Amines, C12-16 alkyl dimethyl, reaction products with ethyl chloroacetate, 2-(2-aminoethylamino)ethanol and 2-propenoic acid

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(2-carboxyethyl)({2-[(2-carboxyethyl)(2-hydroxyethyl)amino]ethyl})azanium; [({2-[(2-carboxyethyl)(2-hydroxyethyl)amino]ethyl}carbamoyl)methyl](dodecyl)dimethylazanium; ethanol

IUPAC Name:

(2-carboxyethyl)({2-[(2-carboxyethyl)(2-hydroxyethyl)amino]ethyl})azanium; [({2-[(2-carboxyethyl)(2-hydroxyethyl)amino]ethyl}carbamoyl)methyl](dodecyl)dimethylazanium; ethanol

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld
- Age at study initiation: approx. 6 weeks at start of treatment
- Weight at study initiation:-
- Fasting period before study:-
- Housing: 5 animals per sex per cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +- 3°C
- Humidity (%):30 - 70 %
- Air changes (per hr):-
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Duplo samples of formulations prepared during week 2, 8 and 13 were taken for analysis of accuracy and homogeneity (16 samples in total).

Duration of treatment / exposure:

90 days

Frequency of treatment:

Once daily for at least 90 days, 7 days per week

Doses / concentrations

Remarks:

Doses / Concentrations:
50 mg/kg/day, 150 mg/kg/day, 450 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on a 14-day range finding study

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION :Yes
- Time schedule for examinations: weekly

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: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at pretest all animals
- Dose groups that were examined: groups 1 and 4 at week 13

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes (overnight)
- How many animals: all animals
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Animals fasted: Yes (overnight)
- How many animals: all animals
- Parameters checked in table [No.1] were examined.

URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 12-13
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity / grip strength / motor activity /

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 2)
ORGAN WEIGHTS: Yes (see table 3)
HISTOPATHOLOGY: Yes (see table 4)

Statistics:

- Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
- 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 when the data could not be assumed to follow a normal distribution.
- The Fisher-Exact test was applied to frequency data.
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. No statistical analysis was performed on motor activity data. 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. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

ALAT and ASAT increased in high dose males

Urinalysis findings:

not specified

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

spleen and liver weight affeted

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

effects in stomach, lungs and kidneys

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY: No mortality occurred during the study period which could be attributed to treatment with the test substance. No clinical signs of toxicity were observed.

BODY WEIGHT AND WEIGHT GAIN: Body weights and body weight gain of treated animals did not show changes that were considered to have arisen as a result of treatment.

FOOD CONSUMPTION AND COMPOUND INTAKE : There were no differences in food consumption between treated and control animals, which were considered to be related to treatment with the test substance.

OPHTHALMOSCOPIC EXAMINATION: There were no ophthalmoscopic findings at pre-dose and in week 13 among the treated animals.

HAEMATOLOGY: There were no changes of haematology parameters that were considered to represent a sign of direct toxicity.

CLINICAL CHEMISTRY: Alanine aminotransferase and aspartate aminotransferase activity values were increased among high dose males. In addition, cholesterol values were reduced among these males.

NEUROBEHAVIOUR: No changes were observed in hearing ability, pupillary reflex, static righting reflex and grip strength in the treated animals, when compared to control animals. The variation in motor activity did not indicate a relation with treatment.

ORGAN WEIGHTS: Spleen and spleen to body weight ratios were reduced among high dose males. In addition, liver to body weight ratios of group 3 and 4 males were lower than controls. Liver weights uncorrected for body weights were reduced in group 3 only, while mean liver weights of group 4
showed no statistically significant reduction. Among high dose females, increased kidney and kidney to body weight ratios were observed.

GROSS PATHOLOGY: Macroscopic findings recorded in this study were considered to be comparable in incidence and severity with the control animals or within the normal range of background alterations that may be seen in untreated animals of this age and strain.

HISTOPATHOLOGY: NON-NEOPLASTIC: An increased incidence of minimal to moderate accumulations of foam cells in the lungs of group 4 males and group 4 females was noted. The forestomach of males and females of group 4 showed minimal to marked squamous hyperplasia (with accompanying
inflammatory changes). These lesions affected the main body of the forestomach. Additionally, minimal to slight cortical tubular basophilia of the kidneys was noted in group 4 females.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

2

Dose descriptor:

NOAEL

Effect level:

75 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: overall effects

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

From the results presented in this report a No Observed Adverse Effect Level (NOAEL) for the substance of 50 mg/kg/day was established. Based on the absence of functional or morphological disturbances supporting the liver weight changes noted for group 3 males, a NOAEL of 150 mg/kg/day is considered.

Executive summary:

A 90 -day oral toxicity study with the substance by daily gavage in the rat was performed. The study was based on the following guidelines: - EEC Directive 87/302/EEC, B Repeated Dose (90 days) Toxicity (orat), 1988.

- OECD 408, Repeated Dose go-day Oral Toxicity Study in Rodents, 1998.

Based on a 16 day range finding study the dose levels were selected to be 0, 50, 150 and 450 mg/kg/day.

The test substance was administered daily for at least 90 days by oral gavage to Wistar rats. One control group and three treated groups were tested, each consisting of 10 males and 10 females.

The following parameters were evaluated:

Clinical signs, functional observations, body weight, food consumption and ophthalmoscopy. At termination: clinical pathology, macroscopy, organ weights and histopathology on a selection of tissues.

RESULTS: Accuracy and homogeneity of formulations of test substance in milli-U water were demonstrated by analyses.

Treatment related findings observed were as follows:

50 mg/kg/day:- No treatment-related findings noted.

150 mglkg/day:- Liver weight reduced (males).

450 mglkg/day:- Alanine and aspartate aminotransferase activity values increased and cholesterol values reduced (males).

- Irregular surface of the forestomach (males and females), with dark red foci or thickening (males); thickened limiting ridge and/or dark red contents of the stomach (females); gray-white foci on the lungs (females).

- Spleen and liver weight reduced (males); kidney weights increased (females).

- Pulmonary foam cell accumulations and squamous hyperplasia of the forestomach (with accompanying inflammatory changes) (males and females); cortical tubular basophilia of the kidneys (females).

CONCLUSION: From the results presented in this report a No Observed Adverse Effect Level (NOAEL) for

the substance of 50 mg/kg/day was established. Based on the absence of functional or morphological disturbances supporting the reduction of liver weights of group 3 males, a NOAEL of 150 mg/kg/day was considered.