather small, for example the differences in absolute body weight in G4R males compared to G1R males were less than 8.5% on both occasions. Food Consumption: No significant changes in food intake were observed in any of the groups during the study period. Functional Observational Battery: Dose-dependent and statistically significant decreases in fore limb and hind limb grip strengths were observed in G3 males (mean fore limb, 677.3 g/f; mean hind limb, 421.9 g/f) and G4 males (mean fore limb, 582.7 g/f; mean hind limb, 408.1 g/f) compared to G1 (mean fore limb, 769.7 g/f; mean hind limb, 462.7 g/f). In addition to this, significant decreases in locomotor activity were observed in G3 males in terms of total movements (mean, 1328.4 counts) compared to G1 (mean, 1455.2 counts) and in G4 in terms of Y-movements, Z-movements, and total movements (mean total movements 1227.0 counts). The above effects in G3 and G4 males were considered to be treatment-related and adverse, however reversible as no significant changes in any FOB parameter were observed in the G4R males. For female rats, significant changes in FOB parameters were limited to a significant decrease in Z-movements in G3 (mean 445.8 counts) compared to G1 (mean 488.4 counts). This effect lacked dose-dependency and was therefore not considered to be treatment-related. No other significant changes in the functional observatory battery (which included sensory reactivity measurement) were observed. Oestrous Cyclicity: No significant change in oestrous cycle length was observed in G2 (mean, 4.18 days), G3 (mean, 4.00 days) or G4 (mean, 4.08 days) compared to G1 (mean, 4.20 days) and no significant changes in oestrous cyclicity were observed. Thyroid Hormone Analysis: In female pups, a dose-dependent and statistically significant increase in TSH levels was observed in G3 (mean, 6015.50 pg/ml) and G4 (mean, 6236.18 pg/ml) compared to G1 (mean, 5233.88 pg/ml). Similarly in female pups, a dose-dependent and statistically significant increase in T4 levels was observed in G3 (mean, 119.05 ng/ml) and G4 (mean, 121.83 ng/ml) compared to G1 (mean, 103.94 ng/ml). In male pups, no significant changes were observed except for a statistically significant decrease in T4 in G3 (mean 102.60 ng/ml) compared to G1 (mean, 109.73 ng/ml) which was not attributed to the test item due to lack of dose-dependency. The observed changes in TSH and T4 levels in the female pups were considered to be potential treatment-related effects but non-adverse in nature as there were no gross or histopathological findings in any of the thyroid glands from the female pups. In adult males, there were no significant changes observed in any of these hormone levels (T4 and TSH). Offspring data: No significant changes in the number of live pups on PND 0, PND 4, and/or PND 13; litter weight on PND 0 and/or PND 4; sex ratio on PND 0 and/or PND 4; male and/or female pup weight on PND 0, PND 4, and/or PND 13; male and/or female AGD; male and/or female normalized AGD were observed at any dose level. No nipple retention was observed in any of the male pups in G1, G2, G3 and G4. All pups in G1, G2, G3 and G4 were also normal upon external examination. Pup survival indexes on PND 4 were 99.9, 92.7, 90.0, and 99.17% in G1, G2, G3 and G4 respectively, with no statistically significant effect observed. Pup survival indexes on PND 13 were 88.9, 78.4, 71.5 and 83.0% in G1, G2, G3 and G4 respectively, with no statistically significant effect observed. Reproductive Performance and Development Indices: Male and female mating indexes were 100, 90, 100, and 90% in G1, G2, G3, and G4 respectively and female fecundity and fertility indexes were 100, 90, 100, and 90% in G1, G2, G3, and G4 respectively. Higher pre-coital intervals were observed in G3 and G4 compared to G1. The number of female rats conceiving at 1-5 days after mating were 6/10, 5/10, 3/10, and 2/10 in G1, G2, G3, and G4 respectively. The number of female rats conceiving at 6-14 days after mating were 4/10, 4/10, 6/10, and 6/10 in G1, G2, G3, and G4, respectively. No remating was needed in G1. One animal each in G2, G3 and G4 conceived during the remating period. The higher pre-coital intervals in G3 and G4 were not considered as adverse since there were no corroborating effects on any other reproductive or developmental parameters such as mating index and fertility index. No significant changes in gestation length were observed in any of the groups. Gestation indexes were 100, 100, 100 and 88.9% in G1, G2, G3, and G4, respectively, i.e. one out of 9 conceived dams in G4 did not deliver any live offspring. The lower gestation index in G4 was not statistically secured and was considered to be within normal biological range. No significant difference in the number of implantations per dam were observed in G2 (mean, 9.7), G3 (mean, 10.7) or G4 (mean, 9.4) compared to G1 (mean, 9.0).  A dose-dependent trend of increased % post-implantation loss was observed in G2 (mean, 6.6%), G3 (mean, 8.5%) and G4 (mean, 9.8%) compared to G1 (mean, 0%). Since the % post-implantation losses in G2, G3 and G4 were less than 10% and could not be corroborated with any other relevant reproductive or developmental parameter in the study, the higher pre-implantation losses in G2, G3 and G4 were not considered to be adverse in nature. Pathology: Haematology: No significant changes in haematology were observed, with the exception of a significant increase in MCH in G2 females compared to G1 females. This effect was not attributed to the test item due to lack of dose dependency. Coagulation Potential Parameters: No significant changes in coagulation parameters were observed in any of the main study groups. A significant decrease in fibrinogen was observed in G4R females (mean, 210.9 mg/dl) compared to G1R females (mean, 266.27 mg/dl). This significant decrease in fibrinogen was considered incidental as it could not be corroborated with any gross or histopathological findings. Clinical Chemistry Parameters:  In male rats the following significant changes were observed: significant increases in urea in G2 (mean, 42.44 mg/dl) and G3 (mean, 42.89 mg/dl) compared to G1 (mean, 35.49 md/dl); significant decreases in creatinine in G3 (mean, 0.58 md/dl) and G4 (mean, 0.57 md/dl) compared to G1 (mean, 0.65 mg/dl); a significant increase in ALT in G2 (mean, 108.10 U/l) compared to G1 (mean, 79.56 U/l); a significant decrease in albumin in G2 (mean, 34.15 mg/dl) compared to G1 (mean, 36.75 mg/dl); and a significant decrease in total bile acids in G4 (mean, 18.45 mmol/l) compared to G1 (mean, 37.81 mmol/l). The observed changes in urea, ALT and albumin in G2/G3 were considered incidental due to lack of dose-dependency. The observed changes in creatinine and total bile acids in G3/G4 were not considered to be adverse effects of treatment due to lack of associated gross or histopathological findings and since no significant changes in creatinine or total bile acids were observed after the recovery period. Terminal Fasting Body Weights and Organ Weights: No significant changes in terminal fasting body weight were observed in the study. In male rats the following significant changes in organ weight were observed: a significant increase in absolute liver weight in G4 (mean, 14.25 gram) compared to G1 (mean, 13.09 gram); a significant decrease in absolute adrenal weight in G4 (mean, 0.08 gram) compared to G1 (mean, 0.10 gram); a significant increase in absolute thyroid with parathyroid weight in G4 (mean, 0.49 gram) compared to G1 (mean, 0.43 gram); a significant increase in relative liver weight in G4 (mean, 3.65%) compared to G1 (mean, 3.21%); and a significant increase in relative testes weight in G4R (mean, 0.98%) compared to G1R (mean, 0.79%). None of the observed changes in G4/G4R were considered to be adverse due to a lack of associated gross or histopathological effects in G4. In female rats, significant changes in organ weight were limited to a significant decrease in absolute kidney weight in G4R (mean 1.93 gram) compared to G1R (mean 1.98 gram) and a significant increase in relative kidney weight in G4 (mean, 0.66%) compared to G1 (mean, 0.51%). Neither change in kidney weight was considered to be adverse due to a lack of associated gross or histopathological effects in G4. Gross Pathology:  Gross findings in the adult males were restricted to one incidence of reddish discoloured liver in G1. In adult females, gross findings were limited to dark secretion at genitalia in one animal in G4, reddish discoloured liver in 3 animals in G1, cysts on both ovaries in one animal in G2 and in one animal in G4, and dark discolouration (autolysis) of all organs in the dam that died on GD 21 in G4. No gross findings were made in any of the pregnancies on PND 13. Histopathology: The microscopic examinations of the animals in G1 and G4 did not show any lesions of pathological significance that could be attributed to the test item. The histopathological findings in adult male rats included: minimal, multifocal dilatation of gastric glands in one G1 rat; minimal involution in the thymus in one G1 rat and in one G4 rat; minimal, multifocal mononuclear cellular infiltration in the alveolar tissue in one G1 rat; mild, multifocal mononuclear cellular infiltration in the alveolar tissue in one G4 rat; minimal mononuclear cellular infiltration in the heart in one G4 rat; mild, multifocal cortical vacuolization in adrenal gland in one G1 rat; unilateral accessory cortical tissue in adrenal gland in two G4 rats; diffused bilateral cell debris in epididymis in one G1 rat; diffused unilateral cell debris in epididymis in one G4 rat; bilateral degeneration/atrophy in seminiferous tubules in one G1 rat; unilateral degeneration/atrophy in seminiferous tubules in one G4 rat; and minimal multifocal presence of multinucleated giant cell in one G4 rat. The histopathological findings in adult female rats included: minimal, multifocal dilatation of gastric glands in one G1 rat and in one G4 rat; minimal involution in the thymus in one G1 rat and in one G4 rat; and unilateral accessory cortical tissue in adrenal gland in one G1 rat. The above microscopic findings observed in the male and female rats were considered incidental as they occurred randomly across G1 and G4, were mild/minimal severity, and/or can be expected for Wistar rats of this age (Elizabeth McInnes, 2012). Formulation analysis: Dose formulation analysis results of performed before treatment (second week of pre-exposure period) and during last week of lactation were within the acceptable range of ± 15 % to the nominal concentration with less than 10% RSD (relative standard deviation). Conclusion: Based on the experimental conditions in this study and toxicological data generated, the following effects levels were established for Dodecan-1-ol, ethoxylated (CAS No.: 9002-92-0) in Wistar rats:

• The No Observed Adverse Effect Level (NOAEL) for general toxicity in the male rats was considered to be 100 mg/kg bw/day.


• The Lowest Observed Adverse Effect Level (LOAEL) for general toxicity in male rats was considered to be 300 mg/kg bw/day based on dose-dependent decreases in locomotor activity at ≥300 mg/kg bw/day.


• The NOAEL for general toxicity in the female rats was considered to be 300 mg/kg bw/day.


• The LOAEL for general toxicity in female rats was considered to be 700 mg/kg bw/day based on dullness observed at 700 mg/kg bw/day in all of the female rats throughout the dosing period.  


• The NOAEL for reproductive and developmental endpoints was considered to be 700 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

700 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch 1

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

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data from peer reviewed journal

Qualifier:

according to guideline

Guideline:

other: As mentioned below

Principles of method if other than guideline:

The developmental toxicity effect of test chemical in Fischer 344 rats was assessed in a two generation of rat.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Fischer 344

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
Source: Parents: Charles River Breeding Laboratories.
F1 Generation: produced as the result of mating of the P generation rats during the conduct of the study.
Age: 4-7 weeks of age

Route of administration:

dermal

Vehicle:

water

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- M/F ratio per cage: one male rat per one female rat.

Duration of treatment / exposure:

not specified

Frequency of treatment:

Daily

Duration of test:

133 Days (F1 generation)

Remarks:

Doses / Concentrations:
0, 10, 100, 250 mg/kg bw/d (1, 10, 25% w/v, 0.5 mL/kg)
Basis:
nominal conc.

No. of animals per sex per dose:

total ;400

F0:240
Control: 30 males, 30 females
1% w/v: 30 males, 30 females
10% w/v: 30 males, 30 females
25% w/v: 30 males, 30 females

F1:160
Control: 20 males, 20 females
1% w/v: 20 males, 20 females
10% w/v: 20 males, 20 females
25% w/v: 20 males, 20 females

Control animals:

yes

Details on study design:

Details of controls: Control animals received water, based on its weekly body weight

Maternal examinations:

All rats were observed for viability at least twice daily throughout the study.
Pups were observed daily for mortality and physical abnormalities.
No mortalities were observed in the F0 generation
5 deaths were noted in the F1 generation (1 in control, 4 in different treatment groups).

Ovaries and uterine content:

No data available

Fetal examinations:

Litter size, sex of pups, and number of live and stillborn pups. Pups were observed daily for mortality and physical abnormalities

Statistics:

Organ weights, LDH-X activity, litter size, sex ratio, and survival index were compared to controls by a one-tailed Dunnett’s t-test at p < 0.05.
Fertility indices were analyzed by a one-tailed Fisher’s exact test. Mating indices were analyzed by Chi-square with continuity correction.
Sperm counts and survival indices were analyzed by the nonparametric Kruskal-Wallis test

Indices:

No data available

Historical control data:

No data available

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Both male and female rats in the 10% and 25% groups had dry flaking skin. Discoloration of the application sites was noted in all treated rats, including the controls.

Dermal irritation (if dermal study):

no effects observed

Description (incidence and severity):

Individual skin irritation scores for all rats in all dose groups was zero (0) throughout the F0 and F1 generations.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the F0 adults, significantly lower (p < 0.05) body weights of the 25% group were observed only during certain intervals when compared to the control .In the F1 adults, body weights of males in the 25% group were lower but were only statistically significant (p < 0.05) at days 21, 28, and 35 during the dosing period. Body weights of the 25% females were slightly lower from days 14-1 19 during the dosing period when compared to the controls.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In the F0 generation, the absolute and relative organ weights were comparable to the controls.
In the F1 generation, organ weight differences were observed sporadically. Since there were no macroscopic or microscopic histopathologic changes in any internal organs, these organ weight changes are considered to be of no toxicologic significance. There were no significant differences in testicular histology for any treated rats compared to the controls in these two generations.

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Number of abortions:

not specified

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

not specified

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

There were no compound-related effects on fertility index and mean gestational length of the F0 and F1 females.

Other effects:

not specified

Details on maternal toxic effects:

Maternal toxic effects:no effects. Remark: There were no compound-related effects on maternal body weights at any dose groups during the gestational and lactational periods in the F, and F, generations

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Remarks on result:

other: No toxic effects were observed

Abnormalities:

not specified

Localisation:

not specified

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Description (incidence and severity):

In the F1 and F2 pups, there were no differences in organ weight or morphology.

Skeletal malformations:

not specified

Visceral malformations:

not specified

Other effects:

not specified

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects. Remark: no dose-related trend in sex ratio alteration was seen at the 250 mg/kg/day

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: no observed adverse effects

Remarks on result:

other: No developmental toxic effects were observed

Abnormalities:

not specified

Localisation:

other: not specified

Developmental effects observed:

not specified

Treatment related:

not specified

EFFECT ON OFFSPRING IN TWO-GENERATION DERMAL STUDY

 

Concentration(%)	0	1	10	25	0	1	10	25
	F1 GENERATION				F2 GENERATION
Mean live litter size
Day 0	8.2	9.5	7.8	6.3	6.8	8.3	8.0	8.3
Day 28	7.6	8.6	6.9	5.9	6.6	8.0	7.4	7.6
Sex ratio (M/F)
Day 4	0.85	0.67	0.89	0.82	0.85	0.79	1.08	1.00
Body weights (g)^a
Day 1	5.4	5.4	5.4	5.6	5.6	5.6	5.7	5.7
Day 28	59.1	59.1	58.5	60.6	55.9	57.9	59.3	57.8
Survival index (%)
Day 1	100	99	99	98	98	99	98	97
Day 28^b	100	100	95	99	98	100	98	98

 

^aAdjusted for litter size.

^bExpressed as a%of the pups alive on day 4 after culling to 10 pups per litter

 

Conclusions:

No Observed Adverse Effect Level (NOAEL) for developmental toxicity was considered to be 250mg/kg bw/day. When male and female rats were treated with test chemical via dermal application

Executive summary:

The two generation reproduction toxicity study of test chemical was performed on male and femaleFisher 344 rats. A group of 30 weanling rats of each sex were dermally exposed to 1 mL/kg bw test chemical at concentrations of 0, 1, 10 or 25% w/v three times a week except during the mating periods. This treatment equals exposure levels of about 0,10, 100 or 250 mg/kg bw/d. Clinical observations were made daily during treatment and mating periods and twice daily during gestation and lactation.

Male and female body weights were taken weekly up to the time of mating and females weekly during gestation and lactation. F0 females were weighed weekly after day 28 of lactation. Pups were weighed as a litter on days1,4, 7,21, and 28 of lactation and individually on day 28 of lactation. After 119 days of dosing the F0 rats and133days of dosing the F1 rats, females were housed with males of the same treatment group. In the first week of the F1 mating period,30males and30females in each dose group were paired. Only proven males with positive matings were used in the following 2 weeks. In the first week of the F1 mating period, 20 males and 20 females in a 1: 1 ratio were cohabitated, and sibling or half-sibling matings were avoided. Only proven fertile males were used for the subsequent mating.

Each male rat in the F0 and F1generations was killed after breeding, and the left testis was used for enumeration of sperm heads and measurement of lactate dehydrogenase isozyme (LDH-X) activityLitters were caged with their mothers for at least 21 days after birth.Atparturition, the following observations were made: litter size, sex of pups, and number of live and stillborn pups. Pups were observed daily for mortality and physical abnormalities. On day4of lactation, any litter with more than 10 pups was culled to 10 by using a table of random numbers. Individual litter weights of the F0 and F1 generations were determined. Gross necropsies were performed on all F0 and F1 parents and selected F1 and F2 pups (5/sex/dose group). Histopathologic examinations included all reproductive organs, all lesions in F0 parents, and all tissues of the selected F1 pups. Organ weights and histopathologic examination of the F1reproductive organs, liver, spleen, heart, kidney, and lung of the F1 parents, and selected F2 pups in the highest dose group and the vehicle control group were performed. No mortalities were observed in the F0 generation Deaths were noted in the F1 generation(1in control,4in different treatment groups). At necropsy, the posterior pharynx of each rat was filled with moist, finely ground, and compacted food material that occluded the laryngeal opening of the rats. Death apparently was caused by asphyxiation and was not considered to be compound related. Both male and female rats in the 10% and25%groups had dry flaking skin. Discoloration of the application sites was noted in all treated rats, including the controls. Individual skin irritation scores for all rats in all dose groups was zero (0) throughout the F0 and F0 generations. In the F0 adults, significantly lower (p < 0.05) body weights of the 25% group were observed only during certain intervals when compared to the control .In the F1 adults, body weights of males in the 25% group were lower but were only statistically significant (p < 0.05) at days 21, 28, and35during the dosing period. Body weights of the 25% females were slightly lower from days14-1 19during the dosing period when compared to the controls. In theF0parental males, testis weights were not affected by the treatment, whereas the number of sperm per testis and per gram of testis were significantly lower in the 25% group when compared to the controls Values of LDH-X enzyme activity in all treated groups were comparable to the control. However, in the F1 parental males, all the noted parameters in all dose groups were comparable to the controls. The lower numbers of sperm in the testis of the high-dose F1 males were within the normal historical control range for this laboratory. Therefore, the difference is not considered to be a treatment-related effect. In the F0 generation, the absolute and relative organ weights were comparable to the controls. In the F1 generation, organ weight differences were observed sporadically. Since there were no macroscopic or microscopic histopathologic changes in any internal organs, these organ weight changes are considered to be of no toxicologic significance. There were no significant differences in testicular histology for any treated rats compared to the controls in these two generations. Mating indices were73.3%, 83.3%, 83.3%,and93.3%in the F0 generation and65.0%, 70.9%.82. 1%, and83.8%in the F1 generation of the 0, 1, 10, and 25% groups, respectively. The incidences appeared to be higher in the treated groups than the control in both generations but were not significantly different from the control group by Chi-square test with continuity correction. The lower mating performance of the control rats is within the historical range for the Fischer strain. There were no apparent morphologic differences in any affected rats to explain their apparent infertility. There were no compound-related effects on fertility index and mean gestational length of the F0 and F1 females. The fertility indices were77.3%, 64.0%. 76.0%,and53.6%in the F0 generation and88.5%,67.9%, 87.5%,and77.4%in the F1 generation of the 0, 1, 10, and 25% groups, respectively. Mean gestational length in all dose groups in the F0 and F1 generations was approximately 22 days. No compound-related effects on litter size, survival index, sex ratio, or body weights of pups in the F1 and F2 generations were observed during lactation. There were no compound-related effects on maternal body weights at any dose groups during the gestational and lactational periods in the F1and F2generations. There was no compound-related effect on development of the pups. Eye opening was observed at approximately16.1-16.9days for the F,pupsand 17.2-17.6 days postnatally for the F, pups in all dose groups. In the F1 and F2 pups, there were no differences in organ weight or morphology. HenceNo Observed Adverse Effect Level (NOAEL) for developmental toxicity was considered to be 250mg/kg bw/day. When male and femalerats were treated withtest chemical via dermal application.

 

 

 

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

chronic

Species:

rat

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

In the OECD 422 study performed with Wistar rats, NOAEL for reproductive and developmental toxicity was found at 700 mg/kg bw/day. Adverse effects in general health included significant decreases in locomotor activity in male rats treated at 300 and 700 mg/kg bw/day and dullness in all rats (both male and females) treated at 700 mg/kg bw/day. On this basis, the substnace is not regarded to be intrinsically toxic to reproduction or development and the substance is therefore considered to be classified as Not Classified for Toxicity to reproduction, as per Regulation EC 1272/2008.