Benzoic acid, 4-[[[4-[(1-oxo-2-propenyl)oxy]butoxy]carbonyl]oxy]-, 2-methyl-1,4-phenylene ester

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:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Reference 3

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

only male animals tested

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

- Physical state: powder / grey-white
- Analytical purity: 95.8% (per weight; HPLC)
- Lot/batch No.: ZD 1151/ 31 (production: 03-Dec-1997)
- Stability under test conditions: the stability of the test substance in olive oil DAB 10 for a time period of 4 hours was confirmed
by analysis. The homogeneity of the test substance preparations was provided by stirring. Additionally the homogeneity was confirmed by analysis.
- Storage condition of test material: refrigerator, protected from light.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: [CRL:WI (GLXIBRL/HAN) IGS BR] rats were supplied by Charles River, Sulzfeld, Germany, on March 7, 2000
- Age at study initiation: 42±1 days. Only animals free from clinical signs of disease were used for the study.
- Weight at study initiation: the males were in the range of 118.1 - 152.9 g (group mean: 137.9 g) and the females were in the range of 96.2 - 123.8 g (group mean: 113.8 g)
- Housing: The animals were single-housed in fully air-conditioned rooms in Stainless steel wire mesh cages Stainless steel wire mesh cages, type DK-lll (Becker & Co., Castrop-Rauxel, FRG; floor area about 800 cm2). Motor activity measurements were conducted in Polycarbonate cages with wire covers (Ehret, Emmendingen, FRG; floor area about 800 cm2) and small amounts of absorbent material. The animals were identified clearly by ear tattoo. The animal room was completely disinfected using a disinfector (AUTEX, fully automatic, formalin-ammonia-based terminal disinfector). The floor and the wails were cleaned once a week. The cleansing liquid used was water containing about 0.1% lncidin (Henkel, Düsseldorf, FRG).
- Diet, ad libitum: basic maintenance diet for mice and rats (9433 LL meal, from Eberle Nafag AG, Gossau, Switzerland). The feed used in the study was assayed for chemical and microbiological contaminants. In view of the aim and duration of the study the contaminants occurring were seen as not likely to influence the results.
- Water, ad libitum: drinking water (from water bottles). In view of the aim and duration of the study the contaminants occurring were seen as not likely to influence the results.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Central air-conditioning guaranteed a range of 20 - 24 degrees
- Humidity (%): 30 - 70 %
- Photoperiod (hrs dark / hrs light): 12/12 hours (6.00 am - 600 pm/ 6.00 pm - 6.00 am)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was administered as a suspension in olive oil. To prepare the suspensions, the appropriate amount of test substance was weighed, depending on the dose group, filled up to the desired volume with olive oil and mixed using a high speed sonicator. During the administration the test substance preparations were kept homogenous with a magnetic stirrer. The test substance preparations were prepared daily.

VEHICLE
- Justification for use and choice of vehicle (if other than water): as the test substance is not stable in the diet and could not be administered in water, olive oil was selected as vehicle.
- Concentration in vehicle: 0.8, 4 and 20 g/100 ml respectively for 20, 200 and 1000 mg/kg bw dose groups
- Amount of vehicle (if gavage): 5 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance at room temperature in the vehicle was verified by HPLC reanalysis.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
40, 200 and 1000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

10 (in 0 and 1000 mg/kg bw dose groups) and 5 (in 40 and 200 mg/kg bw dose groups)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In a pretest study the test substance was administered to groups of 3 male and 3 female rats at a dose level of 1000 mg/kg bw/day for 1 week. Food consumption, body weight, clinical signs and macroscopic findings were recorded. No signs of toxicity were observed. Thus, the following dose levels were selected for the present study: 1000 mg/kg bw: as high dose (the highest dose level to be tested in a limit-test) for non-toxic substances; 200 mg/kg bw (as mid dose) and 40 mg/kg bw (as 10w dose).
- satellite groups: 5 male and 5 female animals in the control and in the highest dose groups
- Rationale for selecting satellite groups: recovery examination
- Post-exposure recovery period in satellite groups: 14 days

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption: Yes, 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: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes, see below

HAEMATOLOGY: Yes, carried out towards the end of the administration period

CLINICAL CHEMISTRY: Yes, carried out towards the end of the administration period

URINALYSIS: Yes, carried out towards the end of the administration period

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: detailed clinical examinations in an open fleld observation (OFO; following parameter were examined, including some clinical examinations: behavior during handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmus, feces (appearance/consistency), urine, pupil size) were conducted prior to the start of the administration period and weekly thereafter. A functional observational battery (FOB; posture, tremor, convulsions, abnormal movements, impairment of gait, general observations [all other abnormal findings]) and measurement of motor activity was carried out after 4 weeks of treatment
- Dose groups that were examined: all groups
- Battery of functions tested: sensorimotor or reflex tests: approach response, touch response, vision (visual placing response), pupillary reflex, winking reflex, pinna reflex, audition (startle response), olfaction, examination of catalepsy (descending from box), coordination of movements (righting response), behavior during handling, vocalization, pain perception (tail pinch), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test

OTHER: the animals were observed for mortality at least once daily.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
All animals were assessed by gross pathology, followed by histopathological examinations.

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY
No animal died during the study.
Slight salivation was seen in two high dose females prior to daily gavage between days 12 and 20 and in one high dose female after gavage on day 14. Because this even was reversible during treatment and its occurrence limited to very few animals of only one sex, these findings were assessed as being incidental rather than representing a toxicologically relevant finding.
BODY WEIGHT AND WEIGHT GAIN
No substance related effects were obtained. The significantly increased values of body weight change in low dose females on days 14 and 21 were assessed as being incidental, due to the lack of a dose-response relationship.

FOOD CONSUMPTION
No substance related effects were obtained. The significantly increased value of group 3 males on day 34 (e.g. during the recovery period) was assessed as being incidental.

FOOD EFFICIENCY
No substance related effects were obtained. The significant increased values of high dose females on day 14 and the significantly decreased values of low dose males on day 27 were clearly incidental in nature.

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

CLINICAL CHEMISTRY
Compound-related differences in clinical pathology 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
- Functional observational battery:
some deviations from zero values were obtained during home cage observations (animal not walking during the observation period on day 26 and 27), open field observations (no defecation/urination during the observation period), sensorimotor tests/reflexes (no reaction to approach response, no reaction to touch response). Grip strength of forelimbs was also significantly decreased in males of group 1. Due to the lack of a dose-response relationship, these effects were assessed as being incidental.
- Assessment of results during FOB: as all findings were equally distributed between treated groups and control or as only single animals were affected, they were clearly incidental in nature.
- Motor activity measurement: regarding the overall motor activity (summation of all intervals) no significant deviations were seen in treated males or females.
Comparing the single intervals with the control groups, there was one isolated significantly increased value in high dose females. Due to the isolated occurrence, this was assessed as being incidental.

GROSS PATHOLOGY AND ORGAN WEIGHTS
- Absolute weights: the mean terminal body weight and the mean weight of the kidneys was slightly although significantly increased in females of the 10w dose group. This was regarded incidental. The other mean absolute weight parameters of the animals of the main groups (FI) did not show significant differences when compared with the concurrent control group. Accordingly, the mean absolute weight parameters of the animals of the recovery groups did not show significant differences when compared with the concurrent control group.
-Relative weights (related to terminal body weight): the mean relative weight parameters (when related to terminal body weight) of the animals of the main groups did not show significant differences when compared with the concurrent control group. Again, the mean relative weight parameters (when related to terminal body weight) of the animals of the recovery groups did not show significant differences when compared with the concurrent control group.
- Gross lesions: only a few gross lesion were noted in the glandular stomach (black erosion/ulcer in the mucosa of two males and three females of the high dose and one female of the control group) and in the liver (focal constriction in a mid dose male and a high dose female rat). In the recovery groups only a few gross lesion were noted in the liver (focal constriction in a high dose female and a small beige focus in a high dose male). They were both regarded incidental and unrelated to treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
- The grossly noted erosion/ulcer in the mucosa of the glandular stomach was not confirmed histopathologically in all cases. However, they should be comparable to the same gross lesion that was confirmed histopathologically in two high dose females as minimal superficial erosion(s) in the mucosa. All other gross lesions were correlated with a meaningful microscopic finding. They were all regarded to be of spontaneous origin and not related to treatment.
- Microscopic findings were only noted in the Iungs (focal lymphoid cell infiltration), heart (focal necrosis), liver (capsular scar), kidneys (focal calcification in the cortico-medullary area), adrenal cortex (extracortical nodule) and uterus). They were either single observations, or they occurred in control animals only, or they were recorded at comparable incidence and graded severity in control and high dose males and/or females.
- After a 4-week application period, there was no indication of a morphologic affection of the organs of the central or peripheral nervous system, the reproductive system, and the immune system.

Recovery groups:
- Of the two grossly noted lesions, only the focal constriction in the liver of a high dose male was confirmed histopathologically. The beige focus in the liver of a high dose male lacked a microscopic correlate. However, both findings were anyhow regarded to have developed unrelated to treatment. No other microscopic investigations were performed.
- After a 2-week recovery period, there was no indication of an affection of the organs of the central or peripheral nervous system, the reproductive system, and the immune system.

OTHER FINDINGS
There were statistically significant intergroup differences in the results of clinical pathology testing. These deviations are marginal, incidental or inconsistent, when compared with the other sex, or lack dose-response relationship. Accordingly, these findings are considered to be of no toxicological significance.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no relevant toxicologically finding observed.

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Conclusion:

- Slight salivation was observed in the high dose females and in one high dose female after gavage, but was reversible / limited to very few animals of only one sex, and therefore assessed as being incidental rather than representing a toxicologically relevant finding.

- There were no treatment related weight changes, gross lesions or microscopic findings in the animals of the main groups or in the animals of the recovery groups. All gross lesions and all microscopic findings recorded in the animals of the main groups and of the recovery groups were either single observations, occurred in control animals only, or recorded at comparable incidence and graded severity in control and high dose males and/or females and hence were all regarded to be unrelated to treatment.

- There was no indication of organs effects of the central or peripheral nervous system, the reproductive system, and the immune system, neither when looking on the weight parameters determined, nor when reflecting the gross lesions or microscopic findings in the organs of the nervous system (brain, spinal cord, sciatic nerve), the reproductive system (testes, ovaries, prostate gland, uterus), or the immunocompetent organs (spleen, thymus, mesenteric and mandibular lymph nodes, Peyer's patches of the jejunum).

- Thus, the no observed adverse effect level (NOAEL) under the conditions of this study was 1000 mg/kg bw per day.