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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 Oct 1987 - 08 Feb 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report date:
1989

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted 2018
Deviations:
yes
Remarks:
methodological deficiencies (please refer to "Principles of methods if other than guideline")
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted 1981
Deviations:
no
Principles of method if other than guideline:
Rats were housed individually instead of in groups per sex, phytoestrogens were not determined in the diet, no lipid analysis was done in blood, no weight was determined for epididymides, prostate, uterus, ovaries, thymus, pituitary gland, thyroid and heart, brain histopathology was not specified, mammary glands were not examined in histopathology, T4, T3 and TSH were not assessed, sensory reactivity was not investigated
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
428-040-8
EC Name:
-
Cas Number:
138261-41-3
Molecular formula:
C9H10ClN5O2
IUPAC Name:
2-chloro-5-{[2-(nitroimino)imidazolidin-1-yl]methyl}pyridine

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
common species, used often for toxicological studies, historical data are available
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Winkelmann, Borchen, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 5-6 weeks
- Weight at study initiation: 64 - 102 g (males), 63 - 91 g (females)
- Fasting period before study: not applicable
- Housing: individually caged in Makrolon7 type II cages on low-dust wood granulate (supplied by
Ssniff Spezialdiaten GmbH, Soest, Germany) during trial period
- Diet: acclimatisation period: AltrominR 1324 pellets, study period: AltrominR 1321 meal, supplied by Altromin GmbH, Lage, Germany, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 10 days

DETAILS OF FOOD AND WATER QUALITY: food was regularly checked for contaminants and spot-checked, tap water was of drinking quality (Drinking Water Statute of 22.5.86, Federal Office Gazette No. 16, issued on 28.5.86, page 760, with effect from 1.10.86)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): approx. 50
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 28 Sep 1987 To: 08 Feb 1988

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet: weekly
- Mixing appropriate amounts with: AltrominR 1321 meal (supplied by Altromin GmbH, Lage, Germany), supplemented with 1% groundnut oil to prevent dust formation
- Storage temperature of food: room temperature
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Both concentration of the acitve substance in the feed as well as homogenity were tested before, during and at the end of the study. The first analysis determined the concentrations to be 164, 618 and 2380 ppm (Oct 1987), the last 153, 606 and 2540 ppm (Jan 1988), the mean was 129, 599 and 2410 ppm. During analytical checks on the target contents, a content of only 60 % was detected midway through the study in the 150 ppm group. No error could be found in the records from when the feed mix was weighed out. Analysis of 2 reserve samples prior to this point and 2 thereafter yielded correct target contents. A potential source of error for this low finding could not therefore be identified. Evaluation of the results for this dose group is thus not questionable.

Homogenity was also verified.
Duration of treatment / exposure:
13 weeks (up to 96 days, minimum 91 days)
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
150 ppm
Remarks:
corresponding to 14.0 and 20.3 mg/kg bw/day for males and females, respectively
Dose / conc.:
600 ppm
Remarks:
corresponding to 60.9 and 83.3 mg/kg bw/day for males and females, respectively
Dose / conc.:
2 400 ppm
Remarks:
corresponding to 300.2 and 422.2 mg/kg bw/day for males and females, respectively
No. of animals per sex per dose:
10 (plus 10 in recovery group)
Control animals:
yes
Details on study design:
- Dose selection rationale: A pilot feeding study was performed administering 0, 120, 600 or 3000 ppm to Wistar rats for 14 weeks. At 3000 ppm, weight reduction was marked, evidence of liver damage and enlarged testicular tubules occurred. At 600 ppm, body weight was slightly reduced. The NOAEL was set to 120 ppm. Therefore, concentrations of 150, 600 and 1000 ppm were selected for this study
- Fasting period before blood sampling for clinical biochemistry: no
- Satellite groups: a recovery group of 10 animals/sex was included in the study for the control and high dose group
- Post-exposure recovery period in satellite groups: 4 weeks
Positive control:
not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at leat twice daily (once at weekends and bank holidays)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least twice daily, detailed examinations were carried out weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: 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: No

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule: at start of the study and prior to necropsy (before collection of blood sample)
- Parameters checked: pupillary reflex of both eyes, area around the eye and the anterior regions of the eye were assessed, the pupils were dilated with Mydriaticum-Roche eye-drops and the refractive sections of the eye and the fundus were examined (using an indirect ophthalmoscope)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 5 and 14 weeks (all animals) and in Week 17 for the satellite groups
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: No
- How many animals: all animals
- Parameters checked: differential blood count, erythrocyte count and morphology, haemoglobin concentration, haematocrit, leucocyte count, MCH (mean corpuscular haematology), MCHC (mean corpuscular haemoglobin concentration), MCV (mean corpuscular cell volume), platelet count, thromboplastin time, reticulocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 5 and 14 weeks (all animals) and in Week 17 for the satellite groups
- Animals fasted: No
- Anaesthetic used for blood collection: No (for glucose determination), yes for all other parameters (ether)
- How many animals: all animals
- Parameters checked: alkaline phosphatase, aspartate-aminotransferase, alanine aminotransferase, lactate dehydrogenase (LDH), creatine kinase, glucose, albumin, bilirubin, cholesterol, creatinine, total protein, urea, triglycerides, phosphate, calcium, potassium, sodium, chloride, cholinesterase activity (in plasma, erythrocytes and brain (for brain, only 5 of 10 animals per groups were tested)

PLASMA/SERUM HORMONES/LIPIDS: Yes (see above)

URINALYSIS: Yes
- Time schedule for collection of urine: after 14 weeks (all animals) and in Week 17 for the satellite groups
- Metabolism cages used for collection of urine: not specified
- Animals fasted: Yes, during collection (over a period of 16 h)
- Parameters checked: blood, bilirubin, glucose, ketone bodies, pH, protein, urobilinogen, sediment, specific gravity (density), volume, creatinine, protein

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Organ weight was reported for brain, testes (both), liver, spleen, kidneys (both) and adrenal glands (both)

HISTOPATHOLOGY: Yes
Organs/tissues that were fixed in Bouin's solution: aorta, eyes (one or both), eyelids, caecum, colon, duodenum, femur, brain, bladder, Harder's glands, ureter, urethra, skin, heart, testes, pituitary, ileum, jejunum, larynx, bone marrow (in femur and sternum), head, liver, lymph nodes (mesenteric and mandibular), stomach, mammary glands, spleen, muscles (thigh), epididymis, adrenal glands, sciatic nerve, optic nerve, kidneys, oesophagus, tattooed auricles, ovaries, oviduct, pancreas, prostate, rectum, residual intestine, spinal cord (cervical, thoratic and lumbar), seminal vesicle, thyroid, salivary glands, sternum, thymus (if present), trachea, extraorbital lacrimal glands, uterus, vagina, tongue
Organs that were fixed in 10% buffered formaldehyde solution: one lobe of the liver and a lung
Other examinations:
None
Statistics:
Arithmetic group means, standard deviation and, sometimes, upper and lower confidence limits (1 - a = 95% and 1 - a = 99%) were calculated for medical laboratory tests, measurement, body weight, feed and water intake and organ weights. Groups were compared using the significance test (U test, two-sided) according to Mann, H.B. and Whitney, D.R. and after Wilcoxon, F. (significance was set to p<0.05).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
not applicable
Mortality:
no mortality observed
Description (incidence):
not applicable
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 600 ppm: slight reduction in body weight in males (8%)
- 2400 ppm: statistically significant lower body weight (14-16%, males and females) until the end of the study period, not reversible during the recovery period

Summarized data can be found in Attachment 1 in the attached background material.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- 2400 ppm: 33% (males) to 42 % (females) increased food consumption relative to body weight, not reversible during the recovery period (males and females still consumed more feed than the control animals in the last week of the follow-up period)

Summarized data can be found in Attachment 2 in the attached background material.
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
not applicable
Ophthalmological findings:
no effects observed
Description (incidence and severity):
not applicable
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- 2400 ppm: slightly longer thromboplastin times and depressed thrombocyte counts, both findings were only partially reversible within the recovery period; plasma, erythrocyte and brain cholinesterase activities were not affected to a toxicologically significant extent in males and females, lower platelet count at Week 5 and 14, reaching statistical significance only in females in Week 5. This finding was reversible in the recovery group.

The effect on platelet counts and thromboplastin times suggest a weak impairment of blood clotting. Prolonged thromboplastin times were recorded even after the recovery period.

Occasional statistically significant differences were found for reticulocyte count, erythrocyte parameters (red cell count, mean cell volume, cellular haemoglobin content, mean cell haemoglobin concentration), haematocrit and haemoglobin concentration but these changes were small and not of toxicological relvance since all individual data were within the normal degree of scatter and not dose-dependent.. Moreover, leucocyte count was decreased in treated females but no dose-relationship was observed, all individual values were within the relatively broad reference range for this parameter and this finding was not reported for the previous range-finding study, so the finding was considered incidental.

Summarized data can be found in Attachment 3 and 5 in the attached background material.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- 150 ppm: reduced protein concentration in males (-3.6% in Week 14)
- 600 ppm: reduced protein concentration in males (-6.1% in Weeks 5, -4.8% in Week 14)
- 2400 ppm: slight but statistically significant increase in alkaline phosphatase activity in males in Week 5 (+14.6%) and significantly raised levels of ALAT activity in Week 14 only (+24.5%), lower triglycerides in males after 14 (-51.9%) and 17 (-14.7%) weeks, and in females after 17 weeks (-17.6%), reduced protein level in males (-5.1% in week 5, -7.6% in Week 14) and females (-7.7% in Week 5, -5.4% in Week 14), reduced albumin levels in males (-2.9%) and females (-6.0%) in Week 14.

The slightly depressed protein, albumin and cholesterol levels are indicatve for functional liver impairment

Other statistically significant differences were not considered of toxicological concern because they were slight and/or transient. These included raised creatinine concentration in females (600 and 2400 ppm) only in Week 5, and in males in Week 14 (2400 ppm), increased and decreased creatine phosphokinase activity for males (2400 ppm, Week 5) and females (2400 ppm recovery group), respecitvely. Also, differences in electrolyte concentrations in serum or plasma were not severe enough to be considered toxicologically relevant. Cholinesterase activity in erythrocytes was increased for males and females in Week 5 and for males in Week 14 and 17 but this was considered statistically but not biologically significant.

Summarized data can be found in Attachment 4 and 5 in the attached background material.
Urinalysis findings:
no effects observed
Description (incidence and severity):
not applicable.
Behaviour (functional findings):
not examined
Description (incidence and severity):
not applicable
Immunological findings:
not examined
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
- 2400 ppm: reduced absolute liver weight for males (-11.6%) and females (-8.5%), reduced absolute kidney weights for males (-13.7%) and females (-9.1%), reduced absolute weights of adrenal glands of females (-15.5%), increased relative weights of brain for males (+13.0%) and females (+11.6%), spleen for females (+39.3%), and testes for males (+14.1%)

These findings were considered to be secondary effects to reduced body weight and therefore not directly treatment-related. However, since clincial chemistry investigations (see above) and histopathological findings (see below) show hepatic alterations in males caused by treatment, the lower liver weight is indicative for a treatment-related effect.
Gross pathological findings:
no effects observed
Description (incidence and severity):
not applicable
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- 600 ppm: slight changes in the plasma of hepatocytes (3/10 males), interpreted as margina functional increase
- 2400 ppm: round cell infiltrates in liver (all males in 14 weeks group), isolated cell necrosis in liver (8/10 males in 14 weeks group), focal necrosis (4/10 males in 14 weeks group), higher incidence of cytoplasmic changes and swollen liver cell nuclei in males in 14 weeks group; no morphological effects were visible in males at the end of the recovery period thus suggesting reversible liver effects
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
not applicable
Other effects:
not examined
Description (incidence and severity):
not applicable
Details on results:
Historical reference values can be found in Attachment 6.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
150 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no adverse effects observed at this concentration
Remarks on result:
other: corresponds to 14.0 mg/kg bw/day
Key result
Dose descriptor:
LOAEL
Effect level:
600 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
clinical biochemistry
food consumption and compound intake
haematology
histopathology: non-neoplastic
other: effects indicative for liver hepatotxicity were reversible within the recovery group
Remarks on result:
other: corresponds to 60.9 mg/kg bw/day
Key result
Dose descriptor:
NOAEL
Effect level:
600 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no adverse effects observed at this concentration
Remarks on result:
other: corresponds to 83.3 mg/kg bw/day
Key result
Dose descriptor:
LOAEL
Effect level:
2 400 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
clinical biochemistry
food consumption and compound intake
Remarks on result:
other: corresponds to 422.2 mg/kg bw/day

Target system / organ toxicity

Key result
Critical effects observed:
no
Lowest effective dose / conc.:
600 ppm
System:
hepatobiliary
Organ:
liver

Applicant's summary and conclusion

Conclusions:
The study was performed under GLP conditions and according to OECD TG 408 (adopted 1981). Deviations to the current version (adopted 2018) are minor. Thus, the study is considered reliable and valid. Due to decreased body weight gain, NOAELs of 150 and 600 ppm were derived for males and females, respectively (corresponding to 14.0 and 83.3 mg/kg bw/day in males and females, respectively). Reversible hepatotoxic effects were evident at 2400 ppm (corresponding to 300.2 mg/kg bw/day).