1-Octanamine, N,N-dimethyl-, N-oxide

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18th October 2007 and 4th March 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N,N-dimethyl-N-octylhydroxylamine

EC Number:

607-854-9

Cas Number:

2605-78-9

Molecular formula:

CH3(CH2)7N(CH3)2O

IUPAC Name:

N,N-dimethyl-N-octylhydroxylamine

impurity 1

Reference substance name:

Dimethyl(octyl)amine

EC Number:

230-939-3

EC Name:

Dimethyl(octyl)amine

Cas Number:

7378-99-6

Molecular formula:

C10H23N

IUPAC Name:

N,N-dimethyloctan-1-amine

impurity 2

Reference substance name:

Methanol

EC Number:

200-659-6

EC Name:

Methanol

Cas Number:

67-56-1

Molecular formula:

CH4O

IUPAC Name:

Methanol

impurity 3

Test material form:

liquid: viscous

Specific details on test material used for the study:

- Analytical purity: 82.3%
- Purity test date: Not available
- Lot/batch No.: GN-8
- Expiration date of the lot/batch: Not available
- Appearance: Amber coloured, extremely viscous liquid
- Storage: In the dark at room temperature
- Additional information on test material used for the study: It was attempted to purify the substance to a purity of >80% by using an alternative synthetic route, as opposed to the usual commercial route of synthesis which results in a purity of the substance of approximately 40% in water. This alternative synthetic route generated the substance at 82.3% purity, however it also generated an impurity (methanol at 4.0%) which is not present in the commercial substance. Based on the classification and labelling of methanol, it is considered that this additional impurity would not have an influence on any of the endpoints discussed in this dossier other than the acute oral toxicity study. Therefore all studies (including the one covering this endpoint) contained in this dossier (other than the acute oral toxicity study) were conducted on the 82.3% pure substance containing the 4.0% methanol impurity. The acute oral toxicity study has been conducted on the commercially generated substance at a purity of approximately 40% in water.

Test animals

Species:

rat

Strain:

other: Sprague-Dawley Crl:CD (SD) IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited Margate Kent
- Age at study initiation: Approximately 5-8 weeks old
- Weight at study initiation: Males weighed between 168-205g and females weighed between 148-185g
- Housing: Housed in groups of 5 by sex in solid bottom cages with stainless steel mesh lids with Environmental enrichment in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd. Cheshire UK.)
- Diet: Ad libitum Pelleted diet of (Rodent 5LF2 (Certified) Diet, BCM IPS Limited London UK)
- Water: Mains drinking water (Water bottles weighed daily to measure intake.)
- Acclimation period:7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±2°C
- Humidity (%): 15%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours dark, 12 hours low intensity flourescent lighting

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS

-Test substance prepared in water.

VEHICLE
- Concentration in vehicle: 150, 30, 3 and 0 mg/ml administered to 4 groups of 6 rats
- Amount of vehicle (if gavage): 5ml/kg/day of solutions administered

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Standard and sample solutions were analysed by gas chromotography using an external standard technique. Test material formulations weere diluted with acetonitrile to give a final, theoretical test material concentration of approximately 0.1mg/ml. For the standards, these were prepared in acetonitrile at a nominal concentration of 0.1mg/ml.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
750mg/kg/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
150mg/kg/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
15mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

5 males, 5 females per dose (4 groups of 6).

Control animals:

yes

Details on study design:

- Dose selection rationale: Range finding study used to determine the maximum tolerated dose up to 1000 mg/kg/day with an 11 day study. The highest tolerated dose was determined to be 750mg/kg/day
-Dose administered according to body weight as 5ml/kg via a stainless steel dosing cannula, attached to a graduated syringe

Range finding study:
A range finder study was used to find the maximum tolerated dose level (up to 1000mg/kg/day). 2 of the 3 males within the range finding test for the concentration 1000mg/kg/day died prematurely on Day 4 out of 8, the remaining male was found to have macroscopic abnormalities after the 8 day exposure with small seminal vesicles. No abnormalities were detected in females of this dose or in 750 or 500mg/kg/day. Reductions in body weight were noted in 1000 mg/kg/day of both sexes and to a lesser extent in both sexes of the 750mg/kg/day, no significant loss was observed in the 500mg/kg/day groups. Clinical observations were noted in all groups, though the symptoms were significantly less severe in the 500g/kg/day testing groups.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to dosing, immediately post dosing and one hour after dosing. After which an additional observation is made daily 5 hours post dosing on weekdays.


BODY WEIGHT: Yes
- Time schedule for examinations: Recorded on test initiation and weekly thereafter, a final measurement is taking prior to sacrifice.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes


WATER CONSUMPTION: Yes
- Time schedule for examinations: Daily visual inspections of water bottles, and a gravimetrical measurement on week 3.


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 28 end of study
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: All test animals and controls
- Parameters checked in table [No.1] were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 28 end of study
- Animals fasted: No
- How many animals: All test animals and controls
- Parameters checked in table [No.1] were examined.


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to treatment on days 4, 11, 18 and 25
- Dose groups that were examined: All dose groups and controls
- Battery of functions tested: sensory activity, grip strength and motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

effects observed, treatment-related

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

effects observed, treatment-related

Ophthalmological findings:

effects observed, treatment-related

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY:
Two unscheduled deaths were recorded, one male, one female. The female died on day 7 in extremis following severe clinical signs. The male died on day 29 prior to terminal kill. Both deaths were animals from the 750mg/kg/day testing group.


BODY WEIGHT AND WEIGHT GAIN:
Serious clinical abnormalities were observed in the female which died prematurely


FOOD CONSUMPTION:
A noticeable reduction in food consumption was noted in the 750mg/kg/day male testing group, however no reduction in food consumption was noted in the 750 mg/kg/day female testing group.

FOOD EFFICIENCY:
Food efficiency reduction was noticeable within the 750mg/kg/day male testing group, however no other reduction was noted in other testing groups.

WATER CONSUMPTION:
An increase in water consumption was noted within both male and female testing groups for 750 mg/kg. No other abnormal water intake was noted in all other testing groups and controls.

HAEMATOLOGY:
Both male and female testing groups for 750mg/kg/day displayed reduced white blood cell counts though only statistically significant in the males. Both males and females in this group showed a decrease in lymphocytes (p≤ 0.01 and p≤0.05 respectively). Females within the 750mg/kg/day showed an increase in neutrophil count. Lower erythrocyte, haemoglobin and haematocrit counts were recorded in both males and females of the 750 mg/kg/day testing group, however this was only considered statistically significant within the female testing group (p≤ 0.01). An increase was recorded for reticulocyte cells within both male and female testing groups for the concentrations 150mg/kg/day and 750mg/kg/day (males p≤0.05 and females p≤0.01). Males treated with 15 and 150mg/kg/day displayed a decrease in leucocyte counts, specifically within lymphocytes (p≤0.01). Platelet counts displayed a reduction in females of the 15mg/kg/day group (p≤0.01) however this reduction was not attributed to the test substance as no other dose related responses were noted.


CLINICAL CHEMISTRY:
Animals of either sex within the 750mg/kg/day testing group displayed an increase in alanine aminotransferase (p≤0.01) was detected along with a slight decrease in total protein, cholesterol and inorganic phosphate. An increase in glucose levels was noted, however this was not statistically significant. Males within this group displayed a statistically significant increase in the levels of urea and albumin/globulin ratio. Females from this group displayed a decrease in chloride and calcium ions. At 150mg/kg/day both males and females were observed to have a slight increase in alanine aminotransferase levels, however this was only statistically significant for males. An increase in aspartate aminotransferase was noted in the males of the 150mg/kg/day group and though statistically significant, this was minimal (p≤0.05) and so not attributed to the test substance due to the absence of a dose related response. A decrease in inorganic phosphate levels was also noted in the males of this group. No abnormalities were observed in the animals of the control or the 15mg/kg/day tests.

NEUROBEHAVIOUR:
During weeks 3 and 4 one male displayed noisy respiration and also one female during week 4 displayed noisy respiration and slight salvitation, both from 750mg/kg/day testing group. An increase in urination was also noted within the 750mg/kg/day groups; in the males during weeks 2, 3 and 4 and also in the females during weeks 3 and 4. No effects were observed withing the other treatment groups when compared to the control animals. Within the 750mg/kg/day tests, the females showed a slight but statistically significant reduction in overall mobile activity (p≤0.05). No treatment related differences in sensory reactivity were observed.


ORGAN WEIGHTS
Females within the highest concentration group had a statistically significant increase in absolute bodyweight-relative liver and kidney weight in comparison to the control animals (p≤0.05). However, females treated with 150 and 15 mg/kg/day displayed a reduction in kidney weights, however this was not attributed to the test substance (due to a control value being higher than normally expected.) Males treated with 750mg/kg/day were noted to have reductions in the weights of the brain and epididymis both absolute and relative to terminal bodyweight (p≤0.05). These were not considered to be due to systemic toxicity due to the lack of histopathological correlates.

GROSS PATHOLOGY
The female treated with 750mg/kg/day killed in extremis displayed pathological abnormalities. Gaseous distension of the gastro-intestinal tract and sloughing of the gastric epithelium of the stomach was observed. The 750mg/kg/day male which died on day 29 had a notably dark liver and thymus, sloughing of the non-glandular region of the stomach and red discolouration fo the right eye. No macroscopic abnormalities were observed for the remaining animals, with the exception of one control female which had a malformed liver, however this was considered to be a congenital defect.

HISTOPATHOLOGY: NON-NEOPLASTIC
Centrilobular hepatocyte enlargement was recorded in the animals of the testing groups 150 and 750mg/kg/day in both sexes and also within two females in the 15mg/kg/day. Increases in extramedullary haemopoiesis and haemosiderin pigment accumulation were seen in males of the 750mg/kg/day testing group which were considered to be higher than normal in laboratory maintained rats. The increrase in 750mg/kg/day female extramedullary haemopoiesis and haemosiderin was not attributed to the treatments. Abnormalities in the kidneys of the females in the 750mg/kg/day group were observed with hypertrophy of the collecting duct epithelium. No significant pathology was observed in the male which died prematurely which could have contributed to the death.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 150 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: overall toxicity

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

See attachment.

Applicant's summary and conclusion

Conclusions:

1-Octanamine, N, N-dimethyl-,N-oxide administered for 28 days via oral gavage to Sprague-Dawley rats with a maximum concentration of 750 mg/kg/day resulted in obvious treatment related changes at all dose levels. However the changes seen at 150 mg/kg/day and 15 mg/kg/day were not considered to represent adverse health effects and so the NOAEL or No Observed Adverse Effect Level was determined at 150 mg/kg/day.

Executive summary:

Introduction:        The study was designed to investigate the systemic toxicity of the test material. It complies with the requirements for notification of a new chemical substance in the EC and follows the testing method described in Commission Directive 96/54/EC (Method B7) and OECD Guidelines for Testing of Chemicals No. 407 "Repeated dose 28 Day Oral Toxicity Study in Rodents" (Adopted 27th July 1995).

Methods:       The test material was administered by gavage to three groups, each of five male and five Sprague-Dawley Crl:CD (SD) IGS BR strain rats, for up to twenty-eight consecutive days, at dose levels of 15, 150 and 750 mg/kg/day (incorporating a correction factor for 82.3% purity). A control group of five males and five females was dosed with vehicle alone (distilled water).

Clinical signs, functional observations, bodyweight development and food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all surviving animals at the end of the study.

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues were performed.

Results:

Mortality: One female treated with 750 mg/kg/day was killed inextremison day 7. One male treated with 750 mg/kg/day was found dead on day 29 prior to terminal kill.

There were no further unscheduled deaths during this study.

Clinical Observations: Animals of either sex treated with 750 mg/kg/day displayed increased salivation soon after dosing from Day 2 until the end of the treatment period. Isolated instances of increased salivation were also evident prior to dosing, and up to one hour after dosing during the treatment period. Noisy respiration was also prevalent at this dose level during the course of treatment, together with instances of laboured and decreased respiration and signs of staining of the external body surface.

The interim death female treated with 750 mg/kg/day showed signs of increased salivation following dosing from the first day of treatment, and noisy respiration was observed from Day 2. Laboured respiration was also observed on Day 6 and by Day 7, this animal displayed noisy, laboured and gasping respiration, together with tip-toe gait, hunched posture and a distended abdomen. This animal was subsequently terminated on Day 7. The interim death male did not show any signs of distress although increased salivation and noisy and laboured respiration were recorded for this animal during the treatment period.

Incidents of increased salivation were observed for animals of either sex treated with 150mg/kg/day, together with an isolated instance of noisy respiration seen for one female.

No clinical observations were detected at 15mg/kg/day.

Bodyweights:       A reduction in bodyweight gains was evident for males treated with 750 mg/kg/day throughout the treatment period. Females treated with 750mg/kg/day also showed a reduction in body weight gain when compared to controls, however, this was confirmed to week 1 only.

No adverse effect on bodyweight gains was detected for animals of either sex treated with 150 or 15 mg/kg/day.

Food consumption:       Reductions in dietary intake and food efficiency were evident for males treated with 750mg/kg/day during the first week of treatment.

No adverse effect on dietary intake was detected for females treated with 750 mg/kg/day or for animals of either sex treated with 150 or 15 mg/kg/day when compared to controls.

Water consumption:       An increase in water consumption was evident for animals of either sex treated with 750 mg/kg/day from Day 7 onwards.

No adverse effects on water intake were detected for animals of either sex treated with 150 or 15 mg/kg/day.

Behavioural Assessments: Weekly open field arena observations revealed instances of noisy respiration and increased salivation for individual animals treated with 750 mg/kg/day animals during Weeks 2 to 4.

No treatment-related effects were detected for animals of either sex treated with 150 or 15 mg/kg/day.

Sensory Reactivity: No treatment-related effects were detected for treated animals when compared to controls.

Functional Performance tests: A slight reduction in overall mobile activity was observed for 750mg/kg/day females when compared to controls.

No such effects were evident for 750 mg/kg/day males or for animals of either sex treated with 150 or 15 mg/kg/day.

Haematology:       Animals of either sex treated with 750 mg/kg/day showed reductions in leucocyte counts, specifically in the lymphocyte fractions. Females also showed an increase in neutrophil counts. Reductions in erythrocytes counts, haemoglobin and haematocrit counts were also observed for animals of either sex at this dose level, together with increases in reticuloyte counts.

Animals of either sex treated with 150 mg/kg/day also displayed increases in reticulocyte counts when compared to controls. Males treated with 150 mg/kg/day showed reductions in leucocyte counts, specifically in the lymphocyte fraction, which extended into the male 15 mg/kg/day dose group.

No treatment-related effects were detected for females treated with 15 mg/kg/day.

Blood Chemistry:       Increase in alanine aminotransferase were detected for animals of either sex treated with 750 mg/kg/day together with reductions in chloride and calcium ions for females. Animals of either sex also showed a slight increase in glucose and reductions in total protein and cholesterol levels when compared to controls. Males treated at this dose level also showed an increase in albumin/globulin ratio and blood urea levels in comparison to controls.

Animals of either sex treated with 150 mg/kg/day showed an increase in alanine aminotransferase.

No treatment-related effects were apparent for animals of either sex treated with 15 mg/kg/day

Necropsy: The decedent female treated with 750 mg/kg/day displayed gaseous distension of the gastro-intestinal tract and sloughing of the gastric epithelium of the stomach. The 750 mg/kg/day decedent male showed a dark liver and thymus, sloughing of the non-glandular region of the stomach and red discolouration of the right eye.

No macroscopic abnormalities were detected for the remaining treated animals at terminal kill.

Organ Weights: Females treated with 750 mg/kg/day showed increases in liver and kidney weights, both absolute and relative to terminal body weights.

Histopathology: The following treatment-related changes were observed:

- liver:       Centrilobular hepatocyte enlargement was seen in relation to treatment for animals of either sex treated with 750 and 150 mg/kg/day and possibly also for females treated with 15 mg/kg/day.

-Spleen:       Higher grade rates of severity of extramedullary haemopoiesis and haemosiderin pigment accumulation were seen in relation to treatment for males treated with 750 mg/kg/day but not at any other dose level.

Kidney:       Hypertrophy of the collecting duct epithelium was seen in relation to treatment among females treated with 750 mg/kg/day but not at any other treatment level and not among males.

Conclusion:      The oral administration of 1 -Octanamine, N, N-dimethyl-, N-oxide to rats by gavage for a period of up to twenty eight consecutive days at a maximum dose level of 750 mg/kg/day (incorporating a correction factor for 82.3% purity) resulted in treatment-related changes at all treatment levels. The effects detected at 150 and 15 mg/kg/day were considered not to represent adverse health effects and as such, a No Observed Adverse Effect Level (NOAEL) was established at 150 mg/kg/day.