Benzoic acid, 2-[4-(diethylamino)-2-hydroxybenzoyl]-, hexyl ester

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000-03-14 to 2000-07-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

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)

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)

Deviations:

no

Principles of method if other than guideline:

Cited version of the EU method: EG Commission Directive 87/302/EEC of 18 November, 1987; Part B: Methods for the determination of Toxicity; Subchronic oral Toxicity Test; 90-day repeated oral dose using rodent species; Official Journal oft he European Communities No. L 133, pp. 8- 11, 1988

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

443-860-6

EC Name:

-

Cas Number:

302776-68-7

Molecular formula:

C24 H31 N O4

IUPAC Name:

hexyl 2-[4-(diethylamino)-2-hydroxybenzoyl]benzoate

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Wistar rats Cr1: WI (GLX/BRL/HAN) IGS BR were supplied by Charles River Sulzfeld, Germany.
- Age at study initiation: 42+/- 1 days.
- Weight at study initiation: The body weight of the males was in the range of 111.0-141.1 g (group mean: 130.4 g) and the body weight of the females was in the range of 102.4 - 123.7 g (group mean: 114.6 g).

- Housing: The rats were housed singly in type DK III stainless steel wire mesh cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area about 800 cm2). Underneath the cages, waste trays were fixed containing absorbent material (type 3/4 dustfree embedding, supplied by SSNIFF, Soest, Germany). Motor activity measurements were conducted in Polycarbonate cages with wire covers from Ehret, Emmendingen, Germany (floor area about 800 cm2) and small amounts of absorbent material (see above). The animals were housed in a fully air-conditioned room.
- Diet: Basic maintenance diet for mouse/rat, 9433 LL Meal, ad libitum, supplied by Eberle Nafag AG Gossau, Switzerland.
- Water: Drinking water from bottles ad libitum
- Acclimation period: 10 days (including randomization and ophthalomological examination).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 30 - 70% relative humidity
- Photoperiod (hrs dark / hrs light): 12 /12

IN-LIFE DATES: From: 2000-03-24 (first administration, males and females) To: 2000-06-24 (At the end of the administration (last administration 2000-06-23) period all animals were sacrificed after withdrawal of food for about 16 - 20 hours.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION:
The test substance was weighed and thoroughly mixed with a small amount of food. Corresponding amounts of food, depending on the dose group, were added to this premix in order to obtain the desired concentration. Mixing was carried out for 10 minutes in a Ruberg (EM 100) laboratory mixer.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analysis confirmed the correctness of the concentrations. The recovery rates were within a range of 95.7% -105.9% of the target concentrations.

Duration of treatment / exposure:

90 days

Frequency of treatment:

The test substance was administered daily via the diet.

Doses / concentrations

Remarks:

Doses / Concentrations:
0; 600; 3000; 15000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

10 rats per sex per dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Basis was a pre- test study with 5000 ppm and 15000 ppm in 3 male and 3 female Wistar rats for two weeks. Therefore 15,000 ppm was selected to be the highest concentration. This dose is equivalent to a test substance intake of > 1g/kg body weight (bw).

- Rationale for animal assignment: Before the start of the administration period, male and female rats were allocated to the test groups according to weight. The list of randomization instructions was compiled by a computer.

Positive control:

No positive control

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
Home cage observation: The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) from Mondays to Fridays and once a day (in the morning) on Saturdays, Sundays and public holidays.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter.
The following parameters were examined:
1. behavior during "handling"
2. fur
3. skin
4. posture
5. salivation
6. respiration
7. activity/arousal level
8. tremors
9. convulsions
10. abnormal movements
11. impairment of gait
12. lacrimation
13. palpebral closure
14. exophthalus
15. feces (appearance/consistency)
16. urine
17. pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were determind weekly.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water consumption was observed daily by visual inspection of the water bottles.

OPHTHALMOSCOPIC EXAMINATION: Yes
Prior to the start of the administration period the eyes of all animals were examined for any changes using an ophthalmoscope after administration of a mydriatic (Pharma Stulln GmbH, Germany). At the end of the study, the animals of the high dose group and controls were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Towards the end of the administration period, in the morning.
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: All surviving animals

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Towards the end of the administration period.
- Animals fasted: Yes
- How many animals: All surviving animals

URINALYSIS: Yes
- Time schedule for collection of urine: Towards the end of the administration period.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

NEUROBEHAVIORAL EXAMINATION: Yes
- Time schedule for examinations: Towards the end of the administration period.
- Dose groups that were examined: All animals were examined.
- Sensory activity, grip strength, motor activity, and reflex tests as part of Functional observational battery (FOB):

The animals were removed from the open field and subjected to following sensorimotor or reflex tests:
1. approach response
2. touch response
3. vision ("visual placing response")
4. pupillary reflex
5. winking reflex
6. pinna reflex
7. audition ("startle response")
8. olfaction
9. examination of catalepsy ("descending from box")
10. coordination of movements ("righting response")
11. behaviour during "handling"
12. vocalization
13. pain perception ("tail pinch")
14. grip strength of forelimbs
15. grip strength of hindlimbs
16. landing foot-splay test

Motor activity assessment:
Motor activity was measured on the same day as FOB was performed. The measurement was performed in the dark using the Multi-Varimex-System (Columbus Instruments Int. Corp., Ohio, USA) with 4 infrared beams per cage. During the measurement the animals were kept in Polycarbonate cages with absorbent material. The animals were put into the cages in a randomized order. The measurements started at about 1.30 p.m. and the number of beam interrupts was counted over 12 intervals, each lasting 5 minutes. Measurement did not commence at the same instant for all cages; the period of assessment for each animal started when the first beam was interrupted by pushing the cage into the rack (staggered start). Measurements ended exactly 60 minutes thereafter. During the measurements the animals received no food and no water.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see below in "any other information on materials and methods")
HISTOPATHOLOGY: Yes (see below in "any other information on materials and methods")

Other examinations:

Immediately after necropsy and organ weight determination the right testis and cauda epididymis were taken from all male animals. The following parameters were determined: Sperm motility, sperm morphology, sperm head count (cauda epididymis), sperm head count (testis).
Sperm morphology and sperm head count (cauda epididymis and testis) were evaluated for the control and for the highest dose group, only.
The ovaries were also fixed and examined.

Statistics:

(1) Statistics of clinical examinations
- Parameters examined: Food consumption, body weight, body weight change, food efficiency
Statistical test: The DUNNET test (two sided)
- Parameters examined: Feces, rearing, grip strength length hindlimbs, grip strength length hindlimbs, foot-splay test, motor activity
Statistical test: KRUSKAL-WALLIS test (two sided). Post test: Wilcoxon-test.

(2) Statistics of clinical pathology
- Parameters examined: Clinical pathology parameters, except differential blood count
Statistical test: KRUSKAL-WALLIS test (two-sided). Post test: MANN-WHITNEY U test (two sided).
- Parameters examined: Urinalysis, except volume, colour, turbidity and specific gravity
Statistical test: FISHER's exact test
- Parameter examined: Sperm analysis
Statistical test: WILCOXON-Test (one-sided)

(3) Statistics of pathology
- Parameters examined: Weight parameters
Statistical test: KRUSKAL-WALLIS test (two-sided). Post test: WILCOXON test.

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY
One male animal of the maximal dose group (15000 ppm) was sacrificed moribund on day 49 after showing the following distinct clinical symptoms:
A rapidly increasing palpable mass in the abdomen, a red discoloration of urine was diagnosed from day 28 onwards, piloerection from day 42 onwards and a severe reduction in the general condition of the animal on day 49.
The reason for the bad clinical condition was predominantly spontaneously occurring kidney lesions. This was not related to treatment.

One control female showed a "scrotum-like" palpable mass in the skin with missing vaginal opening from day 63 onwards. One male of group 1 showed red discoloured urine and anogenital region smeared with urine from day 77 onwards, one female of group 2 showed exophthalmia from day 77 onwards, and in one female of group 3 a tooth anomaly was seen from day 28 onwards. However, due to the isolated occurrence, all these findings were assessed as being incidental.


BODY WEIGHT AND WEIGHT GAIN
No significant change

FOOD EFFICIENCY
Food efficiency on day 84 was significantly increased in females of the dose groups that received 3000 and 15000 ppm of test substance.
However, due to the isolated occurrence, this was assessed as being incidental.

FOOD CONSUMPTION AND COMPOUND INTAKE (feeding study)
The approximate, mean daily test substance intake in mg/kg body weight over the entire study period was as follows:
No of test group / Concentration in the diet (ppm) / Mean daily test substance intake in mg/kg bw (males) / mean daily test substance intake in mg/kg bw (females):
1 / 600 / 51.7 / 59.3
2 / 3000 / 250.2 / 288.0
3 / 15000 / 1248.8 / 1452.1

OPHTHALMOSCOPIC EXAMINATION
No substance-related effects were obtained. All findings were spontaneous in nature and equally distributed between treated animals and controls.

HAEMATOLOGY
There are no treatment-related changes in the hematology parameters measured.

CLINICAL CHEMISTRY
Compound-related differences in clinical chemistry parameters were not evident at any dose level in either males or females.

URINALYSIS
No treatment-related changes were found in urinalyses of either sex.

NEUROBEHAVIOUR
Regarding the results obtained during open field observations, sensorimotor and reflex tests, all findings were assessed as being incidental, as they occurred in single animals, only, or were equally distributed between treated groups and controls.
Rearing was statistically significantly increased in males group 2 and 3 (3000 and 15000 ppm dose groups). However, the relevance of an increase as a relevant finding is doubtful. This is confirmed by the fact that no effect was seen in females, and no changes in motor activity were observed. This finding was therefore assessed as being incidental.
Regarding the overall motor activity and the single intervals, no substance-related effects were seen.

ORGAN WEIGHTS
Absolute weights:
The mean spleen weight was slightly although significantly decreased in females of the mid dose (3000 ppm test substance) group (- 34%). This was not regarded to be treatment-related.
The other mean absolute weight parameters did not show significant differences when compared with the control group.
Relative weights (related to terminal body weight):
The mean weight of the liver was significantly increased in males (+ 7%) and in females (+ 10%) of the high dose (15000 ppm teat substance) group.
The mean weights of testes (+ 9%, high dose group) and heart (females, low dose group, + 15%) were significantly increased, whereas the mean weight of the spleen (mid dose group in females) was significantly decreased (- 31%). This was not regarded to be treatment-related.
The other mean relative weight parameters did not show significant differences when compared with the control group.

GROSS PATHOLOGY
The majority of the few gross lesions noted occurred in a high dose male rat that was killed prematurely 49 days after start of treatment. They consisted of dark red discoloration of the lungs, severely enlarged kidneys that revealed a granular surface, slight dilation of the ureters and moderately reduced organ sizes of seminal vesicles and prostate gland. They are interpreted to be of spontaneous origin and to have no relationship to treatment.
A few other gross lesions were noted in the glandular stomach (erosion/ulcer and hyperemia), liver (cyst), kidneys (cyst), uterus and vagina (dilation), thymus (reduced organ size), iliac lymph nodes (enlarged) and skin (sparse hair). With one exception (uterine dilation in two females of the low dose group), they were all single observations and they are all interpreted to have no relationship to treatment.


HISTOPATHOLOGY: NON-NEOPLASTIC
No treatment-related microscopic findings were detected in any of the organs investigated.
No morphologic correlate was obtained that may account for the significantly increased mean relative weight of the liver in males and in females of the high (15000 ppm test substance) mid (3000 ppm) dose group.
Same is true for the significantly increased mean relative weight of the testes of the animals of the high dose group.
Finally, no microscopic finding was obtained in the high dose group that may account for the significantly decreased mean absolute and relative spleen weights of females of the mid dose group or the significantly increased mean relative heart weight of female rats of the low dose group.
All microscopic findings recorded were either single observations, or they were recorded at a low incidence, or they occurred in control animals only, or at comparable incidence and graded severity in control and high dose males and/or females.

OTHER: Sperm analysis
Sperm examinations revealed no test compound-related changes.

Effect levels

Dose descriptor:

NOAEL

Effect level:

>= 15 000 ppm

Sex:

male/female

Basis for effect level:

other: >= 1249 mg/kg bw/d (males) >= 1452 mg/kg bw/d (females)

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

No remarks