1-Propanamine, 3-(C9-11-isoalkyloxy) derivs., C10-rich, acetates

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 June 2016 - 20 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

September 1998

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Version / remarks:

August 1998

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Chemical Substances Control Law 1973, Notification of Mar. 31 2011 by MHLW (0331 No.7), METI (H23.03.29 SeiKyoku No. 5) and MOE (No. 110331009)

Version / remarks:

March 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dd. 03 November 2015

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3-((C9-11-iso,C10-rich)alkyloxy)propan-1-amine

EC Number:

939-485-7

Molecular formula:

Not applicable

IUPAC Name:

3-((C9-11-iso,C10-rich)alkyloxy)propan-1-amine

Test material form:

liquid

Details on test material:

- Lot/batch No.: 890000543374
- Appearance: Clear light yellow liquid
- Test item storage: At room temperature container flushed with nitrogen
- Purity/composition correction factor: No correction factor required
- Expiration date of the lot/batch: 10 February 2019
- Density: 0.8518 g/cm3 at 20°C
- Purity test date: 2016-02-16
- Analytical purity: 93.6%
- Purity/composition correction factor: No correction factor required
- Test item handling: No specific handling conditions required
- Solubility in water: Dispersible
- Stability in water: Stability for at least 6 hours at room temperature under normal laboratory light conditions is confirmed over the concentration range 0.5 - 5 mg/mL

Specific details on test material used for the study:

Purity/composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Specific gravity/density: 0.8518 g/cm3 at 20°C
Solubility in water: Dispersible
Stability in water: Stability for at least 6 hours at room temperature under normal laboratory light conditions is confirmed over the concentration
range 0.5 - 5 mg/mL

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han) (outbred, SPF-Quality)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: 143-164 g (males); 111-136 g (females)
- Fasting period before study: No
- Housing: Group housing of 5 animals per sex in Macrolon cages (MIV type, height 18 cm) with sterilized sawdust as bedding material and paper as cage-enrichment (except during locomotor activity monitoring, where animals were housed individually in a Hi-temp polycarbonate
cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water (max. 2 hours)
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), except during motor activity and overnight (for a maximum of 24 hours) before sacrifice
- Water: Free access to tap water
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16.9-23.3
- Humidity (%): 44-90
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 17 June 2016 To: 20 September 2016

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing, and were homogenized to visually acceptable levels. Adjustment was made for specific gravity of the test item (0.8518 g/cm3).

VEHICLE
- Amount of vehicle: 10 mL/kg body weight. Actual dose volumes were calculated weekly according to the latest body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of formulations were analyzed for homogeneity (highest and lowest concentration, in weeks 1, 6 and 13) and accuracy of preparation (all concentrations, in weeks 1, 6 and 13). Additionally, low dose formulation was analyzed for accuracy and homogeneity of preparation in week 7 based on the low accuracy of this group in week 6. The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration for suspensions. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.

Duration of treatment / exposure:

At least 90 days

Frequency of treatment:

Once daily, 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

15 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

50 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose levels were selected based on the results of a combined 28-day/repro-screening study (OECD 422) with the same test item (OECD 422 results summarized in ection 7.5.1.).

Positive control:

no.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards, detailed clinical observations were made in all animals after dosing. Once prior to start of treatment and at weekly intervals.

NEUROBEHAVIOURAL EXAMINATION: Yes, functional observations
- Time schedule for examinations: during Week 12 of treatment
- Animals that were examined: first 5 animals/sex/group
- Battery of functions tested:
hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength (mean of three measurements per animal), locomotor activity (total movements and ambulations)

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION:
- Weekly

WATER CONSUMPTION: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations:
at pretest: all animals (including spare animals);
at week 13: control and high dose group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all animals
- All parameters according to OECD guideline were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all animals
- All parameters according to OECD guideline were examined.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The following organ weights and terminal body weight were recorded from the surviving animals on the scheduled day of necropsy:
Adrenal glands, spleen, brain, testes, epididymides, thymus, heart, uterus (including cervix), kidneys, prostate, liver, seminal vesicles including coagulating glands, ovaries, thyroid including parathyroid

HISTOPATHOLOGY: Yes
The following slides were examined by a pathologist:
• All tissues collected at the scheduled sacrifice from all control and high dose animals.
• All tissues from the animal which died after blood sampling.
• Thyroid glands of all males of low and mid dose groups, based on (possible) treatment-related changes in this organ at high dose.
• All gross lesions.

Statistics:

The following statistical methods were used to analyze the data:
• If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
• The Fisher Exact-test was applied to frequency data.
• The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No toxicologically relevant clinical signs were noted during daily detailed clinical observations or during weekly arena observations. Salivation seen after dosing among the test item treated animals was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to the taste of the test item rather than a sign of systemic toxicity. The occurrence of rales in a few animals at 50 mg/kg bw/day was very occasional and not considered toxicologically relevant at the observed incidences. Other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the levels of incidence observed, these were considered to be unrelated to treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female at 50 mg/kg bw/day was found dead prior to necropsy after blood sampling. This animal showed no signs of imminent death and no other animals of the same group showed any toxicologically relevant clinical signs. Therefore, this death was not considered test item related, but to be the result of the blood sampling procedure.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight and body weight gain of treated animals remained in the same range as controls over the study period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before or after correction for body weight remained similar to controls over the study period.

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No ophthalmological findings were noted that were considered treatment-related. The nature and incidence of ophthalmology findings noted during pretest and in week 13 were similar among the groups, and occurred within the range considered normal for rats of this age and strain. These findings were therefore considered to be unrelated to treatment with the test item.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant changes in haematology parameters distinguished treated from control animals:
Higher relative eosinophil counts in females at 50 mg/kg bw/day; Lower reticulocyte levels in males at 50 mg/kg bw/day.
Other haematological parameters of treated rats were considered not to have been affected by treatment.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes were noted in clinical biochemistry parameters.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

At 50 mg/kg bw/day, forelimb grip strength of males was significantly reduced, while the significant reduction seen in 5 mg/kg bw/day females did not occur in a dose-dependent manner. Pupillary reflex was affected in one low dose male (slow pupillary reflex), however as this effect was only seen in one low dose animal, it should not be considered toxicologically relevant. All other animals showed normal pupillary reflexes. Hearing ability and static righting reflex were also normal in all examined animals. Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test item-related alterations in organ weights.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related gross observations. All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Examination of the thyroid glands revealed an increased incidence and severity of follicular cell hypertrophy at a dose of 50 mg/kg bw/day. Minimal follicular cell hypertrophy was seen in 4/10, 5/10, 6/10 and 4/10 males dosed at respectively 0, 5, 15 and 50 mg/kg bw/day. Slight follicular cell hypertrophy was not seen in control and low dose males, but in 1/10 and 4/10 males dosed at respectively 15 and 50 mg/kg bw/day. The slight follicular cell hypertrophy in the right thyroid gland of one animal at 15 mg/kg is most likely a compensatory response to the agenesis of the left gland. Based on the nature and mild severity, this finding is considered to be non-adverse. There were no other test item-related histological changes. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain.

Histopathological findings: neoplastic:

no effects observed

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

>= 50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

The concentrations analysed in the formulations of the low dose group used for dosing in week 1 and week 13 were in agreement with target concentrations (i.e. mean accuracies between 95% and 112%). For the low dose group formulation of week 6, a mean accuracy of 34% was obtained, which falls outside the 85-115% criterion range. No irregularities were noted in dose preparations in this period, however a possible cause could be at the point of drawing the sample for analytical control. Additional analysis on the low dose group formulation was performed in week 7. The mean accuracy of these formulations were 85% and fell within the criterion range. Since the single discrepancy was only noted in the low dose formulation, this does not affect the study validity and outcome.

The mean accuracies of mid and high dose formulations were 96-108%. No test item was detected in the control group formulations.

The formulations of low and high dose groups were homogeneous (i.e. coefficient of variation ≤ 5.5%).

Applicant's summary and conclusion

Conclusions:

A 90-day repeated dose toxicity study was conducted with 3-(Isodecyloxy)-1-Propanamine according to OECD/EC guidelines and GLP principles. Since no adverse effects were seen up to and including the highest dose tested, a NOAEL of at least 50 mg/kg bw/day was established.

Executive summary:

The 90 day study (OECD 408) was performed using dose-groups of 0, 5, 15 and 50 mg/kg, based on results from earlier 14-day RF and OECD 422 repeated dose and reproduction screening studies. The test substance was administered dissolved in water by gavage daily for 90 days.

The following parameters were evaluated: clinical signs daily; functional observation tests in week 12, body weight and food consumption weekly; ophthalmoscopy at pretest and in week 13; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

 

Results: One female at 50 mg/kg was found dead prior to necropsy after blood sampling. This animal showed no signs of imminent death and no other animals of the same group showed any toxicologically relevant clinical signs. Therefore, this death was not considered test item related, but to be the result of the blood sampling procedure.

Besides a dose related increase of incidence of salivation after dosing, no toxicologically relevant clinical signs were noted during daily detailed clinical observations or during weekly arena observations.

No adverse effects were observed in any of the parameters examined in this study across all treatment groups. Functional observation tests revealed lower forelimb grip strength for males at 50 mg/kg. This finding was considered not to represent an adverse effect on neurobehaviour, since this was not supported by clinical observations or other functional observation tests (including hindlimb grip strength), no supportive morphological correlates in examined neuronal tissues were recorded, and since the mean forelimb grip strength value was within the range considered normal for rats of this age and strain.

During histopathology examination, an increased incidence and severity of follicular cell hypertrophy was observed in the thyroid glands of 50 mg/kg males. Given the nature and the mild severity, this finding is considered to be non-adverse.

Slight, possibly treatment-related, changes in haematological parameters were noted at 50 mg/kg. This included higher relative eosinophil levels in females and lower relative reticulocyte levels in males, which occurred in the absence of concurrent treatment-related morphological changes, signs of allergic responses or changes in related hematological parameters and were within the normal range for animals of this strain and age. In this context, these findings are considered non-adverse.

 

Conclusion: From the results presented in this report a No Observed Adverse Effect Level (NOAEL) for 3-(Isodecyloxy)-1-Propanamine of at least 50 mg/kg was established.

Higher doses were not tested since it was expected that a dose level higher than 50 mg/kg was not tolerated (based on previous results).