Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
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
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
OECD Guidelines for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity Study in Rodents" (adopted 03 October 2008).

Also compatible with Commission Directive 96/54/EC (Method B7) and
Commission Regulation (EC) No 440/2008 of 30 May 2008, test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)
Deviations:
yes
Remarks:
Minor protocol deviations / errors described - Clinical observations, Organ weights, Hematology
GLP compliance:
yes (incl. certificate)
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Specific details on test material used for the study:
Identification : Hydroxypropyl, 2-, trimethylammonium formate
Physical State/Appearance : Colorless to light blue liquid
Purity : 95.25% active in water
Batch Number : chernil.20160628.B
Label : DABCO TMR-2 Catalyst solvent free
Date Received : 24 October 2016
Storage Conditions : Room temperature, in the dark
Expiry Date : 28 June 2017

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Wistar Han™:RccHan™:WIST
Sex:
male/female
Details on test animals and environmental conditions:
The animals were housed in groups of five by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding.
A pelleted diet (Rodent 2014C Teklad Global Certified Diet, Envigo RMS (UK) Limited., Oxon, UK) was used. A certificate of analysis of the batch of diet is provided.
Mains drinking water was supplied from polycarbonate bottles attached to the cage.
The animals were housed in a single air-conditioned room.
The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.
Environmental conditions were continuously monitored.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Administration by gavage using a stainless steel cannula attached to a disposable plastic syringe.
Vehicle:
polyethylene glycol
Details on oral exposure:
The test item was administered daily, for twenty-eight consecutive days. Control animals were treated in an identical manner with 4 mL/kg of Polyethylene glycol 400.
The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Representative samples of test item formulations were taken (four times during the course of the study, twice for the daily preparations and on both occasions for the weekly preparations) and analyzed for concentration of Hydroxypropyl, 2-,trimethylammonium formate.
The results indicate that the prepared formulations were within ± 25#% of the nominal concentration confirming accurate formulation.

Duration of treatment / exposure:
The test item was administered daily, for twenty-eight consecutive days.
Frequency of treatment:
Once per day.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control group
Dose / conc.:
30 mg/kg bw/day (nominal)
Remarks:
Low Dose Group
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Intermediate doese group
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High does group
No. of animals per sex per dose:
5 males and 5 females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
The study was performed according to the study plan and was designed to investigate the systemic toxicity of the test item, by repeated oral administration to th rat for a period of twenty-eight consecutive days at dose levels of 30, 300 and 1000 mg/kg bw/day. A control group was dosed with vehicle alone, Polyethylene glycol 400.
Dose levels were selected, in collaboration with the sponsor based on available toxicity data which included a 7 day range finding toxicity study in the rat (Envigo Study Number: PN22WW) where dose levels of up to 1000 mg/kg bw/day were well tolerated. The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item.
Positive control:
No

Examinations

Observations and examinations performed and frequency:
All animals were examined for overt signs of toxicity, ill-health or behavioral change immediately before dosing, up to thirty minutes post dosing and one hour after dosing.
Sacrifice and pathology:
All animals were sacrificed on day 29. No mortalities were seen during the 28 day dosing period.
Other examinations:
All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed.
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry,Absolute Organ Weights, Body Weight-Relative Organ Weights.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no adverse effects of treatment at any dose level.

One male animal treated with 1000 mg/kg bw/day exhibited noisy respiration for one day only (Day 29).
Increased salivation was noted in one male animal treated with 1000 mg/kg bw/day on Day 15 and Day 22.

Such observations are commonly observed in this type of study and are generally considered to be due to an irritant/unpalatable nature of the test item and/or formulation.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related adverse effects on body weight of treatment at any dose level.

Male animals from all treatment groups generally exhibited reduced body weight gains when compared to control throughout the treatment period with all groups achieving statistical significance during Week 3 (p<0.05).
All treated male animals showing a reduction in overall body weight gains of 11.2%, 13.1% and 11.6% for low, intermediate and high dose group animals respectively.
These effects on body weight did not show any dose relationship and ultimately did not have any impact on the animals in terms of clinical condition, haematological, blood chemical or histopathological findings,these effects on body weight were considered to be non-adverse in nature.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were no adverse effects of treatment on food consumptions for any treated group when compared to controls.

Marginal reductions in food consumption were noted for male animals treated with 1000 mg/kg bw/day or 300 mg/kg bw/day as overall food consumptions for these two groups were 5.5% and 4.7% lower than control respectively. No such effects were noted in male animals treated with 30 mg/kg bw/day or in any treated female group as overall food consumption for treated females actually exceeded control.

Any differences in food conversion efficiency when compared with controls were considered to be due to intergroup differences in terms of respective body weight gains and/or food consumption for these animals.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
There were no adverse effects of treatment on water consumptions for any treated group when compared to controls.

Marginal reductions in water intake for all treated males was noted during the treatment period but without a dose relationship, reductions of 16%, 5% and 8% were noted for animals treated with 30, 300 or 1000 mg/kg bw/day respectively. No such effects were noted in any treated female.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically significant effects were detected at any dose level in animals of either sex.

At the end of the dosing period, group mean reticulocytes in all treated animals were statistically significantly higher (p<0.01) than controls with a dose relationship being noted. However, all values obtained were within the historical control data ranges.

Group mean hemoglobin concentration was statistically significantly increased (p<0.05) in female animals treated with 1000 mg/kg bw/day, however, a dose relationship was not apparent and only one value for this dose group was outside of the historical control data ranges.

Statistically significant increases in hematocrit were apparent across all female treatment groups, a dose relationship was not apparent. With the exception of one value in females treated with 30 mg/kg bw/day which was higher than the historical control data range, all values lay within the expected parameters.

Since the majority of values for treated animals lay within the historical control data ranges and there were no histopathological correlates, it is considered that there were no toxicologically significant effects detected in the hematological parameters
examined.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
When compared with controls, males across all treatment groups showed statistically significantly higher (p<0.01) glucose concentrations, these did not show a dose relationship and with the exception of one value in males treated with 30 mg/kg bw/day which was higher than the historical control data range, all values lay within the expected parameters.

No such effects were detected in any treated female animals.

Since the majority of values for treated male animals lay within the historical control data ranges and there were no histopathological correlates, it is considered that there were no toxicologically significant effects detected in the blood chemical parameters examined.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no treatment-related intergroup differences at any dose level.
There were no treatment-related changes in functional performance at any dose level.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No statistically significant effects were detected in the organ weights measured.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No changes were noted which could be attributed to the administration of the test item.
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Details on results:
Mortality
There were no unscheduled deaths during the study.

Clinical Observations
There were considered to be no adverse effects of treatment at any dose level.

Behavioral Assessment
There were no treatment-related intergroup differences at any dose level.

Functional Performance Tests
There were no treatment-related changes in functional performance at any dose level.

Sensory Reactivity Assessments
There were no intergroup differences at any dose level considered to be related to treatment with the test item.

Body Weight
There were no adverse effects of treatment at any dose level.

Food Consumption
There were no adverse effects of treatment on food consumptions for any treated group when compared to controls.

Water Consumption
There were no adverse effects of treatment on water consumptions for any treated group when compared to controls.

Hematology
No toxicologically significant effects were detected at any dose level in animals of either sex.

Blood Chemistry
No toxicologically significant effects were detected at any dose level in animals of either sex.

Necropsy
No macroscopic abnormalities were detected.

Organ Weights
No statistically significant effects were detected in the organ weights measured.

Histopathology
No changes were noted which could be attributed to the administration of the test item.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
haematology
clinical biochemistry
behaviour (functional findings)
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The oral (gavage) administration of Hydroxypropyl, 2-, trimethylammonium formate to male and female Wistar Han™:RccHan™:WIST strain rats at dose levels of up to 1000 mg/kg bw/day resulted in no adverse effects being noted.
As such, 1000 mg/kg bw/day is considered to be the `No Observed Adverse Effect Level' (NOAEL) for systemic toxicity within the confines of this type of study.
Executive summary:

To determine the potential for oral toxicity using the Acute Toxic Class Determination. This study was designed to comply with the standards set forth in the current OECD Guidelines for the Testing of Chemicals, Guideline 423. Guideline 423 is referred to in OPPTS 870.1000 as an acceptable method

to assess lethality within a dose range. The test article was assigned to a toxic category based on the mortality results and significant clinical signs of toxicity up to the Category 4 value tested according to the

current Globally Harmonized System of Classification and Labeling of Chemicals (GHS).

The test item was administered by gavage to three groups, each of five male and five female Wistar Han™:RccHan™:WIST strain rats, for twenty-eight consecutive days, at dose levels of 30, 300 and 1000 mg/kg bw/day. A control group of five males and five females was dosed with vehicle alone (Polyethylene glycol 400).

Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Hematology and blood chemistry were evaluated for all animals at the end of the study.

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed.

Mortality:

There were no unscheduled deaths during the study.

Clinical Observations:

There were considered to be no adverse effects of treatment at any dose level.

Behavioral Assessment:

There were no treatment-related intergroup differences at any dose level.

Functional Performance Tests:

There were no treatment-related changes in functional performance at any dose level.