N-benzyl-N-C16-18 (even numbered)-alkyl-N-methyl-C16-18 (even numbered)-alkyl-1-aminium chloride

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2010-05-11 to 2010-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

other: draft report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.43 (Neurotoxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Quaternary ammonium compounds, di-C16-18 (even numbered)-alkylmethylbenzyl chlorides

IUPAC Name:

Quaternary ammonium compounds, di-C16-18 (even numbered)-alkylmethylbenzyl chlorides

Constituent 2

Reference substance name:

Quaternary ammonium compounds, Benzylbis(hydrogenated tallow alkylalkyl)methyl, chlorides

IUPAC Name:

Quaternary ammonium compounds, Benzylbis(hydrogenated tallow alkylalkyl)methyl, chlorides

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Breeder: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: 6 weeks old
- Weight at study initiation: males: mean body weight of 244 g (range: 229 g to 254 g), females: mean body weight of 205 g (range: 189 g to 221 g)
- Fasting period before study: no
- Housing: individually housed in suspended wire-mesh cages.
- Diet (e.g. ad libitum): free access to SSNIFF R/M-H pelleted maintenance diet
- Water (e.g. ad libitum): free access to bottles containing tap water (filtered with a 0.22 µm filter)
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): 50 +/- 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h / 12 h

IN-LIFE DATES: From: 19 May 2010 To: 16 June 2010.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a suspension in the vehicle.

Preparation of the highest concentration (mother formulation):
- the vehicle was warmed up in a water bath to +80°C,
- appropriate amounts of test item were weighed,
- ~ 5 g of warm oil was slowly added to the test item,
- the mixture of vehicle and test item was warmed up in a water bath to +80°C under magnetic stirring for 20 minutes,
- the required amount of vehicle was slowly added to obtain the final concentration,
- the mixture of vehicle and test item was warmed up in water bath to +80°C under magnetic stirring for between 20 and 30 minutes.

Preparation of the low and intermediate concentrations:
- the required volume of warm mother formulation was taken,
- this was slowly added to the required amounts of warm vehicle to obtain the final chosen concentrations.

Formulations were kept under magnetic stirring at +40°C and delivered to the study room in water at +40°C . All formulations were administered within 4 hours of preparation.

VEHICLE
- Concentrations in vehicle: 0, 5, 10 and 25 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- nature of analytical method: GC-FID
- test item concentrations: the concentration of the test item in samples of control and test item dosage forms prepared for use on day 1 and on a day in week 4 was determined.
- homogeneity and stability: before the start of treatment the suitability of the proposed preparation process was confirmed by analysis of the
homogeneity and stability.
In CIT/Study No. 36408 AHS, a range of dosage forms was prepared at levels which covered the lowest and highest concentrations proposed for use
in this study, and then stored at +40°C in a water bath for 4 hours in order to cover the maximum duration of the administration period.
After preparation of the dosage forms, duplicate samples were taken from three levels of the container (top, middle and bottom) and then analyzed
for homogeneity and stability within 4 hours.

Duration of treatment / exposure:

28 days.
Day 1 corresponds to the first day of the treatment period.

Frequency of treatment:

Daily.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
50 mg/kg/day
Basis:
other: nominal ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
100 mg/kg/day
Basis:
other: nominal ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
250 mg/kg/day
Basis:
other: nominal ingested

No. of animals per sex per dose:

5 males and 5 females per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose-levels were selected in agreement with the Sponsor, based on the results of a preliminary study (CIT/Study No. 36439 TSR) where the test
item was administered daily by the oral route for a 7-day period to rats. The test item was administered to three groups of four male and four female
Sprague-Dawley rats at dose-levels of 300, 500 or 800 mg/kg/day under a constant dosage-volume of 10 mL/kg. A control group of four males and four females received the vehicle, corn oil, under the same conditions.
No mortality occurred during the study. Ptyalism was recorded at all test item dose-levels with a similar incidence. This clinical sign, commonly
observed when a test item is administered by gavage, was considered not to be adverse. Breathing difficulties (abdominal and/or loud) were noted in all animals given 500 or 800 mg/kg/day (from day 3 or from day 2, respectively), and in 4/8 animals at 300 mg/kg/day (mainly between days 5 and
8). Several animals given 500 or 800 mg/kg/day showed signs of poor clinical condition [including piloerection, thin appearance, hunched posture,
hypoactivity, soiled mouth, urogenital and/or head, swelling of the abdomen, soft feces (in males only) and/or half closed eyes (in females only)] with a dose-related onset and incidence. Piloerection and thin appearance were also noted in few males at 300 mg/kg/day.
A marked mean body weight losses were noted in 300, 500 and 800 mg/kg/day males over the whole treatment period, which resulted in a lower
body weights on day 7. In females, body weight loss was noted only at 800 mg/kg/day, with lower severity than in males.
Marked lower mean food consumption was noted in all test item-treated males. In females a similar effect was noted at 800 mg/kg/day, but it was less marked than in males. A similar trend was noted in females given 300 or 500 mg/kg/day between days 4 and 7.
The macroscopic examination revealed higher adrenal gland weights in males given 500 or 800 mg/kg/day and in females given 800 g/kg/day.
Enlargement of the adrenal glands was recorded in one female given 800 mg/kg/day, and this correlated with higher adrenal weight. Distention of a
portion from the stomach to the colon was noted from 500 mg/kg/day, with a higher incidence at 800 mg/kg/day. Reduced thymus size was
observed in one male treated at 500, and in one male and one female at 800 mg/kg/day. In addition, reduced spleen size was seen in one female given 800 mg/kg/day.

- Rationale for animal assignment (if not random): computerized stratification procedure.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day during the acclimation period and at least twice a day during the treatment period

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the beginning of the treatment period and then once a week until the end of the study

BODY WEIGHT: Yes
- Time schedule for examinations: once before group allocation, then on the first day of treatment and once a week until the end of the study

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment period

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the treatment period

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once at the end of the treatment period. This examination included detailed clinical observation, reactivity to manipulation and different stimuli and motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
A complete macroscopic post-mortem examination was performed on all study animals. This included examination of the external surfaces, all
orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and
the neck with its associated organs and tissues.
Special attention was accorded to the stomach and intestines.

HISTOPATHOLOGY: Yes
A microscopic examination was performed on:
- all tissues listed in the Tissue Procedure Table for the control and high-dose animals (groups 1 and 4) sacrificed at the end of the treatment period,
- all macroscopic lesions from all low- and intermediate-dose animals (groups 2 and 3) sacrificed on completion of the treatment period.

According to the microscopic results of the highest-dose group, the adrenals of low- and intermediate dose animals were examined.

ORGAN WEIGHTS: see table below.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
Mortality:
No mortality occurred during the study.

Clinical signs:
Breathing difficulties (loud or abdominal breathing) were noted in 2/5 females given 250 mg/kg/day, mainly in the second half of the treatment
period (between days 10 and 22) and until the end of treatment period (days 27 or 29). They were attributed to the test item treatment.
Hypersalivation (recorded as ptyalism) was noted in both sexes in all groups (except in control females). This clinical sign appeared mainly during
the second part of the treatment period, excepted for three animals treated at 250 mg/kg/day (two males and one female) for which it was noted from day 1 or 3 until the end of the treatment period. As the incidence was similar in all groups, hypersalivation was considered not to be test item-related.

BODY WEIGHT AND WEIGHT GAIN
Body weight was unaffected by the test item treatment.

FOOD CONSUMPTION
Food intake was not disturbed by the test item treatment.

HAEMATOLOGY
No test item-related findings were noted at hematology investigation.

CLINICAL CHEMISTRY
Statistically significant, higher alanine aminotransferase activity was noted in males and females given 250 mg/kg/day (119 vs. 48 IU/L in males, and 73 vs. 42 IU/L in females; 2.5-fold and 1.7 fold respectively). This finding was attributed to the test item treatment.

URINALYSIS
No abnormalities were noted in the urine parameters.

NEUROBEHAVIOUR
No test item treatment-related abnormalities were noted at the Functional Observation Battery.

ORGAN WEIGHTS:
At 250 mg/kg bw, a statistically significant increase in adrenal weights was observed in both sexes. This increase in adrenal weight correlated with a slight hypertrophy (grade 2 on a maximum scale of 4) of adrenal cortical cells in 4 out 5 males and in 1 out of 5 females. All the remaining animals of this dose-group showed a minimal hypertrophy (grade 1). At 50 and 100 mg/kg/day, a slight but not dose-related increase in adrenal weights was observed.
Low thyroids weight were seen in males given 50, 100 or 250 mg/kg/day. These changes were at a similar magnitude at all doses, without any
microscopic correlates and thus, a relationship to treatment was considered to be unlikely.

GROSS PATHOLOGY:
No treatment-related macroscopic changes were noted in rats given the test item without solvent at 50, 100 or 250 mg/kg/day for 4 weeks.

HISTOPATHOLOGY:
At 250 mg/kg bw, the increase in adrenal weight correlated with a slight hypertrophy (grade 2 on a maximum scale of 4) of adrenal cortical cells in 4 out 5 males and in 1 out of 5 females. All the remaining animals of this dose-group showed a minimal hypertrophy (grade 1). At 50 and 100 mg/kg/day, a minimal hypertrophy of adrenal cortical cells (grade 1) was observed in some animals. One female of the control group showed also a minimal hypertrophy of adrenal cortical cells (grade 1). In the absence of associated degeneration necrosis, this change was not considered to be adverse.
Minimal Marginal increased severity of mononuclear cell infiltration in the ventral part of the prostate was seen in animals given 250 mg/kg/day
when compared to control animals. As this change is commonly encountered in rats of this age at a similar severity, a relationship to treatment was
considered to be unlikely.

Effect levels

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

The test item concentrations in the administered dosage forms analyzed in weeks 1 and 4 remained within an acceptable variation range of -7.7% to +1.2% when compared to the nominal values.

Applicant's summary and conclusion

Conclusions:

The NOAEL of the test substance was determined to be 100 mg/kg bw/day in male and female rats.

Executive summary:

The objective of this study was to evaluate the potential toxicity of the test item following daily oral administration (gavage) to rats for 4 weeks according to OECD guideline 407 and EU method B.43. The study was conducted in compliance with the principles of Good Laboratory Practice regulations.

Three groups of five male and five female Sprague-Dawley rats received the test item by oral administration (gavage) at dose‑levels of 50, 100 or 250 mg/kg/day for 4 weeks. A constant dosage-volume of 10 mL/kg was used.

In addition, five males and five females received the vehicle only (corn oil) under the same experimental conditions and acted as controls.

Samples of dosage forms administered on day 1 and on a day 22 were analyzed for test item concentration.

The animals were checked daily for mortality and clinical signs. In addition, a detailed clinical examination was performed once before the beginning of the treatment period and then once a week until the end of the study. A Functional Observation Battery was performed at the end of the treatment period (day 27).

Body weight was recorded once before beginning of the treatment period, then on the first day of treatment and once a week until the end of the study. Food consumption was recorded once a week during the study. Hematology, blood biochemistry and urine investigations were performed on day 29.

On completion of the treatment period, the animals were euthanized and submitted to a full macroscopic post-mortem examination. Designated organs were weighed and selected tissue specimens were preserved. A microscopic examination was performed on designated tissues from the control and high-dose groups. According to the microscopic results of the highest-dose group the adrenals of low- and intermediate‑dose animals were examined.

 

The test item concentrations in the administered dosage forms analyzed in weeks 1 and 4 remained within an acceptable variation range of -7.7% to +1.2% when compared to the nominal values.

No mortalities occurred during the study.

At 250 mg/kg bw/ day, loud and abdominal breathing were noted in 2/5 females during the second part of the treatment period. A statistically significant increase in alanine aminotransferase activity was noted in males and females (2.5 -fold and 1.7 -fold, respectively). There was a statistically significant increase in adrenal weights in both sexes which correlated with a slight hypertrophy (grade 2 on a maximum scale of 4) of adrenal cortical cells in 4 out 5 males and in 1 out of 5 females. All the remaining animals of this dose-group showed a minimal hypertrophy (grade 1). At 50 and 100 mg/kg/day, no adverse test item-related effects were noted. A slight but not dose-related increase in adrenal weights was observed. Minimal hypertrophy of adrenal cortical cells (grade 1) was noted in some animals. One female of the control group showed also a minimal hypertrophy of adrenal cortical cells (grade 1). In the absence of associated degeneration necrosis, this change was not considered to be adverse.

Consequently, under the experimental condition of this study, the No Observed Adverse Effect Level (NOAEL) was set at 100 mg/kg/day.