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Description of key information

Repeated dose toxicity: Oral


In an OECD 422 study performed in Wistar rats, NOAEL in male rats was found at 100 mg/kg bw/day based on dose-dependent decreases in locomotor activity at 300 and 700 mg/kg bw/day whereas NOAEL in female rats was found at 300 mg/kg bw/day based on dullness in all animals treated at 700 md/kg bw/day. All of these effects, including dullness observed in all males treated at 700 mg/kg bw/day, showed complete reversibility during the recovery period.


 


Repeated dose toxicity: Inhalation


According to Annex IX of the REACH regulation, testing by the inhalation route is appropriate only if exposure of humans via inhalation is likely. Taking into account the low vapour pressure of the substance Dodecan-1-ol, ethoxylated (CAS No; 9002-92-0 EC No; 500-002-6) which is reported as 7.5X10-11mmHg at 25 C. Thus, exposure to inhalable dust, mist and vapour of the chemical Dodecan-1-ol, ethoxylated (CAS No; 9002-92-0 EC No; 500-002-6) is highly unlikely. Therefore this study is considered for waiver.


 


Repeated dermal study;


The acute toxicity value for Dodecan-1-ol, ethoxylated (CAS No; 9002-92-0 EC No; 500-002-6) (as provided in section 7.2.3) is >2000 mg/kg body weight. Also, given the use of the chemical; repeated exposure by the dermal route is unlikely since the use of gloves is common practice in industries. Thus, it is expected that Dodecan-1-ol, ethoxylated shall not exhibit 28 day repeated dose toxicity by the dermal route. In addition, there is no data available that suggests that Dodecan-1-ol, ethoxylated shall exhibit repeated dose toxicity by the dermal route. Hence this end point was considered for waiver.


 

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
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
Details on route of administration:
The oral route is the primary route of choice based on practical considerations, e.g., the likelihood of achieving the maximal systemic uptake of the test item. The oral route also represents a physiological route of exposure in humans.
Vehicle:
water
Remarks:
Milli-Q
Details on oral 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.
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 grouos (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.
Positive control:
None
Observations and examinations performed and frequency:
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.

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.
Sacrifice and pathology:
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.
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.
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.
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
Description (incidence and severity):
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 pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Neuropathological findings:
not examined
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).
Key result
Dose descriptor:
LOAEL
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
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
gross pathology
haematology
histopathology: neoplastic
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios
Key result
Dose descriptor:
LOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
behaviour (functional findings)
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
gross pathology
haematology
histopathology: neoplastic
histopathology: non-neoplastic
mortality
organ weights and organ / body weight ratios
serum/plasma hormone analyses
Key result
Critical effects observed:
no

Table 1 - Summary of Clinical Signs – MALES















































GroupsDose(mg/kg bw/day)SexNo. of animals/Total no. treated animalsClinical Signs
G10M10/10Normal
G2100M10/10Normal
G3300M10/10Normal
G4700M10/10Dullness
G1R0M5/5Normal
G4R700M5/5Dullness/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)SexNo. of animals/Total no. treated animalsClinical Signs
G10F10/10Normal
G2100F10/10Normal
G3300F10/10Normal
G4700F10/10Dullness
G1R0F5/5Normal
G4R700F5/5Dullness/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)1815222835*
G10Mean381.21391.04409.80431.82434.71-
SD22.0227.1829.8628.2232.70-
N1010101010-
G2100Mean386.22401.05415.16431.97430.50440.83
SD19.4921.0627.0026.8726.140
N10101010101
G3300Mean381.54392.34401.19413.95420.33477.88
SD18.8423.0331.7140.7131.850
N10101010101
G4700Mean380.02383.45399.12416.72420.52434.88
SD20.5824.4432.8434.6340.3765.70
N10101010102

          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)181522293642
G1R0Mean366.08381.01389.13409.96418.02429.49430.36
SD13.7816.4915.0015.0618.2417.9019.66
N5555555
G4R700Mean365.35365.04367.58*380.50381.58393.20*393.79*
SD15.0321.3413.9426.9332.7724.9921.97
N5555555

 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)FromTo1881515222228Abs Gain128TotalGain %128
G10Mean9.8318.7722.022.8753.4914.03
SD9.206.157.3911.0120.695.47
n101010101010
G2100Mean14.8214.1116.81-1.4744.2811.53
SD11.118.885.338.5120.535.49
n101010101010
G3300Mean10.808.8412.776.3838.7910.14
SD12.409.3912.3913.3422.365.82
n101010101010
G4700Mean3.4315.6717.613.8040.5010.48
SD9.2110.908.6110.2023.255.74
n101010101010

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)FromTo188151522222929363642Abs Gain142Total% Gain142
G1R0Mean14.938.1220.838.0711.460.8764.2817.69
SD8.888.772.8410.814.9928.7923.766.85
N55555555
G4R700Mean-0.31*2.5412.921.0811.620.5928.44*7.77*
SD9.217.9013.857.3317.235.8212.323.31
N55555555
           

           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)1815
G10Mean261.80266.32269.88
SD14.5310.3512.36
n101010
G2100Mean263.45264.30268.54
SD15.1113.8314.03
n101010
G3300Mean261.96263.63270.25
SD15.9011.188.60
n101010
G4700Mean261.39261.98270.04
SD15.1115.5817.55
n101010

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)181522293642
G1R0Mean259.92258.53263.06267.27272.31280.37279.72
SD17.8924.5817.0917.1019.7318.1221.01
N5555555
G4R700Mean260.65260.07267.26272.77274.60279.54276.16
SD16.4514.259.648.8512.5610.0811.63
N5555555

Key: Values are in Mean ± SD


 


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

















































































































GroupsDose(mg/kg bw/day)FromTo18815Abs. Gain115Total% Gain115
G10Mean 4.523.568.083.22
SD 7.958.0011.494.46
N 10101010
G2100Mean 0.854.245.092.06
SD 7.257.8312.954.96
N 10101010
G3300Mean 1.676.628.293.34
SD 5.374.628.683.44
N 10101010
G4700Mean 0.598.068.653.32
SD 5.907.079.103.36
N 10101010

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)FromTo188151522222929363642Abs Gain142Total% Gain142 
G1R0Mean-1.384.534.205.058.06-0.6519.817.74 
SD11.8710.854.689.142.083.1416.956.58 
N55555555 
G4R700Mean-0.587.195.511.834.94-3.3815.516.11 
SD2.845.834.235.254.083.8010.194.29 
N55555555 
            

           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
12
G10Mean26.9828.97
SD1.571.60
N1010
G2100Mean27.6928.52
SD1.631.15
N1010
G3300Mean27.3727.00
SD1.421.02
N1010
G4700Mean25.4028.00
SD1.042.11
N1010

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)123456
G1R0Mean28.2628.6128.3427.6828.9624.37
SD1.601.561.562.060.972.12
N555555
G4R700Mean25.6225.3628.3226.6828.7026.70
SD1.502.042.481.512.051.45
N555555

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
 12
G10Mean17.8018.20
SD1.301.82
N1010
G2100Mean17.8016.39
SD1.460.55
N1010
G3300Mean18.8017.02
SD1.300.72
N1010
G4700Mean19.6116.82
SD1.160.85
N1010

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 periodRecovery period
GroupsDose(mg/kg bw/day)1-88-1515-2222-2929-3636-42
G1R0Mean18.3016.6717.3618.0218.9417.35
SD1.521.552.022.081.001.56
N555555
G4R700Mean19.3316.0019.0217.6718.6917.35
SD1.500.991.001.200.550.64
N555555

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)
0714200-77-1414-200-20
G10Mean271.74295.37324.17385.3522.1328.8061.18112.10
SD15.0918.5324.6138.008.919.3318.0625.34
N1010101010101010
G2100Mean278.27292.79322.91389.3313.0727.1159.7899.96
SD19.1018.8518.2625.496.179.9326.4839.90
N99999999
G3300Mean277.05296.53323.93393.3319.4827.4069.40116.28
SD10.407.3713.9827.597.5010.5818.9730.31
N1010101010101010
G4700Mean270.09286.76321.97390.7515.0031.6961.90108.59
SD19.3521.7524.6928.729.5414.4825.2640.79
N99999999

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)FromTo077141420020
G10Mean22.7023.8022.9523.15
SD2.932.051.362.11
N10101010
G2100Mean20.5920.8220.3620.59
SD7.487.507.287.42
N9999
G3300Mean22.6624.5623.3023.51
SD1.141.871.941.65
N10101010
G4700Mean20.4421.0820.7320.75
SD7.457.627.557.54
N9999

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)
04120 - 44 - 12
G10Mean318.24325.95338.847.7012.89
SD24.6520.1519.7315.428.67
N1010101010
G2100Mean300.91310.54329.589.6319.04
SD32.8133.3630.594.2135.44
N99999
G3300Mean309.26315.19326.635.9311.44
SD17.1815.6618.3710.385.68
N1010101010
G4700Mean298.67304.03312.725.368.68
SD30.9031.0829.357.215.31
N88888

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 - 44 - 12
G10Mean26.4022.51
SD2.251.13
N1010
G2100 Mean27.0722.81
SD8.661.03
N99
G3300 Mean27.3823.06
SD0.831.11
N1010
G4700 Mean20.8918.75
SD11.289.95
N88

Key: Values are in Mean ± SD


 


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




















































































Sex: Male  Parameters
GroupsDose(mg/kg bw) T4TSH
ng/mLpg/mL
G10Mean113.415216.43
SD10.58844.31
N1010
G2100Mean106.795477.67
SD12.50760.63
N1010
G3300Mean118.365899.04
SD3.57691.23
N1010
G4700Mean119.335865.42
SD5.36431.13
N1010

Key: Values are in mean ± SD


 


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




















































































Sex: Male  Parameters
GroupsDose(mg/kg bw) T4TSH
ng/mLpg/mL
G10Mean109.735566.75
SD3.58679.53
N1010
G2100Mean109.525496.24
SD4.77262.92
N99
 G3300     Mean102.60*5555.52
SD3.96580.24
N99
G4700Mean111.095511.55
SD6.98544.40
N88

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) T4TSH
ng/mLpg/mL
G10Mean103.945233.88
SD13.53677.14
N1010
G2100Mean109.025577.88
SD4.46335.05
N99
 G3300     Mean119.05*6015.50*
SD10.27366.68
N99
G4700Mean121.83*6236.18*
SD5.31363.91
N88

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 cycleNo. of animals with abnormal cycle 
 
G1 04.20100 
G21004.18100 
G33004.00100 
G47004.08100 

 


 


 


 


 


 


TABLE 13 - SUMMARY OF FUNCTIONAL OBSERVATION BATTERY (MALES)
























































































ParameterGroups (n)
G1G2G3G4G1RG4R
Home cage observations
Posture
Sitting or standing normally343332
Standing on hind limbs212123
Asleep (lying on side)000100
Respiration
Normal555555
Clonic involuntary movement
None/normal555555
Tonic involuntary movement
None/normal555555

 


 


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




























































































ParameterGroups (n)
G1G2G3G4G1RG4R
Vocalization
None/normal555555
Convulsions
Absent – N555555
Hand-held observations
Reactivity
Easy/ normal (animal does not resist)555555
Handling
Squeaks or does not squeak but exhibit mild resistance; easy to handle/ normal 555555
Palpebral closure
Open555555
Lacrimation
No lacrimation/ normal555555

 


 


 


 


 


 


 


 


 


 


 


 


 


 


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

























































































ParameterGroups (n)
G1G2G3G4G1RG4R
Salivation
No salivation/ normal555555
Piloerection
None/ normal555555
Perineum Wetness
N: Absence of wetness555555
Body Temperature Range (ºC)35.9 - 36.735.8 – 36.635.8 – 36.835.8 – 36.735.4-36.735.2-36.8
Open Field Activity
Clonic involuntary movement
None/ Normal555555
Tonic involuntary movement
None/ Normal555555

 


 


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









































































































































ParameterGroups (n)
G1G2G3G4G1RG4R
Gait
N:Head is horizontal, abdomen rises slightly above floor, and body moves up and down slightly during walking555555
Movements
Normal555555
Arousal
Normal (keeps guard up and engages in exploratory activity)555555
Tremors
None555555
Occurrence of stereotype000000
Abnormal behaviour000000
Number of Defecations (Mean)0.80.40.80.61.21.0
Number of Urinations (Mean)2.02.42.62.81.81.6
Number of Rearings (Mean)7.46.64.84.85.67.8
Sensory Reactivity Measurements
Approach Response
Slowly approaches, sniffs and pulls back/ normal555555
Touch Response
Slowly approaches, sniffs and pulls back/ normal555555

 


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































































































































ParameterGroups (n)
G1G2G3G4G1RG4R
Eyelid reflex
Blink/ Normal555555
Pinna reflex
Auricle twitches/ normal555555
Sound response
Mild reaction, hears sound/normal555555
Tail pinch response
Looks back, moves forward and lightly squeaks/normal555555
Pupillary reflex
Contracts555555
Aerial righting reflex
Lands on four limbs/ normal555555
Tail limb reflex
Present (Y)555555
Abdominal tone
Normal (proper hardness)555555
            

 


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































































































ParameterGroups (n)
G1G2G3G4G1RG4R
Limb tone
Normal (proper hardness)555555
Landing Foot Splay (Mean ± SD (mm))64.13 ± 5.2969.07 ± 10.2365.60 ± 2.7875.00 ± 7.7758.13 ±10.9651.47 ±10.45
Grip strength fore limb (Mean ± SD (g/f))769.67 ±21.54737.73 ±30.34677.33 * ±22.56582.73 * ±15.50652.80 ±84.30657.53 ±103.95
Grip strength hind limb (Mean ± SD (g/f))462.73 ±17.39458.40 ±11.39421.93 * ±10.79408.07 * ±5.56470.07 ±58.58458.13 ±48.69
Locomotor activity (Mean ± SD)
X - Movements541.40 ±51.59529.80 ±58.32510.60 ±42.58527.60 ±37.96686.60 ±74.52687.40   ±14.24
Y - Movements520.00 ±39.14519.00 ±28.11483.20 ±49.48409.60 *±11.84680.60 ±13.24707.40 ±44.09
Z - Movements393.80 ±54.12420.80 ±37.51334.60 ±44.38289.80 * ±40.60740.20 ±45.39750.40 ±104.65
Total Movements (X+Y+Z)1455.20 ±48.241469.60 ±48.461328.40 * ±67.931227.00* ±60.762107.40 ±67.932145.20 ±126.23

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


 


TABLE 14 - SUMMARY OF FUNCTIONAL OBSERVATION BATTERY (FEMALES)















































































ParameterGroups (n)
G1G2G3G4G1RG4R
Home cage observations
Posture
Sitting or standing normally332343
Standing on hind limbs223112
Respiration
Normal555455
Clonic involuntary movement
None/normal555455
Tonic involuntary movement
None/normal555455

 


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




























































































ParameterGroups (n)
G1G2G3G4G1RG4R
Vocalization
None/normal555455
Convulsions
Absent – N555455
Hand-held observations
Reactivity
Easy/ normal (animal does not resist)555455
Handling
Squeaks or does not squeak but exhibit mild resistance; easy to handle/ normal 555455
Palpebral closure
Open555455
Lacrimation
No lacrimation/ normal555455

 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


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

























































































ParameterGroups (n)
G1G2G3G4G1RG4R
Salivation
No salivation/ normal555455
Piloerection
None/ normal555455
Perineum Wetness
N: Absence of wetness555455
Body Temperature Range (ºC)35.70-37.1035.80-37.0035.80-38.0035.90-37.0035.80-36.7035.00-36.70
Open Field Activity
Clonic involuntary movement
None/ Normal555455
Tonic involuntary movement
None/ Normal555455

 


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









































































































































ParameterGroups (n)
G1G2G3G4G1RG4R
Gait
N:Head is horizontal, abdomen rises slightly above floor, and body moves up and down slightly during walking555455
Movements
Normal555455
Arousal
Normal (keeps guard up and engages in exploratory activity)555455
Tremors
None555455
Occurrence of stereotype000000
Abnormal behaviour000000
Number of Defecations (Mean)1.42.21.82.31.21.0
Number of Urinations (Mean)1.81.62.21.82.01.8
Number of Rearings (Mean)7.27.46.87.09.27.6
Sensory Reactivity Measurements
Approach Response
Slowly approaches, sniffs and pulls back/ normal555455
Touch Response
Slowly approaches, sniffs and pulls back/ normal555455

 


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

















































































































ParameterGroups (n)
G1G2G3G4G1RG4R
Eyelid reflex
Blink/ Normal555455
Pinna reflex
Auricle twitches/ normal555455
Sound response
Mild reaction, hears sound/normal555455
Tail pinch response
Looks back, moves forward and lightly squeaks/normal555455
Pupillary reflex
Contracts555455
Aerial righting reflex
Lands on four limbs/ normal555455
Tail limb reflex
Present (Y)555455
Abdominal tone
Normal (proper hardness)555455

 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


 


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































































































ParameterGroups (n)
G1G2G3G4G1RG4R
Limb tone
Normal (proper hardness)555555
Landing Foot Splay (Mean ± SD (mm))63.87 ±13.6366.27 ±7.3871.40 ±7.8362.25 ±10.3967.6716.9150.00*↓ ±3.57
Grip strength fore limb (Mean ± SD (g/f))675.00  ±16.37684.53  ±63.09651.20 ±12.30635.17 ±79.98504.20 ±46.28610.40 ±104.35
Grip strength hind limb (Mean ± SD (g/f))410.67 ±2.12427.27 ±24.23394.93 ±12.79396.50 ±49.29582.20 ±121.91536.60 ±46.64
Locomotor activity (Mean ± SD)
X - Movements518.40 ±43.48522.80 ±49.34505.40 ±21.37467.00 ±42.17616.80 ±61.90650.60 ±44.09
Y - Movements486.60 ±18.43478.40 ±41.58457.80 ±26.66510.25 ±45.04659.00 ±41.25677.80 ±63.50
Z - Movements488.40 ±20.17450.00 ±13.56445.80 * ±25.90444.00 ±37.35705.00 ±91.36765.20 ±87.85
Total Movements (X+Y+Z)1493.40 ±43.431451.20 ±37.381409.00 ±45.461421.25 ±123.351980.80 ±170.342093.60 ±172.15

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


 


TABLE 15 – SUMMARY OF TERMINAL FASTING BODY WEIGHT AND ABSOLUTE ORGAN WEIGHTS (g) – MALES






































































































































































GroupsDose(mg/kg/day)Fasting Body Weight And Absolute Organ Weights (g)
 FBWLiver KidneysSpleenAdrenalsHeartThymusBrain
(g)(g)(g)(g)(g)(g)(g)(g)
G1 0Mean409.3713.093.041.240.101.330.372.07
SD29.790.420.190.230.000.100.060.20
N55555555
G2100Mean411.9212.962.841.280.101.370.352.03
SD18.200.270.290.250.010.130.070.07
N55555555
G3300Mean45.3013.192.861.310.091.280.311.98
SD18.440.060.210.270.010.070.090.16
N55555555
G4700Mean390.6514.25*3.501.250.08*1.360.402.03
SD37.081.230.180.130.010.240.070.13
N55555555

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)
 FBWTestesEpididymisProstrate + seminal vesicles with coagulation glandsThyroid with parathyroid
 (g)(g)(g)(g)(g)
G10Mean421.444.061.613.530.43
SD32.430.190.140.210.07
N1010101010
G2100Mean420.523.891.643.550.48
SD25.530.390.160.260.06
N1010101010
G3300Mean407.114.021.593.440.47
SD35.240.310.230.380.04
N1010101010
G4700Mean409.803.871.553.460.49*
SD39.210.380.140.250.05
N1010101010

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


 


TABLE 16 – SUMMARY OF TERMINAL FASTING BODY WEIGHT AND ABSOLUTE ORGAN WEIGHTS (g) – FEMALES






































































































































































GroupsDose(mg/kg/day)Fasting Body Weight And Absolute Organ Weights (g)
 FBWLiver KidneysSpleenAdrenalsHeartThymusBrain
(g)(g)(g)(g)(g)(g)(g)(g)
G1 0Mean323.9613.421.650.880.091.090.271.82
SD7.440.430.320.200.010.080.050.05
N55555555
G2100Mean321.3112.981.951.040.081.120.251.91
SD31.370.670.200.180.010.070.030.11
N55555555
G3300Mean321.9113.532.120.860.251.130.341.92
SD8.761.040.300.450.380.100.020.10
N55555555
G4700Mean305.4813.172.011.080.091.070.271.85
SD33.920.470.180.110.010.040.110.10
N44444444

Key: Values are in Mean ± SD















































































































GroupsDose(mg/kg/day)Fasting Body Weight And Absolute Organ Weights (g)
 FBWOvariesUterusThyroid with parathyroid
 (g)(g)(g)(g)
G1 0Mean323.950.230.750.47
SD19.430.020.070.06
N10101010
G2100Mean299.460.240.690.47
SD38.360.020.050.04
N10101010
G3300Mean312.090.220.720.47
SD17.940.020.050.06
N10101010
G4700Mean301.140.240.740.50
SD24.910.030.080.03
N8888

Key: Values are in Mean ± SD


 


TABLE 17 – 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
 FBWLiver KidneysSpleenAdrenalsHeartThymusBrainTestesEpididymesProstate+seminal vesicles with coagularting glandsThyroid with parathyroid
 (g)(g)(g)(g)(g)(g)(g)(g)(g)(g)(g)(g)
G1R0Mean421.8813.022.891.080.101.520.362.033.331.463.260.47
SD17.360.850.210.240.010.150.080.050.400.270.620.06
N555555555555
G4R700Mean383.6212.982.471.080.091.320.342.033.751.522.930.49
SD22.851.210.160.070.010.120.070.150.410.120.460.06
N555555555555

Key: Values are in Mean ± SD


 


TABLE 18 – 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
 FBWLiver KidneysSpleenAdrenalsHeartThymusBrainOvariesUterusThyroid with parathyroid
 (g)(g)(g)(g)(g)(g)(g)(g)(g)(g)(g)
G1R0Mean268.328.621.980.740.091.090.421.930.200.690.48
SD21.290.720.230.080.010.040.060.110.050.130.02
N55555555555
G4R700Mean267.378.971.93*0.710.101.170.421.980.210.720.50
SD11.960.940.160.100.010.270.120.170.020.070.05
N55555555555
               

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


TABLE 19 – SUMMARY OF RELATIVE ORGAN WEIGHTS – MALES







































































































































































GroupsDose(mg/kg/day)Relative Organ Weights (%)
 LiverKidneysSpleenAdrenalsHeartThymusBrain 
 (%)(%)(%)(%)(%)(%)(%) 
G1 0Mean3.210.750.310.020.330.090.51 
SD0.220.060.070.000.020.010.04 
N5555555 
G2100Mean3.150.690.310.020.330.080.49 
SD0.170.050.050.000.030.010.01 
N5555555 
G3300Mean3.260.710.320.020.320.080.49 
SD0.150.050.060.000.010.020.03 
N5555555 
G4700Mean3.65*0.780.320.020.350.100.52 
SD0.180.040.040.000.080.010.04 
N5555555 

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


                                                                                          















































































































GroupsDose(mg/kg/day)Relative Organ Weights (%) Males
 Testes EpididymisProstrate + seminal vesicles with coagulation glandsThyroid with parathyroid
 (%)(%)(%)(%)
G1 0Mean0.970.380.840.10
SD0.070.040.070.02
N10101010
G2100Mean0.930.390.850.11
SD0.080.020.070.02
N10101010
G3300Mean0.990.390.840.12
SD0.120.040.060.02
N10101010
G4700Mean0.950.380.850.12
SD0.110.040.080.01
N10101010

Key: Values are in Mean ± SD


 


TABLE 20 – SUMMARY OF RELATIVE ORGAN WEIGHTS – FEMALES







































































































































































GroupsDose(mg/kg/day)Relative Organ Weights (%)
 Liver KidneysSpleenAdrenalsHeartThymusBrain 
 (%)(%)(%)(%)(%)(%)(%) 
G1 0Mean4.140.510.270.030.340.080.56 
SD0.140.100.060.000.020.010.02 
N5555555 
G2100Mean4.080.610.330.030.350.080.60 
SD0.520.100.060.000.040.020.05 
N5555555 
G3300Mean4.200.630.310.030.340.110.58 
SD0.300.040.060.000.020.010.03 
N5555555 
G4700Mean4.350.66*0.350.030.350.090.62 
SD0.500.070.040.000.030.050.11 
N5555555 

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
































































































GroupsDose(mg/kg/day)Relative Organ Weights (%) Females
 OvariesUterusThyroid with parathyroid
(%)(%)(%)
G1 0Mean0.070.230.15
SD0.010.040.01
N101010
G2100Mean0.080.230.16
SD0.010.020.03
N101010
G3300Mean0.070.230.15
SD0.010.020.03
N101010
G4700Mean0.080.250.17
SD0.010.020.02
N888

Key: Values are in Mean ± SD


 


TABLE 21 - SUMMARY OF RELATIVE ORGAN WEIGHTS – RECOVERY MALES



































































































































GroupsDose(mg/kg/day)Relative Organ Weights (%) – Recovery Males
 FBWLiver KidneysSpleenAdrenalsHeartThymusBrainTestes EpididymisProstrate + seminal vesicles with coagulation glandsThyroid with parathyroid
 (%)(%)(%)(%)(%)(%)(%)(%)(%)(%)(%)(%)
G1R0Mean421.883.090.680.260.020.360.090.480.790.340.770.11
SD17.360.270.050.070.000.040.020.020.070.060.120.01
N555555555555
G4R700Mean383.623.400.650.280.020.340.090.530.98*0.400.770.13
SD22.850.460.060.010.000.030.010.060.130.040.160.02
N555555555555

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


 


TABLE 22 - SUMMARY OF RELATIVE ORGAN WEIGHTS –RECOVERY FEMALES














































































































































GroupsDose(mg/kg/day)  Relative Organ Weights (%) – Recovery Females
 FBWLiver KidneysSpleenAdrenalsHeartThymusBrainOvariesUterusThyroid with parathyroid
 (%)(%)(%)(%)(%)(%)(%)(%)(%)(%)(%)
G1R0Mean268.323.220.740.280.030.410.160.720.080.250.18
SD21.290.210.100.050.000.030.030.090.020.030.01
N55555555555
G4R700Mean267.373.350.720.260.040.440.160.740.080.270.19
SD11.960.210.050.030.000.110.040.050.010.030.01
N55555555555
               

Key: Values are in Mean ± SD


 


TABLE 23 - SUMMARY OF EXTERNAL AND GROSS PATHOLOGICAL OBSERVATIONS – MALES














































GroupG1G2G3G4
No. of male Animals Observed10101010
 External Observations^
NAD10/1010/1010/1010/10
 Internal Observations^
NAD9/1010/1010/1010/10
Reddish discoloration of Liver1/100/100/100/10

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


 


TABLE 24 - SUMMARY OF EXTERNAL AND GROSS PATHOLOGICAL OBSERVATIONS – (FEMALES AND PROGENY)




















































































GroupG1G2G3G4
No. of Females/Dams10101010
 External Observations^
NAD10/1010/1010/109/10
Dark secretion at genetalia0/100/100/101/10
 Internal Observations^
NAD7/109/1010/108/10
Reddish discoloration of Liver3/100/100/100/10
Cysts on both the ovaries0/101/100/101/10
Autolysis0/100/100/101/10
No. of progeny observed78688256
External Observations (Progeny )^
NAD78/7868/6882/8256/56

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


 


TABLE 25 - SUMMARY OF EXTERNAL AND GROSS PATHOLOGICAL OBSERVATIONS – MALES RECOVERY































GroupG1RG4R
No. of male Animals Observed55
 External Observations^
NAD5/55/5
 Internal Observations^
NAD5/55/5

 


 


 


 


 


 


TABLE 26 - SUMMARY OF EXTERNAL AND GROSS PATHOLOGICAL OBSERVATIONS – FEMALES RECOVERY































GroupG1RG4R
No. of Female Animals Observed55
 External Observations^
NAD5/55/5
 Internal Observations^
NAD5/55/5

 


 


 


 


 


 



TABLE 27: 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 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: 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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
K1
System:
other: Behaviour/function

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

In the OECD 422 study performed in Wistar rats, NOAEL in male rats was found at 100 mg/kg bw/day based on dose-dependent decreases in locomotor activity at 300 and 700 mg/kg bw/day whereas NOAEL in female rats was found at 300 mg/kg bw/day based on dullness in all animals treated at 700 md/kg bw/day. All of these effects, including dullness observed in all males treated at 700 mg/kg bw/day, showed complete reversibility during the recovery period. Since the observed effects on locomotor activity in male rats treated at 300 mg/kg bw/day were not associated with any appearant clinical signs of toxicity or associated gross/histopathological findings, and showed reversibility after cessation of treatment, they were not taken as significant or severe toxicological effects that would justify a classification for STOT RE as per CLP regulation (Section 3.9.2.7.3 in EC 1272/2008). The dullness observed in all male and female rats treated at 700 mg/kg bw/day showed reversibility after cessation of treatment and this clinical finding was observed at a dose level well above the CLP guidance threshold value of <= 300 mg/kg bw/day that would justify a STOT RE 2 classification on the basis of a 28-days repeated dose toxicity study (Refer to "Guidance on the Application of the CLP Criteria, ECHA, 2017, p. 464). Taken together, the substance (CAS 9002-92-0) is regarded to be classified as Not Classified for STOT RE.