Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May - July 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: ordered from Charles River Italia S.p.A., Calco (Lecco), Italy and supplied by Charles River Laboratories, Domaine des Oncins, 69210 Saint Germain Nuelles, France.
-Age/Weight: The animals were approximately 7 to 8 weeks old and weighing 200-225 g for males and 175-200 g for females. After arrival, the weight range for each sex was determined and found to be 216 - 233 g for males and 199 - 209 g for females.
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Housing: in polysulfone solid bottomed cages
- Diet (e.g. ad libitum): 4 RF 21,Mucedola S.r.l., Via G. Galilei, 4,20019 SettimoMilanese (MI), Italy
- Water (e.g. ad libitum): drinking water
- Acclimation period: An acclimatisation period of 33 days was allowed before the start of treatment
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 55 ± 15%
- Air changes (per hr): approximately 15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: 14 May 2019 to 24 July 2019

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 2% aqueous solution of carboxymethylcellulose.
Details on oral exposure:
The required amount of test item was suspended in the vehicle. The preparationswere made daily/weekly (concentrations of 10, 30 and 100 mg/mL) based on stability data obtained.
Concentrations were calculated and expressed in terms of test item as supplied.
The test item was administered orally by gavage at a dose volume of 10 mL/kg body weight.
Control animals received the vehicle alone at the same dose volume.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis was performed in a separate study in order to validate both the analytical method and the preparation procedure and to verify the stability of the preparations (ERBC StudyNo. A3524). The analytical method was validated in the range from 10 to 100 mg/mL. Linearity, accuracy and precision were within the limits stated in ERBC SOPs for suspensions (r >0.98; accuracy 85-115%; precision CV <10%). In the same study, a 28 hour stability at room temperature and a 32 day stability at 2-8°C were verified in the range from 10 to 100 mg/mL.
The proposed preparation procedure for the test item was checked in the range from 10 to 100 mg/mL by chemical analysis (concentration and homogeneity) to confirmthat the method was suitable. All results for all levels were within the acceptability limits stated in ERBC SOPs for suspensions (85-115%) and homogeneity (CV <10%). In the present study, samples of the preparations made on two occasions during the study (Day 1 and again towards the end of the study) were analysed to check the homogeneity and concentration. Chemical analysis was carried out by the Analytical Chemistry Department using a gas chromatography (GC) with flame-ionisation detection (FID). The software used for this activity was Chromeleon 7, version 7.2.2.6394 (Thermo Scientific). Results of the analyses were within the acceptability limits stated in ERBC SOPs for suspensions (85-115% for concentration and CV <10% for homogeneity).
Duration of treatment / exposure:
Males: 30/31 days in total
Females: 7 to 8 weeks of treatment for at least 51 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
actual ingested
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
actual ingested
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
actual ingested
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
The dose-levels were selected in agreement with the Sponsor, and are based on the results of a previous study. In this study, five male and five female Sprague Dawley rats were given the test item at 100, 300 or 1000 mg/kg/day for 2 weeks.
One high dose female animal was sacrificed for humane reasons on Day 14. On the day of sacrifice, the animal appeared moribund. No clinical signs were noted in this animal in the days prior to sacrifice. At necropsy examination, the finding observed was limited to a slightly distended stomach. Due to the absence of signs of toxicity and the single mortality occurred, it was not possibile to correlate the mortality to the toxicity of the test item. No relevant clinical signs were observed during the treatment.
No effects on body weight were observed during the study. No effects on food consumption were observed in males and females receiving dose levels= 300 mg/kg, during the study. Food consumption in females of the high dose group appeared reduced during the second week of treatment. At post mortem examination performed at the end of the study, no treatment-related changes were noted in the animals.
Based on the results obtained in this study, the same dose-levels were used in the present study.
- Rationale for animal assignment: computerized stratification procedure
Positive control:
no (not required)

Examinations

Observations and examinations performed and frequency:
MORTALITY/MORBIDITY:
- Time schedule: at least twice a day during the treatment period.
CLINICAL OBSERVATIONS:
- Time schedule: once a day during the treatment period.
DETAILED CLINICAL SIGNS
- Time schedule: once before the beginning of the treatment period and then at least once a week until the end of the study.
BODY WEIGHT:
- Time schedule: Males will be weighed weekly from allocation to termination. Females will be weighed weekly from allocation to positive identification of mating and on Days 0, 7, 14 and 20 post coitum. Dams will also be weighed on Days 1, 4, 7, 13 post partum and just before to necropsy.
FOOD CONSUMPTION:
- Time schedule: The weight of food consumed by each cage of males and females will be recorded weekly (whenever possible) during the pre-mating period starting from Day 1 of dosing up to mating. Individual food consumption for mated females will be measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Day 7 and 13 post partum starting from Day 1 post partum.
BIOCHEMICAL INVESTIGATIONS:
- Time schedule: on the day of scheduled sacrifice.
Blood samples for haematology, clinical chemistry and coagulation were collected, at the end of treatment period, by random selection from 5 males and 5 females (females with viable litters) of each group, under condition of food deprivation.
Males
Blood samples for haematological investigations, biochemical tests and hormone determination were collected under isoflurane anaesthesia from the retro-orbital sinus. Blood samples for coagulation test (food available) were collected at necropsy from the vena cava under isoflurane anaesthesia. The order of collection was equalised between groups.
Females
As a part of the sacrificial procedure, blood samples for all determinations were withdrawn from the abdominal vena cava under isoflurane anaesthesia. The order of collection was equalised between groups.
The blood samples were divided into tubes as follows:
– EDTA anticoagulant for haematological investigations
– Heparin anticoagulant for biochemical tests
– Citrate anticoagulant for coagulation tests
– No anticoagulant for hormone determination
The measurements performed on blood samples are:
Hematology
– Haematocrit
– Haemoglobin
– Red blood cell count
– Reticulocyte count
– Mean red blood cell volume
– Mean corpuscular haemoglobin
– Mean corpuscular haemoglobin concentration
– White blood cell count
– Differential leucocyte count
· Neutrophils
· Lymphocites
· Eosinophils
· Basophils
· Monocytes
· Large unstained cells
– Platelets
These parameters were analysed by Siemens Advia 120.
No haematological data were available for two male animals, nos. X1140026 (Group 2) and X1140078 (Group 4), since the samples were not analysable (clotted samples).
Coagulation
- Prothrombin time
This parameter was analysed by Instrumentation Laboratory ACL Elite PRO.
Clinical chemistry
– Alkaline phosphatase
– Alanine aminotransferase
– Aspartate aminotransferase
– Gamma-glutamyltransferase
– Urea
– Creatinine
– Glucose
– Triglycerides
– Bile acids
– Total bilirubin
– Total cholesterol
– Total protein
– Albumin
– Globulin
– A/G Ratio
– Sodium
– Potassium
– Calcium
– Chloride
– Inorganic phosphorus
These parameters were analysed by Siemens Advia 1200.
URYNALYSIS
-Time schedule : during the last week of treatment for males only.
Individual overnight urine samples were collected during the last week of treatment from the same male animals selected for the clinical pathology investigations and under the same conditions. Before starting urine collection, water bottles were removed from each cage and each animal received approximately 10 mL/kg of drinking water by gavage, in order to obtain urine samples suitable for analysis.
– Volume (manually recorded)
– Appearance
– Specific gravity
– pH
– Protein
– Glucose
– Ketones
– Bilirubin
– Urobilinogen
– Blood
These parameters were analysed by Menarini Aution Max AX 4280/Aution Eleven AE 4020/Aution Sticks 10EA or ATAGORefractometer (for Specific gravity), according to internal procedures.
The sediment, obtained from centrifugation at approximately 3000 rpm for 10 minutes, was examined microscopically for:
– Epithelial cells
– Leucocytes
– Erythrocytes
– Crystals
– Spermatozoa and precursors
– Other abnormal components
Sacrifice and pathology:
EUTHANASIA:
Parental animals in extremis or killed for humane reasons will be killed under carbon dioxide asphyxiation. Parental animals that have completed the scheduled test period, will be killed by exsanguination under isoflurane anaesthesia.
Parental males
The males will be killed after the mating of all females or after at least 28 days of treatment period.
Parental females
The females with live pups will be killed on Day 14 post partum. The females with total litter loss will be killed on the day of the occurrence of total litter loss
or shortly after.

ORGAN WEIGHTS
Parental animals
From all animals completing the scheduled test period, the organs indicated in Annex 1 will be dissected free of fat and weighed. The ratios of organ weight to body weight will be calculated for each animal. At the discretion of the pathologist, organs may be weighed from animals dying or killed
prior to terminal kill.

PRESERVATION AND FIXATION
Samples of all the tissues listed in Annex 1 will be fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves and Harderian glands, testes and epididymides which will be fixed in modified Davidson's fluid and preserved in 70% ethyl alcohol).

HISTOPATHOLOGY
The tissues required for histopathological examination are listed in Annex 1. In the first instance, the examination will be restricted as detailed below:
a) Tissues specified in Annex 1 from 5 males and 5 females randomly selected (animals evaluated for clinical pathology) in the control and high dose group killed at term;
b) Tissues specified in Annex 1 from all animals killed or dying during the treatment period.
c) All abnormalities in all groups.
Since differences were observed in stomach and liver between control and high dose animals, the examination was extended to:
– Stomach and liver in all males and females of Groups 2 and 3 and in the remaining animals of control and high dose groups.
In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.

Statistics:
Standard deviations were calculated as appropriate. For variables, e.g. body weight, food consumption, clinical pathology parameters and organ weights, the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. Statistical analysis of histopathological findings was carried out by means orthe nonparametric Kolmogorov-Smirnov test. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the nonparametric version of theWilliams test. The criterion for statistical significance was p<0.05. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
See Table 2.
Males
In all males receiving 1000 mg/kg/day (Group 4), piloerection, ataxia and salivation were generally observed during the treatment period. In addition, decreased activity was observed in 7 out of 10 males in few days during the treatment period. Hunched posture (kyphosis) was also observed in a limited number of males (3 out of 10) in sporadic cases during the study. Damaged eye, noted in one male (no. X1140072), was seen in the last days of treatment and was due to the bleeding procedure.
In males receiving 300 mg/kg/day (Group 3), salivation was observed in a single occasion (Day 4 of the pre-mating phase) in 6 out of 10 animals. Hairloss on the muzzle/head observed in one male (no. X1140042) in the last days of treatment was not considered related to the treatment.
No signs of toxicity were observed in males receiving 100 mg/kg/day (Group 2) and in control animals. Scabs and/or injury observed in single animals of low dose group and control were not considered related to the treatment.
Females
In females receiving 1000 mg/kg/day (Group 4), salivation, piloerection, ataxia, decreased activity, hunched posture (kyphosis), semiclosed and/or closed eyes, prone posture and/or lethargic appearance were generally observed during the premating period. Piloerection, salivation, ataxia, decreased activity and hunched posture (kyphosis) were observed during post coitum period. In addition, one female (no. X1140065) showed prone posture in a single occasion (Day 2 of the post coitum period). During post partum period, salivation was noted in all females.
In females receiving 300 mg/kg/day (Group 3), salivation was observed in a single occasion (Day 4 of the pre-mating phase) in 3 out of 10 animals. No clinical signs were observed in the females of the mid-dose group during the post coitum and post partum periods.
No signs of toxicity were observed in females receiving 100 mg/kg/day (Group 2) and in control animals. Hairloss observed during post coitum and/or post partum periods in few animals of low dose group and control were not considered related to the treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
See Table 1.
One female (no. X1140079) receiving 1000 mg/kg/day was sacrificed for humane reasons on Day 2 of the study (premating phase). No clinical signs were observed in the animal after the first administration. The animal appeared moribund and showed salivation prior to sacrifice.
Macroscopically, the findings observed in this animal were firmcontents of caecum and distended urinary bladder with yellow/orange fluid. Microscopically, no relevant changes were observed. The pathological picture of this animal did not allow to establish the cause of the ill status.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See Table 4.
No differences of toxicological significance were noted throughout the study in the body weight and body weight gain of treated males, when compared to the control group.
In female animals, no differences of toxicological significance were noted before pairing, while during the post coitum and post partum periods, in the high dose group only, a slight decrease, significant at statistical analysis, in body weight (ranging from -6% to -8%) was noted, when compared to the control group.
Body weight gain in female animals at the end of the premating period showed an increase, statistically significant in high dose group only. During the post coitum and post partum periods, body weight gain in female animals showed no differences of toxicological significance.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
See Table 6.
Food consumption in treated males was comparable to the control group during the study.
Food consumption in high dose females only was slightly reduced (-17%), during the first week of treatment (premating period) when compared to the control group. During the post coitum and post partum periods, high dose females continued to show reduced food consumption (ranging from -7% to -13%) statistically significant only at the start of the gestation phase and at the end of the treatment period (Day 13 post partum).
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):
See Tables 9 & 10.
An increase of neutrophils was recorded in males dosed at 300 and 1000 mg/kg/day. Compared with controls, changes were 81% and 87%, respectively. Due to the limited severity and the absence of other related changes, this finding was considered to be not adverse.
The other statistically significant differences between control and treated animals (mean corpuscular volume in males, lymphocytes in both sexes) were not dose-related, therefore they were considered to be unrelated to treatment.
Coagulation
No changes were recorded in prothrombin time measured at the end of the study.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
See Table 11.
Compared with controls, fluctuations of some biochemical parameters were recordedin treated animals, such as: increase of albumin and bile acids in males dosed at 1000 mg/kg/day (7% and 178%, respectively), increase of calcium in those receiving 300 and 1000 mg/kg/day (4% and 7%, respectively), increase of sodium in males from all treated groups (1% in all groups) and increase of bilirubin in females dosed at 300 mg/kg/day (8.6 fold).
Change of bilirubin in females was not dose-related, therefore it was considered to be unrelated to treatment. In addition, the increase of bile acids was due to one animal only (no. X1140062), therefore it was considered to be incidental. The other findings recorded were of minimal severity, therefore they were considered to be not adverse.
Urinalysis findings:
no effects observed
Description (incidence and severity):
See Table 12.
No relevant changes were observed in urine analysis performed in males at the end of the treatment period.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
See Table 3, 7 & 8.
Motor activity, grip strength and sensory reactivity to stimuli performed at the end of the treatment period did not show relevant differences between control and treated groups in both sexes with the exception of reduced landing footsplay (first, second trial and mean) recorded in the high dose females. However, the reduced landing footsplay (-38% calculated for the mean value) was considered related to the lower bodyweight of the high dose females at the time of test when compared with control.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See Tables 23, 24 & 25.
No changes were observed on terminal body weight of treated animals, when compared to
the controls.
A statistically significant increase was observed in mean liver weight of animals of both sexes (absolute and/or relative weights), when compared to controls. Such organ weight variation in high dose animals of both sexes could be considered treatment-related. The remaining statistically significant change in absolute and relative mean spleenweight noted in high dose females sacrificed at termwas considered of doubtful interpretation, as not corroborated by histopathological evaluation.
No other organ weight variations were considered toxicologically relevant in treated groups of both sexes, when compared to the controls.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
See Table 26.
The most relevant change observed at necropsy was thickening of the non glandular region of the stomach observed in most high dose rats of both sexes sacrificed at the end of treatment period, whereas in a single instance, it was observed in one mid-dose animal of both sexes (nos. X1140060 and X1140055) and in one control male (no. X1140006).
All other observed changes had comparable incidence in the control and treated groups and/or are known to occur spontaneously in untreated Sprague Dawley rats of the same age, under our experimental conditions.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See Table 27.
Treatment-related changes, associated with the oral administration of 2-OCTANOL, were present in the non glandular region of the stomach of mid- and high dose animals of both sexes and in the liver of high dose males.
- Forestomach: treatment-related changes were seen in the non-glandular region of the stomach (forestomach) and consisted of squamous epithelial hyperplasia (1) associated with hyperkeratosis (orthokeratotic), chronic inflammation and/or oedema in the submucosa, in some instances associated with mucosal ulceration/erosion from minimal to marked degree in all high dose treated animals and of minimal or moderate degree in few instances of the mid-dose animals of both sexes.
- Liver: minimal centrilobular hepatocellular hypertrophy was seen in half of the high dose treated males. Hepatocyte hypertrophy could be associated with microsomal enzyme induction secondary to the exposure to the test item and was distributed mainly in centrilobular areas; hepatocellular cytoplasm showed a pale, ground glass appearance and could be considered an adaptative change which correlated with an increased liver mean weight (2).
The remaining sporadic lesions were considered to be an expression of spontaneous and/or incidental pathology seen in this species.
1) Toxicologic Pathology, 39: 337-347, 2011 - SIEGRID DAMSCH
2) Toxicologic Pathology, 38: 5S-81S, 2010 BOB THOLEN
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Effect levels

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic effects
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
Dose descriptor:
NOEL
Remarks:
Local effects
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The test item, was administered daily by oral gavage to male and female Sprague-Dawley rats, for 2 weeks before mating, during mating, and until sacrifice for males, or through gestation and until Day 14 p.p. for females, at dose-levels of 100, 300 and 1000 mg/kg/day. Based on the results, the NOAEL (No Observed Adverse Effect Level) was considered to be 300 mg/kg/day for systemic toxicity due to clinical signs observed at the high dose-level and 100 mg/kg/day for local toxicity due to microscopic findings noted in the forestomach of animals of the mid- and high-dose groups.
Executive summary:

The test item,2-OCTANOL, was administered daily by to male and female Sprague-Dawley rats, for 2 weeks before mating, during mating, and until sacrifice for males, or through gestation and until Day 14 p.p. for females, at dose-levels of 100, 300 and 1000 mg/kg/day.

All doses (100, 300 and 1000 mg/kg/day) were administered orally, by gavage. The control group received 2% aqueous solution of carboxymethylcellulose. The dose volume was 10mL/kg body weight. Males were treated daily for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 30/31 days. Females were treated for 2 weeks prior to pairing, during pairing,post coitum and post partum periods until Day 13 post partum(for at least 51 days). Four non pregnant females were dosed up to the day before necropsy.

Mortality observed in one female of the high dose group could not to be clearly related to treatment due to the absence of clear pathological findings.

A number of clinical signs indicating toxicity (i.e. salivation, ataxia, hunched and/or prone posture, lethargy) were observed in both males and females treated at 1000 mg/kg/day. Salivation was generally observed in the animals treated at 300 mg/kg/day.

Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item.

No differences of toxicological significance were noted throughout the study in the body weight and body weight gain of treated males. In females of the high dose group, a slight decrease in body weight was noted during the post coitum and post partum periods. An increase in body weight gain in females of the high dose group was recorded at the end of the premating period.

No effects on food consumption were observed in males. In females of the high dose group, food consumption was slightly reduced during the first week of treatment and during the post coitum and post partum periods.

Motor activity, grip strength and sensory reactivity to stimuli performed at the end of the treatment period did not reveal changes attributable to the test item.

Change in neutrophilis (increase) recorded in males treated at dose levels 300 mg/kg/day was considered to be not adverse. No changes were recorded in prothrombin time measured at the end of the study.

Fluctuations of some biochemical parameters (albumin, bile acids, calcium, sodium and bilirubin) in males and/or females were considered to be unrelated to treatment, incidental or considered to be not adverse. No relevant changes were observed in urine analysis performed in males.

No changes were observed on terminal body weight of study animals of both sexes, when compared to the control group. A statistically significant increase was observed in mean liver weight of high dose animals of both sexes.

At macroscopic observation performed at post mortem, the most relevant change was thickening of the non glandular region of the stomach observed in most high dose rats of both sexes sacrificed at the end of treatment period, whereas in a single instance, it was observed in one mid-dose animal of both sexes and in one control male.

Treatment-related changes were present in the non glandular region of the stomach (forestomach) of mid- and high dose animals of both sexes and consisted of squamous epithelial hyperplasia associated with hyperkeratosis (orthokeratotic), chronic inflammation and/or oedema in the submucosa, in some instances associated with mucosal ulceration/erosion.

Treatment-related changes were also noted in the liver of high dose males and consisted of minimal centrilobular hepatocellular hypertrophy indicating a possible microsomal enzyme induction as an adaptive response to the exposure to the test item."

Based on the results, the NOAEL (No Observed Adverse Effect Level) was considered 300 mg/kg /day for systemic toxicity due to clinical signs observed at the high dose-level and 100 mg/kg/day for local toxicity due to microscopic findings noted in the forestomach of animals of the mid- and high-dose groups.