p-[(2-chloroethyl)ethylamino]benzaldehyde

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

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:

no

Test material

Test material information

Constituent 1

Reference substance name:

p-[(2-chloroethyl)ethylamino]benzaldehyde

EC Number:

220-150-2

EC Name:

p-[(2-chloroethyl)ethylamino]benzaldehyde

Cas Number:

2643-07-4

Molecular formula:

C11H14ClNO

IUPAC Name:

4-[(2-chloroethyl)(ethyl)amino]benzaldehyde

Details on test material:

- Name of test substance: N-Chlorethyl,N-Ethyl-4-Aminobenzaldehyde
- CAS No.: 2643-07-4
- Batch identification: 20080616
- Purity: 98.4 corr.area-%
- Homogeneity: homogeneous
- Stability: 16 Jun 2010 (The stability of the test substance under storage conditions over the test period was guaranteed by the manufacturer, and the manufacturer holds this responsibility.)
- Date of production: 16 Jun 2008
- Physical state/appearance: solid/dark blue

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Age at study initiation: 32-34 days
- Weight at study initiation: mean 192 g (males)/144 g (females)
- Housing: 5 animals per cage in H-Temp polysulfonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm²)
- Diet: ad libitum (ground Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water: ad libitum
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was applied as a solution. To prepare this solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, Olive Oil Ph. Eur. was filled up to the desired volume and warmed up to 50°C, subsequently mixed using a magnetic stirrer. The test-substance preparations were produced at least every 7th day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in Olive Oil Ph. Eur. at room temperature for a period of 7 days was proven before the start of the administration period. Homogeneity and concentration control analyses of the test-substance preparations were performed in samples of all concentrations at the start of the administration period.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 2.5, 10, 40 mg/kg/d
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Mortality: twice daily on working days and once daily on Saturdays, Sundays and public holidays; clinical observations: daily before as well as <1 hour and 3-4 hours after the administration

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration period and thereafter at weekly intervals

BODY WEIGHT: Yes
- Time schedule for examinations: Before the start of the administration period in order to randomize the animals. During the administration period the on day 0 (start of the administration period) and thereafter at weekly intervals.

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

WATER CONSUMPTION: Yes
- Time schedule for examinations: Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at sacrifice (study day 29)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all animals

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at sacrifice (study day 29)
- Animals fasted: Yes
- How many animals: all animals

URINALYSIS: Yes
- Time schedule for collection of urine: towards the end of the administration period (study day 26)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: towards the end of the administration period (study day 26/27)
- Dose groups that were examined: all animals
- Battery of functions tested: functional observational battery (home cage observations, open field observations, senorimotor tests/reflexes) and motor activity assessment

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY
No animals were found dead during the study. Salivation was seen shortly after treatment (<1 hour) in 4 males and all females of test group 3 (40 mg/kg bw/d). This finding was observed on day 4 for the first time and afterwards on several days of the study. Piloerection and respiration sounds were observed in 1 female animal of test group 3 (40 mg/kg bw/d) from day 23 until the end of the study. As no other male or female animal of test group 3 (40 mg/kg bw/d) showed similar findings they were assessed as being incidental rather than treatment-related. No effects of toxicological relevance were obtained in test groups 1 and 2 (2.5 and 10 mg/kg bw/d).

BODY WEIGHT AND WEIGHT GAIN
Significant impairment of mean body weights were not observed in any test group. However, at the end of the administration period body weights of male animals in test group 3 (40 mg/kg bw/d) were 7.9% lower compared to the control animals. In addition, body weight change was significantly decreased in male animals of test group 3 (40 mg/kg bw/d) on day 7 (-39.8%) and non-significantly on study days 14 (-15.0%), 21 (-12.2%) and 28 (-20.2%). Although these effects were only marginal, a relation to dosing was assumed. No test substance-related changes were seen for male animals of test groups 1 (2.5 mg/kg bw/d) and 2 (10 mg/kg bw/d) as well as female animals of all test groups. The significantly increased body weight change value in females of test group 1 (2.5 mg/kg bw/d) on study day 14 was assessed as being incidental and not related to treatment.

FOOD CONSUMPTION
Deviations in food consumption were seen in all treatment groups at several time points. Taking effects on body weights into account, a relation to treatment was only assumed for male animals of the test group 3 (40 mg/kg bw/d) since food consumption was decreased by 10.0% on day 21 and by 20.1% on day 28. All other changes were regarded as incidental.

WATER CONSUMPTION
No test substance-related findings in male and female animals of all test groups were observed.

HAEMATOLOGY
No treatment-related changes among hematological parameters were measured. At the end of the study, relative reticulocyte counts were lower in males of test group 3 (40 mg/kg bw/d) compared to controls. This was the only changed red blood cell parameter in dosed rats of this study. Therefore, it was regarded as not adverse (ECETOC Technical Report No. 85, 2002).

CLINICAL CHEMISTRY
At the end of the study, in females of test groups 2 and 3 (10 and 40 mg/kg bw/d) the total protein levels were decreased. This was mainly due to a decrease of the globulins which became significant in rats of both sexes in test group 3 (40 mg/kg bw/d). In males of test group 1 (2.5 mg/kg bw/d) the globulins were also significantly reduced, but this was the only changed parameter in rats of this test group and, therefore, it was regarded as non-adverse (ECETOC Technical Report No. 85, 2002). In rats of both sexes of test group 2 (10 mg/kg bw/d) and, additionally, in male rats of test group 3 (40 mg/kg bw/d) the triglyceride levels were decreased. In dosed female rats the triglyceride levels were not dose-dependently decreased and in males of test group 2 (10 mg/kg bw/d) the triglyceride level changes were the only changed clinical pathology parameter. Therefore, the triglyceride levels decrease in females was regarded as incidental and in males it was estimated as potentially treatment-related, but not adverse. In males of test group 3 (40 mg/kg bw/d) the cholesterol concentrations were decreased. In females of the same test group the creatinine levels were lower compared to controls. In females of test groups 2 and 3 (10 and 40 mg/kg bw/d) the calcium levels were decreased (not significantly in test group 3). In males of test group 3 (40 mg/kg bw/d) the potassium concentrations were higher compared to controls.

URINALYSIS
No treatment-related changes among urinalyses parameters were measured.

NEUROBEHAVIOUR
Functional observational battery: There were no test substance-related findings in male and female animals of all test groups. Any deviations from "zero values" were equally distributed between test substance-treated groups and controls or occurred in single animals only. Therefore, these observations were considered as being incidental (Home cage observations: No test substance-related or spontaneous findings in male and female animals of all test groups during the home cage observation were observed; Open field observations: The open field observations did not reveal any test substance-related findings in male and female animals of all test groups. However, piloerection and respiration sounds were observed in 1 female animal of test group 3 (40 mg/kg bw/d). These findings were assessed as being spontaneous in nature and not related to treatment; Sensorimotor tests/reflexes: No test substance-related or spontaneous findings in male and female animals of all test groups were observed; Quantitative parameters: No test substance-related impaired parameters were observed in male and female animals of all test groups. Frequent vocalization was observed for one female of test group 3 (40 mg/kg bw/d). As this animal showed no other findings it was assessed as being incidental and not related to treatment.)
Motor activity measurement: There were no significant deviations with regard to single intervals and the overall motor activity (summation of all intervals) in male and female animals of all test groups in comparison to the concurrent control group.

ORGAN WEIGHTS
Absolute weights: When compared with control group 0 (= 100%) following mean absolute weights were increased or decreased: Although not significant, the terminal body weight decrease (92%) in males of test group 3 (40 mg/kg bw/d) was regarded to be treatment-related. The prostate weight decrease (77%) in males of the same test group was considered to be secondary to the body weight decrease, since no histopathological correlate was noted. The significant terminal body weight decrease (94%) in females of test group 3 (40 mg/kg bw/d) was regarded as treatment-related. No histopathological correlate was found for the liver weight increase (121%) in females of test group 3 (40 mg/kg bw/d). All other mean absolute weight parameters did not show relevant differences when compared to the control group 0 and were considered to be within the normal range typical for rats of this strain and age.
Relative weights: When compared with control group 0 (= 100%) following mean relative weights were increased: The significant weight increases observed in testes of males (119%) and brain of females (111%) of test group 3 (40 mg/kg bw/d) occurred without histopathological correlate and was regarded as incidental. The liver of females of test group 3 (40 mg/kg bw/d) showed a significant relative weight increase (129%). Although no microscopic correlate was noted, since both absolute and relative weights were increased, a treatment-related effect could not be completely ruled out. All other mean relative weight parameters of treated animals did not show relevant differences when compared to the control groups.

GROSS PATHOLOGY
No gross lesions were observed at necropsy.

HISTOPATHOLOGY
All findings noted were either single observations or were biologically equally distributed between control animals and treated rats. All of them were considered to be incidental and/or spontaneous in origin.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

2.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: clinicochemical parameters

2

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

PATHOLOGY - WEIGHT PARAMETERS:

Absolute weights: When compared with control group 0 (= 100%) following mean absolute weights were significantly increased or decreased (printed in bold):

 

	Male animals			Female animals
Test group (mg/kg bw/d)	1 (2.5)	2 (10)	3 (40)	1 (2.5)	2 (10)	3 (40)
Terminal body weight	101%	97%	92%	102%	97%	94%*
Prostate	105%	85%	77%*
Liver				108%	105%	121%**

* : p ≤ 0.05; **: p ≤ 0.01

 

Relative weights: When compared with control group 0 (= 100%) following mean relative weights of following organs were significantly increased (printed in bold):

 

	Male animals			Female animals
Test group (mg/kg bw/d)	1 (2.5)	2 (10)	3 (40)	1 (2.5)	2 (10)	3 (40)
Testes	106%	113%	119%*
Liver				105%	108%	129%**
Brain				98%	104%	111%**

* : p ≤ 0.05; **: p ≤ 0.01

Applicant's summary and conclusion

Conclusions:

The oral administration of N-Chlorethyl,N-Ethyl-4-Aminobenzaldehyde by gavage over a period of 4 weeks caused incipient signs of general systemic toxicity at dose levels of 10 mg/kg bw/d in female animals (clinicochemical parameters) and 40 mg/kg bw/d in both genders. Therefore, the NOAEL was determined to be 2.5 mg/kg bw/d in female and 10 mg/kg bw/d in male Wistar rats.

Executive summary:

The study was performed according to OECD TG 407 in compliance with GLP. The test substance N-Chlorethyl,N-Ethyl-4-Aminobenzaldehyde was administered for 4 weeks daily by gavage to male and female Wistar rats at dose levels of 2.5, 10 and 40 mg/kg bw/d (test groups 1-3).

Regarding clinical examinations, signs of general systemic toxicity were only observed at a dose level of 40 mg/kg bw/d as there were lower body weights in male animals at the end of the study accompanied with reduced body weight gain. In addition, food consumption was slightly lower during the second half of the administration period. In male animals of test groups 1 and 2 (2.5 and 10 mg/kg bw/d) as well as in female animals of all test groups no impairment of these parameters were observed during clinical examinations. Salivation was seen after dosing nearly all rats of test group 3 (40 mg/kg bw/d) and. From the temporary, short appearance immediately after dosing it is likely, that this finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. This finding was not considered to be an adverse and toxicologically relevant effect. All other clinical parameters as well as FOB and MA did not reveal any treatment-related effects.

Regarding clinical pathology, the common alteration in rats of both sexes of test group 3 (40 mg/kg bw/d) was the decrease of globulin levels. This may include globulin transport proteins because their reduction led to decreased calcium levels in females of test groups 2 and 3 (10 and 40 mg/kg bw/d) and to decreased cholesterol and triglyceride levels in males of test group 3 (40 mg/kg bw/d). The reduced creatinine levels in combination with the decreased globulin levels in females might be due to a decreased formation of skeletal muscles. The reason for the higher potassium levels in males of the test group 3 (40 mg/kg bw/d) could not be elucidated, but a compound-related effect could not be excluded.

Regarding pathology, males and females of test group 3 (40 mg/kg bw/d) showed a decrease in terminal body weight, which was regarded to be a treatment-related effect. In addition, significant increases of absolute and relative liver weights were observed in females of this test group but no histopathological correlate was found for the liver weight increase. Therefore, a treatment-related effect cannot be completely ruled out.

In conclusion, the oral administration of N-Chlorethyl,N-Ethyl-4-Aminobenzaldehyde by gavage over a period of 4 weeks caused incipient signs of general systemic toxicity at dose levels of 10 mg/kg bw/d in female animals (clinicochemical parameters) and 40 mg/kg bw/d in both genders. Therefore, under the conditions of the present study the no observed adverse effect level (NOAEL) was 2.5 mg/kg bw/d in female and 10 mg/kg bw/d in male Wistar rats.