Imidazole

Endpoint summary

Description of key information

In a 90-day oral gavage study Wistar rats were treated with imidazole at dose levels of 0, 20, 60 and 180 mg/kg bw/d. Liver and kidney were identified as the target organs. A NOAEL of 60 mg/kg bw/d was derived.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2001-11-19 to 2002-03-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was conducted in accordance with the OECD Guideline 408 and GLP.

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, 67056 Ludwigshafen, Germany

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Male and female rats (CrlGlxBrlHan:WI), only animals free from clinical signs were used for the study.
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 34 +/- 1 day
- Housing: housed singly in type DK III stainless steel wire mesh cages supplied by Becker & Co., Castrop-Rauxel, Germany
- Diet (ad libitum): Ground Kliba maintenance diet rat/mouse/hamster, meal, supplied by Provmi Kliba SA, Kaiseraugst, Switzerland
- Water: from water bottles (ad libitum)

ENVIRONMENTAL CONDITIONS
- housing: housed in a fully air-conditioned room
- Temperature (°C): 22-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12

IN-LIFE DATES: From: 2001-11-13 To: 2002-02-21

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

VEHICLE
- double distilled water
- Concentration in vehicle: 0, 0.20, 0.60, 1.80 g/100 mL
- Amount of vehicle (gavage): 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration control analyses were carried out in samples of all concentrations before the start of the administration period and with the last test substance preparation.
The stability of the test substance in double distilled water over a period of 8 days at room temperature was tested prior to the start of the
study. Additional, not analyzed, reserve samples were taken every 4 weeks.

Duration of treatment / exposure:

90 d

Frequency of treatment:

The test substance was administered daily by gavage using 3 and 5 ml syringes (Fa. Beckton & Dickinson) for about 13 weeks.

Remarks:

Doses / Concentrations:
0, 20, 60, 180 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

10 male/10 female per sex and dose group

Control animals:

yes, concurrent vehicle

Details on study design:

TEST ORGANISM
A total of 80 animals, 10 rats per dose/sex were used, age 42+/-1 d at start of administration period. Body weight ca. 150g for male
and 120g for female rats. Animals were housed individually.

OVERALL EXPERIMENTAL DESIGN
Animals were given imidazole daily by gavage for 3 months. At the end of the administration period the animals were sacrificed. All surviving animals were sacrificed after a fasting period (withdrawl of food) for at least 16 hours. Clinical examinations, clinical chemistry, gross pathological and histopathological changes were examined in various organs including testes as prescribed by OECD 408. Ophthalmological and
behavioural (functional observation battery, motor activity) examinations were performed. Additionally, sperm parameters were examined.

ADMINISTRATION
Dose levels were selected based on the results of a 4-week range finding study (data filed under 31S0694/00094 but not reported).
Imidazole was dissolved in bidistilled water. Dose volume was 10 ml/kg bw.

Observations and examinations performed and frequency:

The animals were examined for signs of toxicity or mortality at least once a day after treatment. Detailed clinical examinations in an
open field were conducted prior to the start of the administration period and weekly thereafter. A functional observational battery (FOB)
and measurement of motor activity were performed towards the end of administration. Ophthalmological examinations were carried out in all animals before and in control and high dose animals at the end of the administration period. Vaginal smears for estrus cycle determination
of all female animals were prepared and evaluated each day during the last 4 weeks of the study. Clinicochemical, hematological
examinations and urinalyses were carried out towards the end of the administration period. All animals were assessed by gross pathology,
followed by histopathological examinations. Food consumption, water consumption and body weight were determined weekly.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Other examinations:

Functional observational battery (FOB )
A functional observational battery was performed in all animals at the end of the administration period starting at about 10.00 a.m.
The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field
observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of
severity, if applicable. The observations were performed at random.

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-VarimexSystem(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 2.00 p.m. and the number of beam interrupts were 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.

Estrus cycle determination
Vaginal smears for cycle determination were prepared in the morning and evaluated according to the timetable. The differentiation
was conducted according to following stages:
Code Cycle stage Appearance in vaginal smear
1 Diestrus Leukocytes, few nucleated epithelial cells
2 Proestrus Single leucocytes, many nucleated and few horny epithelial cells
3 Estrus Only horny epithelial cells
4 Metestrus Leucocytes, some horny epithelial cells

Sperm analysis
Sperm parameters were determined in all males at termination. Right testis and cauda epididymis were taken and weighed. Sperm count in testis and epididymis, motility and sperm morphology were determined.

Statistics:

Evaluation of clinical data (body weight, bw changes, food and water intake, food efficiency) was done using DUNNETT's test.
Non-parametric one-way analysis using KRUSKALL-WALLIS test was used to evaluate FOB and motor activity data.
If p-values were equal or less than 0.05, WILCOXON-test was also used.
Non-parametric one-way analysis using KRUSKALL-WALLIS test was also used to evaluate clinical pathology parameters.
FISHER'S exact test was included to evaluate some of the urinalysis parameters.
WILCOXON-test was used for evaluation of sperm parameters.
Non-parametric one-way KRUSKALL-WALLIS test and WILCOXON-test was also used to evaluate significant differences in absolute
and relative organ weights.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food efficiency:

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CHEMICAL ANALYSIS
Stability of TS solutions over a period of 8 days was confirmed. Concentration control analyses yielded 92-105% of the nominal values. 
These results demonstrated the correctness of the concentrations of Imidazol in doubly distilled water. No homogeneity analysis were
performed, because the test substance preparations were true solutions.

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One male animal of test group 1 (20 mg/kg bw/day) showed lateral position, salivation, red discoloured urine, red discharge in right eye,
slight pulmonic respiratory sounds and moderate labored respiration on day 86 and was therefore sacrificed on this day. Two males of test group 1 showed alopecia on different parts of the body on a few study days. one male showed piloerection, red crusted eyes and slight
reduced general condition from day 88 till the end of the study.

One male rat of the control group (0 mg/kg bw/day) showed exophthalmia (left eye) from day 16 onwards, moderate skin paleness from day 28, oblique head posture and piloerection from day 37, piloerection from day 38 and died prematurely on day 40.

One female animal of dose group 3 (180 mg/kg bw/day) showed slight salivation on day 90 and 91 as well as red discoloured urine from day17 till 21. Two females of this dose group showed slight to moderate urine-smeared anogenital region on a few days.

All above-mentioned findings were assessed as being incidental and not substance related, because they were equally distributed between control and treated animals and reflected therefore the range of biological variation.

BODY WEIGHT
In male animals of the low dose group (20 mg/kg bw/day) the body weight change was significantly increased (18 %), only on day 28.
In females, body weight change was increased in dose groups 1 to 3 on a few days of the study, with a statistically significantly maximum of 35 % on day 7 in dose group 2. These impairments were assessed as spontaneous in nature and therefore not substance-related.

FOOD CONSUMPTION and EFFICIENCY
Female animals of dose group 1 showed a statistically significant decrease in food consumption (-7.9 %) on study day 14, only.
This single finding is purely accidental and therefore not substance related.
On a few occassions, food efficiency differed statistically significantly in all treatment groups from control (increased/ decreased values).
This was not assessed as being substance related.
REPRODUCTIVE FUNCTION: ESTROUS CYCLE
In the estrus cycle determinations conducted from day 63 to day 91, no substance related effects were obtained.

REPRODUCTIVE FUNCTION: SPERM MEASURES
There occured no treatment-related changes in the sperm parameters measured at all dose levels.

ORGAN WEIGHTS
Absolute weights
The mean liver weight was significantly increased in females of the high dose group (+6.9%).
The mean kidney weight was significantly increased in males of the high (+ 12.2%) and mid dose groups (+13%) and in females of the high dose group (+9.9%).
The mean heart weight was incidentally although significantly increased in females of the mid dose group (+8.7%).
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 liver weight was significantly increased in males (+7.5%) and in females (+2.6%) of the high dose group.
The mean kidney weight was significantly increased in males of the high dose group (+9.1%).
The mean weights of heart and thymus were incidentally although significantly decreased in males of the low dose group (-5.5 or -31.8%, respectively).
The other mean relative weight parameters (when related to terminal body weight) did not show significant differences when compared with
the control group.

GROSS PATHOLOGY
In animals killed as scheduled at study termination, gross lesions were noted in the kidneys (clay colored discoloration and granular surface in control male #5) and thymus (organ size reduced in low dose male #17). They were single observations and they did not show an obvious relationship to treatment.
Male control animal #6 died intercurrently 40 days after start of treatment. The postmortem state of the animals was bad (i.e. it showed clear signs of autolysis). Spontaneous gross lesions were noted in the lungs (dark red discoloration), kidneys (clay colored discoloration and
granular surface) and eyes (unilateral exophthalmia).
Male rat #16 of the low dose group was killed for human reasons 86 days after start of treatment. Gross lesions unrelated to treatment werenoted in the esophagus (abscess) and mandibular lymph nodes (moderately enlarged, unilateral).

HISTOPATHOLOGY
(Four numbers seperated by dashes indicate the number of microscopic findings noted in the control group and low, mid and high dose groups).
Not all of the gross lesions could be correlated with a meaningful microscopic finding: By animal #6 of the control group, in the eyes, no correlate was detected that may account for the grossly noted exophthalmia, and in animal # 6 (male, low dose group) the unilaterally enlarged mandibular lymph node was without a morphologic correlate. All other gross lesions were matched with a meaningful histologic finding.
However regardless of whether or not they had a microscopic correlate, all the gross lesions in animals killed at study termination were considered to have developed spontaneously and unrelated to treatment.
Treatment related microscopic findings were detected in the kidneys. They consisted of a slight or moderate accumulation of alpha2-microglobulin (1/1/1/10) in the epithelia and tubular lumina of the proximal tubules of the renal cortex, exceeding the physiologic minimal or slight focal accumulation of alpha 2-microglobulin at that sight (7/8/9/0). The accumulation of alpha2u-microglobulin was regarded to have caused significanly increased mean absolute and relative kidney weights in males of the high dose group.
By contrast the significantly increased mean absolute kidney weight in mid dose males was regarded to be fortuitous, as there was no dose response relationship, a slight although not significant increase in the mean terminal body weight (+5.4%) and hence no such observation inthe more reliable relative weight, and no histopathological correlate.
Treatment related microscopic findings were also detected in the liver. They consisted of minimal or slight centrolobular hypertrophy of the liver cells in males (0/0/0/9) and in females (0/0/0/2). This finding correlated with the recorded signifiantly increased mean absolute (females) and relative (males and females) liver weights. No further degenerative liver lesions were noted, histopathologically.

Male control animal #6 died intercurrently 40 days after start of treatment. Although the postmortem state of the animals was bad (i.e. it showed advanced post mortem autolysis in many organs), major disease and cause of death were identified to be related to severe chronic
progressive nephropathy (CPN), associated with other morphologic signs indicative for a long lasting history of illness like degeneration in atrophy of several organs (e.g. epididymides, heart, thymus prostate gland, seminal vesicles and coagulation glands).

Male rat #16 of the low dose group was killed for human reasons 86 days after start of treatment due to the consequences of a gavage error with perforation of the esophagus and associated inflammatory reactions in neighboring organs (e.g. sublingual glands, thyroid glands, thymus and trachea).
Indications of a comparable accidental perforation of the esophagus were also noted in male animal #17 of the low dose group (e.g. inflammatory reaction and lymphocyte depletion in the thymus), however, this animal was killed as scheduled at study termination without any
further relevant microscopic findings.

With the exception of the kidneys in high dose male rats, histopathology failed to correlate the other significantly increased organ weights with a meaningful microscopic finding. A relationship of these weight alterations to treatment were, however, denied for the following reasons:

1. slightly although significantly increased mean absolute kidney weight in high dose females (+9.9%).
- no dose-relationship
- slight, although not signficant increase of the mean terminal body weight (+4.4%) and, hence, no such observation in the more reliable relative weight
- no morphologic correlate

2. significantly increased mean absolute heart weight in females of the mid dose group and signficantly decreased mean relative weights of heart and thymus in males of the low dose group, because of:
- no dose-response relationship
- lack of a microscopic correlate in the respective organs of the high dose group.

All other microscopic findings recorded were either single observations, or they were recorded at low incidence, or they occured in control animals only, or at a comparable incidence and graded severity in control and high dose males and/or females.

OPTHALMOSCOPIC EXAMINATION
No substance-related effects noted.

HAEMATOLOGY
No substance-related effects noted

CLINICAL CHEMISTRY
No treatment-related changes except decreased chloride and serum globulins in animals of both sexes at 180 mg/kg/d, and decreased protein and albumin values in high dose females.

URINALYSIS
At the end of the study urine sediments of high dose animals revealed increased numbers of transitional epithelial cells in both sexes.
Most of these cells were degenerated. No other treatment-related changes were noted.

FUNCTIONAL OBSERVATIONAL BATTERY
Deviations from "zero values" were obtained in all animals. However, as all findings were equally distributed between treated groups and controls, were without a dose-response relationship or occured in single animals only, the observations were considered to be incidental.

MOTOR ACTIVITY MEASUREMENT
Regarding the overall motoractivity (summation of all intervals) no substance-related effects were obtained. The single intervals were statistically significantly increased in high dosage males in interval 8, 10 and 12 and decreased in interval 1. The isolated occurrences of the mentioned observations were regarded to have been fortuitous and could not be interpreted as substance related.

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: treatment related adverse effects at 180 mg/kg bw/d; the lesions identifying liver and kidney as the target organs

Critical effects observed:

not specified

Executive summary:

Imidazole was tested in a thorough 90-d study according to OECD 408 including several functional tests (FOB, motor activity, ophthalmological examinations, sperm parameters). Liver and kidney were identified as target organs. At the high dose (180 mg/kg bw/d) significant and substance-related changes noted consisted of centrolobular liver cell hypertrophy in both sexes (9/10 males and 2/10 females); diffuse a2u-microglobulin accumulation in proximal tubules of the renal cortex in male rats; increased absolute (females) and relative (males and females) mean liver weight; increased absolute and relative mean kidney weight in male rats; and blood chemistry changes (decreases in serum protein and albumin in females, and in chloride and globulins in both sexes). No other substance-related finding was noted. This includes the histopathological examination of reproductive organs of both sexes, sperm parameters and estrus cycle examined as an indicator of the integrity of the male and female reproductive organs. Since the substance-related findings described above were limited to the high dose group animals and absent in low dose and intermediate dose group animals, the no observed adverse effect level (NOAEL) for both sexes was 60 mg/kg bw/d in this study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

60 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Subchronic effects of imidazole were determined in a 90-day study in Wistar rats according to OECD TG 408. This study aimed at the examination of systemic and specific organ toxicity, ophthalmologic effects, effects on male and female reproductive organs, and effects on behaviour and sensi-motor capabilities which were examined in a series of tests delineated as the Functional Observation Battery (FOB). Imidazole was given daily by gavage dissolved in water at 20, 60 and 180 mg/kg bw/d. Liver and the male kidney were identified as target organs in the animal groups receiving 180 mg/kg bw/d as substantiated by significantly increased relative liver weights in males (+7.5 %) and females (+2 .6 %) which correlated with minimal to slight centrilobular liver cell hypertrophy in males (9/10 animals affected) and females (2/10), and by a significant increase of absolute and relative kidney weight in high-dose males that was accompanied by an accumulation of alpha 2-microglobulin in the epithelia and lumina of the proximal tubules of the male rat renal cortex . The alpha 2-microglobulin was detected by Mallory Heidenhain staining technique and specificity for alpha 2-microglobulin could be demonstrated by immunohistochemical staining (BASF SE, 2004). The accumulation of microglobulin is considered as a rat-specific phenomenon and has no toxicological relevance for humans. Additionally, significant changes in parameters of blood chemistry were noted in high dose animals as substantiated by decreased serum globulin and chloride in male rats, and total protein, albumin, globulin, and chloride in females. No other substance-related effect was noted in the 90-d study at 180 mg/kg bw/d; i.e. mortality, clinical observation for signs of toxicity, bodyweight, bodyweight development and food consumption, clinical chemistry other than noted above, pathology and histopathology of the numerous organs examined were not affected. Also, no effects were noted during ophthalmologic examinations or the FOB tests. Male and female reproductive organs were not affected (including histopathology), as were sperm quality parameters (sperm number, motility, and morphology were determined in testis, epididymides and estrus cycle). No substance-related effect was noted at the intermediate and at the low dose level. Therefore the no observed adverse effect level (NOAEL) was 60 mg/kg bw per day in both sexes under the conditions of this study (BASF SE, 2002).

Red blood cells were additionally affected in 28-d experiments. Female rats receiving 125 mg/kg bw per day or more and male rats receiving 500 mg/kg bw/d were affected (BASF AG, 1976). The NOAEL was approximately 62.5 mg/kg bw per day. The effect on red blood cells was, however, not confirmed in the 90-day guideline study when rats received up to 180 mg/kg bw per day.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver; urogenital: kidneys

Justification for classification or non-classification

In conclusion the administration of imidazole to rats by gavage for 90 days caused treatment-related findings only at the highest dose level (180 mg/kg bw/d), the lesions identifying the liver and kidneys as the target organs. The NOAEL was 60 mg/kg bw/d and is thus comparable with the NOAEL found in the 28 -day rat study using the same route of administration. Based on the available data imidazole is not subject to classification for repeated dose or specific target organ toxicity according to Regulation 1272/2008/EC.