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Diss Factsheets

Administrative data

Description of key information

Oral administration

key; M-007967-01-1; subchronic (96 days, rat): NOAEL (oral) 14.0 mg/kg bw/day (males) and 83.3 mg/kg bw/day (females)

key; M-027741-02-1; chronic (2 years, rat): NOAEL (oral) 5.7 mg/kg bw/day (males) and 24.9 mg/kg bw/day (females)

key; M-026540-01-1; subchronic (90 days, dog): NOAEL (oral) 23.0 mg/kg bw/day (combined values for both sexes, re-calculated based on body weights and feed consumption)

key; M-027093-01-1; chronic (52 weeks, dog): NOAEL (oral) 15 mg/kg bw/day (males, females)

 

Inhalative administration

key; M-026004-01-1; subacute (4 weeks, rat): NOAEC (inhalation) 5.5 mg/m³ air (males, females)

 

Dermal administration

key; M-025976-01-1; subacute (4 weeks, rabbit): NOAEL (dermal) 1000 mg/kg bw/day (males, females)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

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
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
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
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
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
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).
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.
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
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
600 ppm
System:
hepatobiliary
Organ:
liver
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).
Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jul 1987 - Jul 1989 (M-027741-02-1); Sep 1988 - Sep 1990 (M-027135-01-1)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Version / remarks:
adopted 2018
Deviations:
yes
Remarks:
methodological deficiencies (see 'additional information on materials and methods incl. tables' section)
Qualifier:
according to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Version / remarks:
adopted 1981
Deviations:
no
Principles of method if other than guideline:
Comment on study design

In this RSS, the main study (M-027741-02-1) and a supplementary study (M-027135-01-1) are reported. The supplementary study was performed to support determination of a MTD of the test item in rats. The main study consisted of a control group (0 ppm) and the dose groups 100, 300 and 900 ppm, while the supplementary study consisted of a control group (0 ppm) and a high-dose group (1800 ppm). If not stated otherwise, the procedures in the supplementary study were the same as compared to the main study.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat was chosen since it is a recommended species for chronic toxicological studies in test guidelines.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Winkelmann Experimental Animal Breeder, Borchen, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 4 - 6 weeks
- Weight at study initiation: 56 - 102 g (males), 59 - 89 g (females)(M027741-02-1); 77 - 111 g (males), 68 - 100 g (females)(M-027135-01-1)
- Housing: animals were individually kept in Type II Makrolon cages on low-dust wood shavings.
- Diet: Altromin 1321 flour (Altromin GmbH & Co KG, Lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): about 50
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: Jul 1987 To: Jul 1989 (M-027741-02-1) and From: Sep 1988 To: Sep 1990 (M-027135-01-1)
Route of administration:
oral: feed
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet: weekly
- Mixing appropriate amounts with: standard diet containing 1% ground nut oil to minimise dust formation during mixing
- Storage temperature of food: deposit samples of each mix taken for re-analysis were kept at approx. +4 °C and from Oct 1987 at -20 °C.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For verification of nominal concentration, homogeneity and stability of the test substance in the diet (Altromin 1321 with 1% peanut oil), the test item was analysed by high performance liquid chromatography and UV detection. Verification of nominal concentration was performed 10-times over the course of the study for all 4 dose levels (100, 300, 900 and 1800 mg/kg). Mean analytical concentrations found were 98, 295, 897 and 1790 mg/kg (mean in % of nominal: 98, 98, 100 and 99 for the dose levels 100, 300, 900 and 1800 mg/kg, respectively). For homogeneity and stability checks, analyses of another study were used (report no. T 1025699). The nominal concentrations of 50 and 1000 mg/kg were analyzed for homogeneity and stability. To check for homogeneity, 5 feed mixture samples of 100 to 200 g each were obtained. Three of the 5 samples were selected at random and analyzed. The distribution of active ingredient was homogeneous because the relative standard deviation of the measured values did not exceed 10%. The analytical concentrations found were 51 and 1020 mg/kg at Day 0 and 43 and 880 mg/kg at Day 7 and 42 and 870 mg/kg at Day 14 of storage for the nominal concentrations 50 and 1000 mg/kg, respectively. The active ingredient was stable in the feed mixture over a period of 14 days in consideration of the tolerance range of +- 20% of the nominal concentration.
Duration of treatment / exposure:
up to 24 months (interim section after 12 months)
Frequency of treatment:
continously via the diet
Dose / conc.:
100 ppm
Remarks:
5.7 mg/kg bw/day (males), 7.6 mg/kg bw/day (females)
Dose / conc.:
300 ppm
Remarks:
16.9 mg/kg bw/day (males), 24.9 mg/kg bw/day (females)
Dose / conc.:
900 ppm
Remarks:
51.3 mg/kg bw/day (males), 73.0 mg/kg bw/day (females)
Dose / conc.:
1 800 ppm
Remarks:
102.6 mg/kg bw/day (males), 143.7 mg/kg bw/day (females)
No. of animals per sex per dose:
Terminal sacrifice (24 months): 50
Interim sacrifice (12 months): 10
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: dose selection was based on two subchronic feeding studies in which doses of 0, 120, 600 and 3000 ppm and 0, 150, 600 and 2400 ppm were used, respectively.
Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day (once a day on weekend and public holidays)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a week. Body surfaces, body openings, posture, general behavior, respiration and excretory products were inspected.

BODY WEIGHT: Yes
- Time schedule for examinations: prior to treatment (week 0), at weekly intervals thereafter until and including week 14, and at two-week intervals from week 16 until and including week 102. The body weights were also recorded immediately prior to scheduled necropsy after 12 or 24 months to allow calculation of the relative organ weights.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Food consumptions during weeks 2 and 15 and for four-week intervals starting with week 19 were individually determined for all animals.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: water intake was determined in weeks 14, 27, 40, 53, 66, 79 and 92 for each dose and sex group (M-027741-02-1) and in weeks 2, 16, 29, 42, 55, 68, 81 and 94 for each dose and sex group (M-027135-01-1).

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at the start of the study and after 12 months
- Dose groups that were examined: 0, 900 and 1800 ppm (10 per dose and sex)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 6, 12, 18 and 24 months after initiation of the study
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: No
- How many animals: 10 randomly selected animals from each group
- Parameters checked: Differential blood count, erythrocyte morphology, erythrocyte count, blood haemoglobin concentration, hematocrit, leukocyte count, mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular cell volume (MCV), thrombocyte count, coagulation time, reticulocytes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 6, 12, 18 and 24 months after initiation of the study
- Animals fasted: No
- How many animals: 10 randomly selected animals from each group
- Parameters checked: activities of alkaline phosphatase, optimized aspartate aminotransferase, optimized alanine aminotransferase, creatine kinase, GLDH, cholinesterase (plasma and erythrocyte); glucose, bilirubin, cholesterol, total protein, urea, albumin, inorganic phosphate, sodium, potassium, calcium, chloride

PLASMA/SERUM HORMONES/LIPIDS: Yes, TSH, T3 and T4 (M-027135-01-1)
- Time of blood sample collection: 76th week of study
- Animals fasted: No
- How many animals: 10 randomly selected animals from control and 1800 ppm groups (both sexes)
Serum was obtained for determination of the TSH, T3 and T4 levels. For the TSH assay, positive controls were required. Therefore, 8 µg/kg bw doses of the hormone preparation TRH (Ferring Arzneimittel GmbH, Kiel, Germany) were administered i.p. to groups of 10 male and 10 female rats and blood samples were collected after 30 minutes.

URINALYSIS: Yes
- Time schedule for collection of urine: The urine samples were collected during sampling periods of about 16 hours (overnight) a few days before blood was withdrawn in each case. About 5 mL drinking water was administered to the animals by stomach tube prior to the collection periods.
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes
- How many animals: 10 randomly selected animals from each group
- Parameters checked: blood, glucose, bilirubin, ketone bodies, pH, protein, urobilinogen, sediment (semiquantitative) and specific gravity, volume, protein (quantitative)

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
SACRIFICE: After 12 months all surviving animals selected for the interim post mortem and at the end of the study after 24 months all surviving animals were sacrificed by exsanguination under deep ether anaesthesia.

GROSS PATHOLOGY: Yes
The animals were dissected, and their organs/tissues subjected to thorough pathoanatomical evaluation.

HISTOPATHOLOGY: Yes

The following tissues/organs were sampled for histopathological examination: aorta, eyes, eyelids, cecum, colon, duodenum, extraorbital lacrimatory gland, femur, brain, harderian gland, urinary bladder, ureter, urethra, skin, heart, testes, pituitary, ileum, jejunum, larynx, bone marrow (in femur and sternum), head, liver, lung, lymph nodes (mesenteric and mandibular), stomach, mammary glands, spleen, musculature of thigh, epididymis, adrenals, sciatic nerve, optic nerve, kidneys, esophagus, tattooed ear scoops, ovaries, oviduct, pancreas, prostate, rectum, lower intestine, spinal cord (cervical, thoracic, lumbar), seminal vesicles, thyroid gland, cranial salivary gland, sternum, thymus, trachea, uterus, vagina, tongue, zymbal gland
Optional endpoint(s):
Optional endpoints: No
Other examinations:
Organ weights: The following organs of animals sacrificed for the interim post mortem after 12 months or at the end of the study after 24 months were weighed: brain, heart, liver, lung, spleen, kindeys (both), adrenal glands (both), ovaries (both) and testes (both)
Statistics:
The arithmetic group means and standard deviations were calculated from all individual animal results. Except for the cholinesterase data, the test cohort results were subjected to a two-tailed comparison with those of the control cohort using the significance test ("U" test) of H.B. Mann and D.R. Whitney (Ann. Math. Stat. 18, 50 [1947]) and F. Wilcoxon (Biometrics 1(6). 80 [1945]) at significance levels of a = 5 % and a = 1 %.

The survival curves were analyzed using BMDP Routine 1L. The incidences of the individual status variable intensities have been grouped according to the treatment. Intensities deviating from "died intercurrently/sacrificed in moribund condition" - such as "interim necropsy" or "terminal necropsy" - were treated as censored observations, since the animals had not been observed until occurrence an event, i.e. to the point of "natural" death in these cases. The survival curves were subsequently compared using the generalized Wilcoxon test (Breslow test) suggested by N.E. Breslow, Biometrika 51, 579 - 594 (1979), the weight assigned to each event point being proportional to the size of the pertinent group.

The cholinesterase data were separately assessed for each sex by means of single-factorial variance analysis for each time point.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant increased incidences of findings were observed in the groups receiving the test item up to 1800 ppm. Poor general condition, loss of hair and palpable masses were among the most frequently observed clinical signs noted. No exceptional features in the body surfaces and openings, or with respect to the general behavior, posture, respiration and excretory products could be observed at doses to 1800 ppm. No treatment effect was inferred from the incidence, location and chronology of palpable tissue masses at doses to 1800 ppm.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
M-027741-02-1: Mortality observed was not treatment-related, since incidences were comparable between treatment groups. The cumulative mortalities after the first 12 months of study were 1/60, 1/60, 1/60 and 1/60 (males) and 0/60, 0/60, 3/60 and 1/60 (females) for control, 100, 300 and 900 ppm, respectively. The cumulative mortalities at the end of the study (24 months) were 6/50, 6/50, 6/50 and 6/50 (males) and 13/50, 6/50, 10/50 and 13/50 (females) for control, 100, 300 and 900 ppm, respectively.

M-027135-01-1: Mortality observed was not treatment-related, since incidences were comparable between groups. The cumulative mortalities after the first 12 months of study were 1/60 and 0/60 (males) and 1/60 and 1/60 (females) for control and 1800 ppm, respectively. The cumulative mortalities at the end of the study (24 months) were 10/50 and 5/50 (males) and 13/50 and 10/50 (females) for control and 1800 ppm, respectively.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gain was comparable with that of control rats at doses up to and including 300 ppm. At a dose of 900 ppm, body weight gain of male animals was up to 5% slower and that of females up to 8% slower than in the control group. At a dose of 1800 ppm body weight gains in male and female rats were significantly retarded at all times. The weight declined reached a maximum of 12 % (males) or 11 % (females) in week 10. At the close of the study males were 5 % lighter and females were 11 % lighter than controls.

Due to the significant reduction in body weight, 1800 ppm was regarded as maximum tolerated dose.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
not applicable
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
Water intake was reduced by 13 % in females at 1800 ppm. For all other groups no effects were observed.
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In the control as well as 900 and 1800 ppm group several animals exhibited spontaneous alteration such as focal diffuse clouding of the light-refracting media, foreign boy inclusions or eminences in the cornea. These are common observations made in rats of this breed and age and are not regarded as evidence for an ocultotoxic effect by the test item.
Haematological findings:
no effects observed
Description (incidence and severity):
not applicable
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In the 1800 ppm dose group significantly increased aspartate aminotransferase (males and females) and creatine kinase (females) levels were found at the end of the study.
Endocrine findings:
no effects observed
Description (incidence and severity):
No statistically significant differences between the hormone titers of TSH, T3 and T4 of control and treated animals (1800 ppm) were determined.
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):
M-027741-02-1: After 12 months of study significantly depressed figures for the absolute liver (14 %) and kidney (13 %) weights were observed in the 900 ppm group of female rats. At the end of the study (24 months), these organ weights were still significantly lower compared to control (4 and 5 % for liver and kidney, respectively), but not considered as evidence for organ damage due to the lower deviation between treated and control animals. In male rats only in the high-dose group a decrease in liver weight (13 %) at the interim sacrifice (12 months) was recorded.

Alterations in liver and kidney organ weights are not attributable to liver or kidney damage, but must be seen in direct relation to the reduced body weight gain in the affected dose groups. Histophathological assessment of these organs afforded no evidence for treatment-related lesions.

M-027135-01-1: After 12 and 24 months of study depressed absolute and relative organ weights in the case of several organs, particular the liver, were found in the 1800 ppm group (males and females) compared to controls. These were considered as a result of the decreased body weights rather than evidence for a specific toxic effect by the test item.
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):
M-027741-02-1: In the thyroid glands, mineralized particles (MPs) were found in the colloid of isolated, large-lumen follicles in both sexes at the interim and the final section. Interim section: MPs were observed in the thyroid glands of male rats of all dose groups, but incidence increased dose-dependently from 3/10 (control) to 3/10 (100 ppm), 6/10 (300 ppm) and 10/10 (900 ppm). In female rats the effect was less pronounced and MPs were only found in the top dose (3/10), but in no other group. Final section: MPs in thyroid glands were found in 2/50 (control), 12/50 (100 ppm), 31/50 (300 ppm) and 44/50 (900 ppm) male rats and 11/50 (control), 6/50 (100 ppm), 11/50 (300 ppm) and 27/50 (900 ppm) in female rats. The incidence of 12/50 in the 100 ppm treated males was significantly higher compared to the control animals of this study, however, when compared to historical control data from four chronic studies from with the rats being sacrificed between 1987 and 1990, and histopathological samples analysed retrospectively, no differences were found. 300 ppm is considered as a threshold for this effect in male rats and 900 ppm in female rats. However, such findings occur as spontaneous phenomena in the sense of senile involution of isolated follicles in ageing rats, and thus, the observed effects are regarded as a treatment effect in the sense of premature biological ageing processes. As plasma levels of thyreotropin, triiodothyronine and thyroxine were unaffected by treatment, a toxicological effect on thyroid function is excluded.

No findings exhibiting treatment-related distribution were seen in the other organs at doses up to and including 900 ppm.

M-027135-01-1: In the thyroid glands, mineralized particles (MPs) in the colloid of isolated, large-lumen follicles were found more frequently and colloid aggregation sire more rarely in both sexes at the interim and the final section. Interim section: MPs were observed in the thyroid glands of male rats of all dose groups; 5/10 (control) and 10/10 (1800 ppm). In female rats the effect was less pronounced; 2/10 (control) and 5/10 (1800 ppm). Colloid aggregation: 6/10 (control males) and 0/10 (1800 ppm males), 2/10 (control females) and 0/10 (1800 ppm females). Final section: MPs in thyroid glands were found in 12/50 (control) and 46/50 (1800 ppm) male rats and 3/50 (control) and 38/50 (1800 ppm) in female rats. The incidence of colloid aggregation decreased in male rats from 41/50 (control) to 20/50 (1800 ppm) and in female rats from 22/50 (control) to 7/50 (1800 ppm).
Furthermore, enhance porphyrin accumulation in the Harderian glands and an increased incidence of retinal atrophy were found in female rats of the 1800 ppm group.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
The total number of tumors observed and the incidence of begnin and malignant neoplasms were comparable in all study groups. Most tumors were observed in the pituitary gland, thyroid, mamma and uterus. Their incidence was independent of the dose. Tumors in the liver only occurred in male rats of the 1800 ppm group (one adenoma, one carcinoma and two cholangiocellular carcinomas). However, comparison with historical control data shows that the incidences of hepatic adenomas and carcinomas do not lie outside the reference ranges for these tumors.
Other effects:
not examined
Description (incidence and severity):
not applicable
Key result
Dose descriptor:
NOAEL
Effect level:
100 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 5.7 mg/kg bw/day
Key result
Dose descriptor:
LOAEL
Effect level:
300 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
histopathology: non-neoplastic
Remarks on result:
other: corresponds to 16.9 mg/kg bw/day
Key result
Dose descriptor:
NOAEL
Effect level:
300 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 24.9 mg/kg bw/day
Key result
Dose descriptor:
LOAEL
Effect level:
900 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
histopathology: non-neoplastic
Remarks on result:
other: corresponds to 73 mg/kg bw/day
Key result
Dose descriptor:
other: maximum tolerated dose, MTD
Effect level:
1 800 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: corresponds to 102.6 and 143.7 mg/kg bw/day in males and females, respectively
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
300 ppm
System:
endocrine system
Organ:
thyroid gland
Conclusions:
The studies were performed under GLP conditions and according to OECD TG 453 (adopted 1981). Deviations to the current version (adopted 2018) are minor. Thus the studies are considered reliable and valid. For male rats a NOAEL of 100 ppm (equivalent to 5.7 mg/kg bw/day) and for female rats a NOAEL of 300 ppm (equivalent to 24.9 mg/kg bw/day) was established based on increased incidence of colloid mineralisation at the next higher test concentrations (300 ppm in males and 900 ppm in females, corresponding to 16.9 and 73 mg/kg bw/day, respectively). In the absence of altered plasma hormone levels, an effect on thyroid function can be excluded, as concluded previously in the assessment report (Assessment Report, 2011).
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 1987 – January 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)
Version / remarks:
adopted 1981
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)
Version / remarks:
1998
Deviations:
yes
Remarks:
methodological deficiencies (few parameters of clinical biochemistry and urinalysis not examined, weights of epididymides, uterus and thymus not provided, no histopathology of salivary glands, test substance not calculated as mg/kg bw/day)
GLP compliance:
yes
Limit test:
no
Species:
dog
Strain:
Beagle
Remarks:
Bor:Beag strain
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: F. Winkelmann Breeders, Borchen, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 18 - 20 weeks
- Weight at study initiation: 4.9 - 8.2 kg
- Fasting period before study: not specified
- Housing: all animals were kept in individual cages (floor area about 1.10 x 1.15 m)
- Diet: "Ssniff HH Double-Milled Sole Diet for Dog Maintenance", Ssniff Versuchstierdiäten GmbH, Soest, Germany
- Water: tap water, ad libitum
- Acclimation period: not specified

DETAILS OF FOOD AND WATER QUALITY:
The nutritive composition and contaminant content of the standard diet were routinely spot-checked and determined by analysis. No complaints resulted from these tests. In addition, water analyses were performed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 22
- Humidity (%): 30 - 50
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 7 October 1987 To: sacrifice
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The food-substance mixes were produced once weekly. The test substance was evenly blended with the food. The powdered food (controls) or food-substance mixes (Groups I to III) were blended 1:1 with lukewarm tap water immediately before the animals were fed. A homogeneous food mash was produced from the mixture of food and water by kneading with a kitchen food processor. All animals were given the food mash every morning. The food not consumed by an animal within the period of about 20 - 22 hours until the next feeding time was back weighed.

DIET PREPARATION
- Rate of preparation of diet: once weekly
- Mixing appropriate amounts with (Type of food): standard diet
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the test substance was quantitatively determined by high performance liquid chromatography (HPLC) and UV detection.

Variance between nominal and actual dosage:
Regular analytical monitoring throughout the entire study period ensured that the food-substance mixes actually contained the declared levels of the test substance. Results of the verification of nominal concentration of the test item at 200, 600, 1200 and 1800 mg/kg bw were within the range of 97 - 101 %. Therefore, the variance between nominal and actual dosage was considered acceptable.

Homogeneity analysis:
Homogeneity of the test substance in the diet at 10 and 4000 mg/kg bw was determined. The distribution of active ingredient was considered homogeneous because the relative standard deviation of the measured values did not exceed 10 %.

Stability analysis:
The stability of test substance (nominal concentrations of 10 and 4000 mg/kg bw) in the standard diet with and without water was determined within a storage period of 14 days. The active ingredient was stable in the feed mixture without water over a period of 14 days in consideration of the tolerance range of ± 20 % of the nominal concentration (96 and 89 % active ingredient were determined for 10 and 4000 mg/kg bw, respectively). The active ingredient was stable in the feed mixture with water over a period of 24 hours in consideration of the tolerance range of ± 20 % of the nominal concentration (104 and 99 % active ingredient were determined for 10 and 4000 mg/kg bw, respectively).
Duration of treatment / exposure:
13 weeks (control and test groups)
Frequency of treatment:
once daily, 7 days/week
Dose / conc.:
200 ppm
Remarks:
corresponding to mean achieved doses of 7.8 mg/kg bw/day (combined value for both sexes), recalculated based on weekly food consumption and body weight
Dose / conc.:
600 ppm
Remarks:
corresponding to mean achieved doses of 23.0 mg/kg bw/day (combined value for both sexes), recalculated based on weekly food consumption and body weight
Dose / conc.:
1 800 ppm
Remarks:
1200 ppm from week 4 due to low food consumption, corresponding to mean achieved doses of 42.0 mg/kg bw/day (combined value for both sexes), recalculated based on weekly food consumption and body weight
No. of animals per sex per dose:
4/sex/group
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dosage was established on the basis of results obtained in a pilot range-finding study (M-027014-01-1), in which animals were orally exposed to 200, 1000 and 5000 ppm for 28 days. Adverse effects such as ataxia, tremor and occassionally vomiting were observed at 5000 ppm. All animals treated at 5000 ppm died or were killed for humane reasons. Therefore, 200, 600 and 1800 ppm were selected as the dose levels for the main study (please refer to the endpoint summary for further details).
- Fasting period before blood sampling for clinical biochemistry: Not specified
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: appearance and behavior of all animals was repeatedly checked each day during feeding, grooming, cleaning of the stall and during exercising

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before starting the study and during the third, seventh and 13th weeks of treatment (reflex tests, body temperature)

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: no
- 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: not specified

WATER CONSUMPTION: Yes
It was noted whether group-related variations in the frequency with which it was necessary to refill the water dishes occurred (check of water intake).

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before starting the study and during the seventh and 13th weeks of treatment
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before starting the study and during the third, seventh and 13th weeks of treatment
- Anaesthetic used for blood collection: not specified
- Animals fasted: not specified
- How many animals: all animals
- The following parameters were examined: erythrocyte and leukocyte counts, haemoglobin, haematocrit, MCV, MCH, MCHC and methaemoglobin (Met-Hb) values, thrombocyte count, thromboplastin time (TPT), partial thromboplastin time (PTT), reticulocyte count, differential blood count and the blood sedimentation rate (BSR)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before starting the study and during the third, seventh and 13th weeks of treatment
- Animals fasted: not specified
- How many animals: all animals
- The following parameters were examined: blood sugar, urea, creatinine, total protein, aspartate aminotransferase (AST/GOT), alanine aminotransferase (ALT/GPT), alkaline phosphatase (AP), lactate dehydrogenase (LDH), creatine kinase (CK), bilirubin, cholesterol, triglyceride, glutamate dehydrogenase (GLDH), serum electrolytes sodium, potassium, calcium, chloride, inorganic phosphate and magnesium, thyroxine (T4), triiodothyronine (T3) and the thyroxine binding capacity (TK), N-demethylase, serum protein, cytochrome P-450 and triglyceride

PLASMA/SERUM HORMONES/LIPIDS: Yes, please refer to "clincial chemistry"

URINALYSIS: Yes
- Time schedule for collection of urine: before starting the study and during the third, seventh and 13th weeks of treatment
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, but a massive dose of water (about 250 mL tap water) was administered to the animals by stomach tube prior to their transfer. Animals yielding no urine during repeated six-hour waiting periods in the metabolism cage were left there overnight (about 16 hours) for collection of urine. The animals were provided with food and water during this extended
urine collection period.
- The following parameters were examined: volume of collected urine, specifc gravity, urine specimens were semiquantitatively tested for protein, glucose, blood, bilirubin, ketone bodies, urobilinogen, pH and urine sediments (leukocytes, erythrocytes, epithelial cells, bacteria and crystals)

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. At the end of the study, all animals were sacrificed by exsanguination under Evipan® anesthesia, necropsied and subjected to gross pathological examination. The brain, pituitary, heart, liver, lung, spleen, adrenals, kidneys, pancreas, thyroid, testes, prostate and ovaries were weighed.

HISTOPATHOLOGY: Yes. The following organs were examinded:
adrenal glands, aorta, bone marrow (femur), bones (femur, sternum), brain (cerebrum, cerebellum, brain stem), epididymides, esophagus, eyes, gall bladder, heart (left ventricle with papillary muscles), intestine (duodenum, jejunum, ileum, cecum, colon, rectum), kidneys, liver, lungs, mammary region, mesenteric lymphnode, optic nerves, ovaries, pancreas, parathyroid glands, parotid gland, pituitary gland, prostate, sciatic nerve, skeletal muscle, skin, spleen, spinal cord (cervical, thoracic, lumbar), stomach (fundus, pars pylorica), testes, thymus, thyroid gland, tongue, tonsils, trachea, urinary bladder, uterus any other organs showing changes
Statistics:
The statistical procedure was descriptive due to the small number of animals per group (four males and four females). The calculations encompassed determination of the arithmetic means and standard deviations. Maximum and minimum values were also specified.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- Reflex tests: no pathological findings at any examination date

- Body temperature: no unusual variations among the animals of any group

- Nutrition state:
Overall, control and low dose animals exhibited a normal state of nutrition until the end of the study period.
0 ppm (control): obese state of nutrition (1/4 male) in necropsy
200 ppm: normal to lean state of nutrition (2/4 males) during the seventh week
600 ppm: normal to lean state of nutrition was repeatedly determined (1/4 male and 3/4 females) between the fifth and 13th study weeks
1800/1200 ppm: a depressed state of nutrition was determined (3/4 males and 2/4 females) between the third and 13th study weeks
All mid- and high dose animals exhibited a normal state of nutrition at sacrifice.

- Further clinical signs:
200 ppm: Detetction of blood in the feces (1/4 female) during the first week of the study.
600 ppm: Trembling independent of the feeding time was observed (all animals) up to the fifth week of the study (5/8 animals displayed “trembling” on one and 3/8 animals on two occasions during wk 1, but not at later time points).
1800/1200 ppm: Trembling independent of the feeding time was observed (all animals) up to the fifth week of the study, and even some cases of severe tremblor. From the start of exposure up to Week 5, all animals showed “trembling” or “slight trembling” on one or several occasions (up to six times/week). “Severe” trembling was observed during week 1 at least once, sometimes up to five times during this time. As a consequence, the dietary concentration was lowered from 1800 down to 1200 ppm from Week 5 on. Tremor and/or trembling were no longer observed until the end of the study when feed concentration had been lowered to 1200 ppm. No toxicological relevance based on these findings is considered since the tremblor was not observed in the comparative pilot study (M-027014-01-1) at a dose of 1200 ppm, or in the chronic dog study at levels up to and including 2500 ppm (M-027093-01-1).
independent of the dosage: Vomiting of food mash or mucus was observed in isolated instances as well as loose or pultaceous feces.
Mortality:
no mortality observed
Description (incidence):
not applicable
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
1800/1200 ppm: Lower body weight gains were observed in most animals during the first 3 - 6 weeks of the study as a result of the reduced food consumption. No significant differences in body weight gains compared to the controls were determined after the dose had been reduced from 1800 to 1200 ppm starting with week 4. The state of nutrition at 1800/1200 ppm was relatively more depressed as a result of the low food intakes which particularly occurred during the initial weeks of the study. However, all animals exhibited a normal nutritional state at necropsy. Thus, the depressed state of nutrition during the initial weeks of exposure is assessed as a direct consequence of incomplete food consumption.
For details, please refer to the attached background material, Attachment 1 (body weight data).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
600 ppm: The food consumption at 600 ppm was nearly complete. Repeated instances of incomplete food consumption were observed in 2/4 females during the first half of the study.
1800 ppm: Reduced food intakes during the first three weeks of the study were determined in all animals.
The test substance concentration was therefore reduced from 1800 ppm to 1200 ppm starting with the fourth week of the study.
1200 ppm: Repeated incomplete food consumption was also observed in 2/4 males and 1/4 female even after reduction of test concentration.
Therefore, Pal® dog food (150 g) was repeatedly blended into the food of these dogs to improve their appetite, after which complete food consumption was observed.
The reduced food intake at 600 ppm and 1800/1200 ppm presumably resulted from the unpleasant taste of the test substance to the dogs.
For details, please refer to the attached background material, Attachment 2 (food consumption data).
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not applicable
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
600 ppm: transient, slight corneal erosion (1/4 male)
1800/1200 ppm: transient, slight corneal erosion (2/4 males), brownish serous ocular discharge (1/4 male), pigment spots in the tapetum lucidum (1/4 female) in the preliminary examination and at all other examination dates
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
independent of the dosage: temporary slight increase of leukocyte count, reticulocyte count, thromboplastine time, elevated blood sedimentation in scattered instances
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The following parameters exhibited marginally elevated values:
0 ppm (control): AP (1/4 female) in the third week of the study, GLDH (2/4 females) on study Day 86 and on study Day 15, respectively
600 ppm: AST and ALT (1/4 female) on study Day 86, creatinine (1/4 female) on study Day 86 and 92
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
independent of the dosage: scattered positive tests for blood and erythrocytes were obtained (various animals)
The positive urinary blood results were not confirmed in any animal when urine collection was repeated a few days after the first test. Therefore, the occasional detection of traces of blood in the urine was not assessed as a sign of kidney damage, but rather as result from minor accidental injuries at time of urine collection.
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):
200 ppm: marginally elevated spleen weights (females)
600 ppm: marginally elevated lung weights and ovary weights (females)
1800/1200 ppm: marginally elevated lung weights (males) and spleen weights (males)
As all the values lay within the 2-delta scattering range of historical laboratory control data in all cases, the effects are not considered as indicative for adversity. Further, no concordant findings were determined at histopathology.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
0 ppm (control): tonsils reddening (1/4 male)
200 ppm: tonsils reddening (1/4 male and 2/4 females), red areas of the lungs (1/4 male), tartar (1/4 male)
600 ppm: tonsils reddening (1/4 male), tartar (1/4 male)
1800/1200 ppm: tonsils reddening (1/4 male), red areas of the lungs (1/4 male and 1/4 female), emphysema of the lung (1/4 male and 1/4 female)

All other lesions registered during necropsy were only singular.
To sum up the results, no test substance-related pathological changes could be established.
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
To sum up the results, no histopathological findings attributable to treatment with the test substance were determined. For details on the observed microscopic findings, please refer to the attached background material Attachment 3 (histopathology data).
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
not applicable
Key result
Dose descriptor:
NOAEL
Effect level:
600 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at this concentration
Remarks on result:
other: corresponding to 23.0 mg/kg bw/day (combined value for both sexes)
Key result
Dose descriptor:
LOAEL
Effect level:
1 800 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Remarks on result:
other: corresponding to 42.0 mg/kg bw/day (combined value for both sexes)
Key result
Critical effects observed:
no
Conclusions:
The study was performed under GLP conditions and according to OECD TG 409 (adopted 1981). Deviations to the current version (adopted 1998) are minor. Thus, the study is considered reliable and valid. Haematology, clinical biochemistry, urinalysis, gross pathology, organ weights and histopathology afforded no adverse findings attributable to the test substance treatment. Due to decreased body weight gain and reduced food intake at 600 ppm, the NOAEL was set to 200 ppm, corresponding to 7.8 mg/kg bw/days (combined value for both sexes).
Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 Oct 1987 - 27 Oct 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 452 (Chronic Toxicity Studies)
Version / remarks:
adopted 2018
Deviations:
yes
Remarks:
missing parameters (see section 'additional information on materials and methods incl. tables')
Qualifier:
according to guideline
Guideline:
OECD Guideline 452 (Chronic Toxicity Studies)
Version / remarks:
adopted 1981
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
dog
Strain:
Beagle
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Laboratory Research Enterprises, Inc., Kalamazoo, MI, USA
- Age at study initiation: 4 - 6 months
- Weight at study initiation: 6.6 - 9.2 kg (males), 5.3 - 7.4 kg (females)
- Housing: individually in kennels with at least 2 m² floor space
- Diet: repelleted standard Kliba 335 dog maintenance diet (Klingentalmühle AG, Kaiseraugst, Switzerland), 299 - 301 g per day were presented from 10.00 and withdrawn at 13.00.
- Water: tap water, ad libitum
- Acclimation period: 5 weeks

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 23
- Humidity (%): 40 - 70
- Air changes (per hr): 20
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 21 Oct 1987 To: 27 Oct 1988
Route of administration:
oral: feed
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet: every two weeks
- Mixing appropriate amounts with: the test item was mixed with microgranulated feed. Water (1:10 v/w) was added to aid pelleting by a pelleting machine. The pellets were then dried with warm air for approximately 48 h prior storage.
- Storage temperature of food: at room temperature in disposable paper bags
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For verification of stability, homogeneity and content of the test substance in the feed, samples were analysed by means of high performance liquid chromatography and UV detection before test initiation and at intercurrent 3 monthly intervals (only homogeneity and content). Results of the stability test showed, that the test item is stable at room temperature in dog feed for at least 21 days. The analysis of homogeneity was performed by analyzing samples of each group's feed from three segments of the respective mixing container (top, middle, bottom). The homogeneity varied in the range of mean concentration +- 6 %. The overall mean concentrations of test item found were 95.1 %, 99.8 %, and 101.0 % for the dose groups of 200, 500 and 1250/2500 ppm, respectively.
Duration of treatment / exposure:
52 weeks
Frequency of treatment:
continously via the diet
Dose / conc.:
200 ppm
Remarks:
corresponding to 6.1 mg/kg bw/day
Dose / conc.:
500 ppm
Remarks:
corresponding to 15 mg/kg bw/day
Dose / conc.:
1 250 ppm
Remarks:
the dose was increased to 2500 ppm from week 17 onwards (corresponds to 41 and 72 mg/kg bw/day for 1250 and 2500 ppm, respectively)
No. of animals per sex per dose:
4
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: dose selection was performed based on the results of a 4-week study
- Fasting period before blood sampling for clinical biochemistry: 18 h
Positive control:
None.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at the beginning of the pretest, week 13, 26 and 52
- Dose groups that were examined: all groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the beginning of the pretest, week 13, 26 and 52
- Animals fasted: Yes, animals were fasted for 18h prior to sampling; water was provided.
- How many animals: all animals
- Parameters checked: erythrocyte count, haemoglobin, haematocrit, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelet count, reticulocyte count, nucleated erythrocytes normoblasts, Heinz bodies, methaemoglobin, total leukocyte count, differential leukocyte count, red cell morphology, thromboplastin time, partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the beginning of the pretest, week 13, 26 and 52
- Animals fasted: Yes, animals were fasted for 18h prior to sampling; water was provided.
- How many animals: all animals
- Parameters checked: glucose, urea, creatinine, bilirubin total (BILI. T.), lipids total (LIPIDS T.), cholesterol total (CHOLEST. T.), triglycerides (TRIGL.), aspartate aminotransferase (ASAT/GOT), alanine aminotransferase (ALAT/GPT), lactate dehydrogenase (LDH), creatine kinase (CK), alkaline phosphatase (ALP), gamma-glutamyl-transferase (G-GT), ornithine carbamyl-transferase (OCT), calcium, phosphorus, sodium, potassium, chloride, triiodothyronine total (T3), thyroxine total (T4), protein total. In liver tissue: cytochrome P450 (CYT. P-450), N-demethylase (N-DEMETHYL.), O-demethylase (O-DEMETHYL.)

URINALYSIS: Yes
- Time schedule for collection of urine: at the beginning of the pretest, week 13, 26 and 52
- Metabolism cages used for collection of urine: No. Urine samples were collected using a catheter
- Animals fasted: Yes, animals were fasted for 18h prior to sampling; water was provided.
- Parameters checked: specific gravity, color, appearance, pH, protein, glucose, ketone, bilirubin, blood, urobilinogen, urine sediment

OTHER:
HEARING TESTS: Yes
-Time schedulue for examinations: hearing tests were performed at the beginning of the pretest, week 13, 26 and 52.
Sacrifice and pathology:
SACRIFICE: After 52 weeks all surviving animals were anaesthetized by i.v. injection of Narcoren and then sacrificed by exsanguination.

GROSS PATHOLOGY: Yes
At the post-mortem examination, all organs were examined and all findings recorded.

HISTOPATHOLOGY: Yes

The following tissues/organs were sampled for histopathological analysis: Adrenal glands, aorta, articular cartilage - proximal femoral, bone - femoral head, bone marrow - sternal, femoral, brain - cerebrum, cerebellum, medulla oblongata/pons, caecum, colon, duodenum, epididymides, oesophagus, eyes, gallbladder, heart, illeum, jejunum, kidneys, liver, lungs, lymph nodes - mesenteric, retropharyngeal, mammary gland area, nictitans gland, optic nerves, ovaries, pancreas, pituitary gland, prostate gland, rectum, salivary glands - mandibular, sublingual, sciatic nerve, skeletal muscle, skin, spinal cord - cervical, midthoracic, lumbar, spleen, stomach, testes, thymus, thyroids and parathyroids, tongue, trachea, urinary bladder, uterus with cervix, vagina, zygomatic glands and any gross lesions.
Other examinations:
ORGAN WEIGHTS:
Thei weight of the following organs of all animals were recorded: adrenal glands, brain, heart, kidneys, liver, lungs, ovaries, pancreas, prostate gland, spleen, testes, thyroid gland with parathyroids
Statistics:
The following statistical methods were used to analyse the body weights, food consumption, clinical laboratory data and organ weights.
Univariate one-way analysis of variance was used to assess the significance on intergroup difference. If the variables could be assumed to follow a normal distribution, the DunnettTest (many to one T-test) based on a pooled variance estimate was applied for the comparison between the treated groups and the control groups. The steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Individual values, means, standard deviations and statistics were rounded off before printing. For example, test statistics were calculated on the basis of exact values for means and pooled variances and them rounded-off to two decimal places. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
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, non-treatment-related
Description (incidence and severity):
Slightly different patterns of weight gain were observed between groups, however, this was not associated with administration of the test item since no dose-related trend was observed. See attachment 1 for details.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the top dose, a slight drop of food consumption was observed during week 1 (males) and week 1 and 2 (females) of treatment period, when compared to the pretest period. From week 2 (males) and week 3 (females), food consumption was similar to control in the 1250 ppm group until and including week 16. From week 17 onwards the top dose group animals received 2500 ppm of test item instead of 1250 ppm. During weeks 17 and 18 food consumption in males of this group fell by 5 % and in females by 9 % in weeks 17 - 19 and by 5 % in week 20 compared to intakes determined during week 16 (last week before increase of test item concentration). Following this short period there was no treatment-related effect on food consumption in this group. There was no effect on food consumption at 200 and 500 ppm.

Please refer to attachment 2.
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not applicable
Ophthalmological findings:
no effects observed
Description (incidence and severity):
not applicable
Haematological findings:
no effects observed
Description (incidence and severity):
not applicable
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Changes of clinical biochemistry parameters were only observed in the top dose group. Cholesterol levels were significantly higher in females of the 1250/2500 ppm group compared to control animals in weeks 13 and 26 and slightly higher in week 52. Liver cytochrome P450 enzyme levels were significantly higher in both sexes of the 1250/2500 ppm group than the control figures. See attachments 3 and 5 for details.
Endocrine findings:
no effects observed
Description (incidence and severity):
not applicable
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, treatment-related
Description (incidence and severity):
No effects were found at the dose levels 200 and 500 ppm. In the top dose group (1250/2500 ppm), a slight increase in liver-to-brain weight was found in both sexes, which is considered as adaptive process due to induced metabolism. Details are provided in attachment 4.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Treatment was not associated with any abnormal gross pathology finding.
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Treatment was not associated with any abnormal gross pathology finding.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
not applicable
Other effects:
no effects observed
Description (incidence and severity):
Administration of the test item did not affect hearing ability of the animals in both sexes and all dose groups.
Key result
Dose descriptor:
NOAEL
Effect level:
500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed at this dose level
Remarks on result:
other: corresponds to 15 mg/kg bw/day
Key result
Dose descriptor:
LOAEL
Effect level:
1 250 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
organ weights and organ / body weight ratios
Remarks on result:
other: corresponds to 41 mg/kg bw/day
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
1 250 ppm
System:
hepatobiliary
Organ:
liver
Conclusions:
The study was performed under GLP conditions and according to OECD TG 452 (adopted 1981). Deviations to the current version (adopted 2018) are minor. Thus the study is considered reliable and valid. For male and female dogs, a NOAEL of 500 ppm (equal to 15 mg/kg bw/day) was established based on compound-induced elevated cholesterol and increase of cytochrome P450 enzyme levels at 1250/2500 ppm which were correlated to slightly increased relative liver/brain weights. All effects are considered as indicative of an adaption process based on substance-induced metabolism.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
5.7 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
Four reliable guideline studies are available, conducted in rat and dog, which all fulfill the criteria of key studies.
System:
other: hepatobiliary system based on sub-acute and subchronic data
Organ:
liver

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Feb - Mar 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 412 (28-Day (Subacute) Inhalation Toxicity Study
Version / remarks:
adopted 2018
Deviations:
yes
Remarks:
methodological deficiencies (MMAD exceeds the currently recommended particle size, no BALF analysis or determination of lung burden was conducted, food and water consumption were not measured)
Qualifier:
according to guideline
Guideline:
OECD Guideline 412 (28-Day (Subacute) Inhalation Toxicity Study
Version / remarks:
adopted 1981
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
adopted 1984
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Winkelmann Experimental Animal Breeders, Borchen, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 2-3 months
- Weight at study initiation: 160 - 200 g
- Fasting period before study: not applicable
- Housing: in groups of five in Type III Makrolon® cages with type S 8/15 low-dust wood shavings (Ssniff Spezialdiäten GmbH, Soest, Germany)
- Diet: Altromin® 1324 Maintenance Diet for Rats and Mice (Altromin GmbH, Lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least one week

DETAILS OF FOOD AND WATER QUALITY: Tap water met drinking water standards (Drinking Water Statute of May 22, 1986; Bundesgesetzblatt Part I, page 760), feet was regularly checked for contaminants, spot checked and analyzed

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): approx. 5o
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: dust
Type of inhalation exposure:
head only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 2.37 - <= 5.7 other: µm (for details please refer to "Any information on materials and methods")
Geometric standard deviation (GSD):
1.96
Remarks on MMAD:
Results of particle size analysis are provided under "Any information on materials and methods".

Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: PVC inhalation chamber, 30 cm diameter, 28 cm height (volume approx. 20 liters)
- Method of holding animals in test chamber: Plexiglass tubes (closed, tail of the rat was outside the tube)
- Source of air and method of conditioning air: compressed air was produced with two Model SB 270/15/350D Boge compressors in parallel. Type A 110 compressed air dryer mounted behind the compressors were used for conditioning. The regulated operating pressure of the compressors was 8-10 bars (800 - 1000 kPa)
- System of generating particulates/aerosols: Wright Dust Feeder (5 and 30 mg/m³ air) and a RBG brush-type generator (180 mg/m³ air, only on the first day because of a defect thereafter) or an Exactomat 4200 (180 mg/m³ air, starting on the second day of exposure).
- Temperature, humidity, pressure in air chamber (for details on temperature and humidity please refer to "Additional information on Materials and Methods"):
- Air flow rate: continously monitored, 20-30 L/min
- Air change rate: 30 air exchanges per hour
- Method of particle size determination: aerodynamic particle sizer with a laser velocimeter (TSI APS 3300) for 5 mg/m³, 30 and 180 mg/m³: APS 3300 instrument in conjunction with two dilution stages (TSI Model 3200)
- Treatment of exhaust air: The exhaust air was treated by passing it through a cotton
wool aerosol filter. The cotton wool filters were destroyed by incineration.

TEST ATMOSPHERE
- Brief description of analytical method used: Leybold - Heraeus measuring system, temperature and humidity were determined at 10 min intervals (automatically), the data was compared to appropriate reference data. The sensors were located in the inhalation chamber cover.
- Samples taken from breathing zone: yes

VEHICLE:
- Dust is most appropriate in accordance with potential exposure route for humans. An inhalation toxicity study using an aerosol was considered inadequate because of the low solubility of the active ingredient in nontoxic vehicle substances (maximum technically producible concentration: 69 mg/m³ air).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Gravimetric filter analysis (Sartorius SM 11106 cellulose acetate filter, pore size 0.45 µm). 100, 50 and 30 liters of test atmosphere were sampled in breathing zone for 5, 30 and 180 mg/m³ air, respectively (rate: 4 L/min). If possible, 3 air samples were taken, one at the beginning of the test, one in the middle of the test and one near the end. All concentrations stated below refer to mg test substance (95.2% purity) per m³ air. Recalculation to a 100% active ingredient basis was not performed.
Duration of treatment / exposure:
28 days, 6h per exposure
Frequency of treatment:
daily, 5 times per week
Dose / conc.:
5 mg/m³ air (nominal)
Remarks:
corresponds to 5.5 mg/m³ air as analytical concentration
Dose / conc.:
30 mg/m³ air (nominal)
Remarks:
corresponds to 30.5 mg/m³ air as analytical concentration
Dose / conc.:
180 mg/m³ air (nominal)
Remarks:
corresponds to 191.2 mg/m³ air as analytical concentration
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
- Dose selection rationale: Acute inhalation toxicity studies summarised in the technical dossier under 7.2.2 "Acute toxicity: inhalation" served as range-finding studies (M-027586-01-1). LC50 values > 0.069 mg/L air and > 5.3 mg/L air were derived for aerosols and dust, respectively. Regarding orientative subacute inhalation (dust) with 20, 109 and 505 mg/m³ air, the NOEL was 20 mg/m³ air and the LC 50 > 505 mg/³. From 109 mg/m³ air, body weight was reduced slightly and liver enzyme activity was increased. Other changes were not found. Therefore, 5, 30 and 180 mg/m³ air were set as concentrations for this study.

- Fasting period before blood sampling for clinical biochemistry: only for glucose
Positive control:
not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: before exposure and after, and on exposure-free days
- parameters checked: appearance of visible mucous membranes of eyes and respiratory
tract, general state of muzzle skin and ear scoops, condition of coat, grooming activities, respiration, circulation (as far as evaluation was possible)

BODY WEIGHT: Yes
- Time schedule for examinations: prior to first exposure, then 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: No
- 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: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before first exposure and close to study termination
- Dose groups that were examined: all
- Number of animals per dose: 5 per group and sex
- parameters checked: changes in the retina, vitreous humor, lens, cornea and the outer surface of the eye (analyzed with a Heine indirect ophthalmoscope, pupil dilitation with Mydriatikum Roche® 5-10 minutes prior to examination)
And:
- Time schedule for examinations: daily
- Dose groups that were examined: all
- parameters checked: corneal and light reflexes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: Yes (not for glucose determination)
- Animals fasted: Not specified
- How many animals: all animals
- Parameters checked: Hematocrit, hemoglobin, leukocytes, erythrocytes, mean corpuscular erythrocyte volume (MCEV), mean erythrocyte hemoglobin concentration (MEHC), mean erythrocyte hemoglobin (MEH), thrombocyte count, differential blood count, reticulocytes, Heinz bodies, methemoglobin, thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at time of necropsy
- Animals fasted: no
- for glucose determination: fasted, non-anesthetized animals, in the week before necropsy
- How many animals: all animals
- Parameters checked:aspartate aminotransferase (ASAT/GOT), alanine aminotransferase (ALAT/GPT), glutamate dehydrogenase (GLDH), lactate dehyrogenase (LDH), plasma cholinesterase, alkaline phosphatase, sorbitol dehydrogenase (IDH), albumin, blood sugar, urea, bilirubin, creatinine, total protein, triglyceride, cholesterol, serum protein electrophoresis, T3 (not in all animals), T4, TBC, sodium, potassium, calcium, magnesium, phosphate, chloride, for liver: Cytochrome-P450, N-demethylase, O-demethylase, triglycerides

URINALYSIS: Yes
- Time schedule for collection of urine: overnight (from 8-16 h), one week before necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: sediment composition, pH, protein, glucose, blood, bilirubin, urobilinogen, ketone bodies

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: daily
- Dose groups that were examined: all animals
- Parameters checked: somatomotor system and behavioral pattern (including tremor, convulsions, hypersalivation, dyspnea, diarrhea, lethargy, sedation and coma), central nervous and autonomous symptoms

IMMUNOLOGY: No


BRONCHOALVEOLAR LAVAGE FLUID (BALF): No


LUNG BURDEN: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Organs that were weighed: brain, heart, testes, liver, lung, spleen, adrenals, kidneys, ovaries, pancreas, thyroid, thymus

HISTOPATHOLOGY: Yes
- Number of animals: all
- fixation: 10 % aqueous buffered formaldehyde solution
- staining: hemalum-eosin (HE), additional sections for glycogen and lipid staining were prepared from the liver, bone marrow smears were stained with May-Grünwald solution
- organs/tissues examined: aorta, eyes (including lid), vas deferens, epididymis (including accessory glands), brain (cerebellum and cerebum), skin (rhinarium - muzzle area and mamma area), harderian glands and extraorbital lacrimatory gland, urinary bladder (instillation fixation with Bouin's solution), heart, testes, pituitary gland, intestine (stomach, duodenum, jejunum, ileum, cecum, colon, rectum), bone (femur), bone marrow (femur and sternum), coagulating gland, head (nasopharynx, oropharynx, nasal and paranasal cavities), larynx, liver, lung (with main bronchi, instillation fixation), lymph nodes (mediastinal, cervical / mandibular, mesenteric), mamma, spleen, muscle (quadriceps femoral muscle), paratyroid glands, adrenals, sciatic nerve, kidneys with pelvis, esophagus, ovaries, pancreas, prostate, spinal cord (cervical, thoracic, lumbar), seminal vesicle, salivary glands (head), sternum, trachea, lacrimatory gland, thyroid, thymus, uterus (including tubes), vagina, tongue
Other examinations:
none
Statistics:
Where possible, means and standard deviation were calculated. For animal data, whenever possible, the upper and lower confidence limits at confidence levels of (1-a) = 95 % and (1-a) = 99 % were determined.

For body weight and laboratory tests, groups were compared with the Mann and Whitney rank test (U test) in the modification by Walter. Significance was set to be p < 0.05.

Organ weight was compared with ANOVA (variances between the groups were tested for homogeneity with Box's test). If differences occured, a pairwise post hoc (one and two-tailed) comparison of the groups is performed using the Games and Howell modification of the Tukey - Kramer-test was performed.

Statistical analysis of the urine was only performed for the pH (U-test).

Histological examinations were compared with the pairwise Fisher test. Necropsy findings were compared with the pairwise Fisher test with a preliminary R x C chi square test.
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):
- 180 mg/m³: significantly reduced body weight throughout the study in males (-5.8 to -8.6%) compared to controls

Summarized data can be found in Attachment 1 of the attached background material.
Food consumption and compound intake (if feeding study):
not examined
Description (incidence and severity):
not applicable
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not applicable
Ophthalmological findings:
no effects observed
Description (incidence and severity):
not applicable
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
- 180 mg/m³: HQUICK increase in females (+10%), increased blood coagulation time and elevated total serum bilirubin (females) compared to controls

Not considered toxicologically relevant were slightly decreased thrombocyte counts (-15.1%) in males compared to control animals. Isolated significantly altered values in SEGM, erythrocytes, hemoglobin, MCH and MCHC are considered as incidental findings not indicative for hematotoxiciy as those effects appeared occasionally in one sex without dose-response.

Summarized relevant data can be found in Attachment 2 in the attached background material
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- 30 mg/m³: increased ALAT (+24.7%) and APh (+20.4%) activity in females, decreased CHE activity in females (-26.1%, but not statistically significant), increased N-demethylase activity (+26.6%) in females, decreased a-globulin level in males (-7.4%) and females (-7.1%)
- 180 g/m³: increased ALAT (+70.3%), APh (+46.1%) activity in females, increased GLDH activity in males (+333.3%) and females (+731.6%), increased bilirubin (+33.3%) in females, decreased triglyceride levels in males (-48.8%) and females (-72.1%), decreased CHE activity in females (-28.2%, but not statistically significant), increased O-demthylase activity in males (+83.2%) and females (+21.5%), increased N-demthylase activity in males (+51.2%) and females (+76.5%), increased P450 content in males (+33.8%), decreased a-globulin level in males (-11.6%) and females (-8.2%)

Findings that were not considered toxicologically relevant were changes in urea content, since it is mainly dependent on food consumption and inhalation studies depend on long withdrawal from food and water. Thus, decreased plasma urea levels are a commonly occurrance. Decreased plasma protein levels in males was considered to be due to depressed hematocrit values (slight hypervolemia) determined in this sex and altered phosphorus levels in males and females were considered not biologically significant due to lack of dose-dependency.

Summarized relevant data can be found in Attachment 3, 4 and 5 in the attached background material. A summary of the results can be found in Attachment 9.
Endocrine findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
- 180 mg/m³: significantly higher pH in females compared to controls
Behaviour (functional findings):
no effects observed
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, treatment-related
Description (incidence and severity):
- 30 mg/m³: increased relative liver weight in females compared to controls (+8.8%, not statistically significant)
- 180 mg/m³: increased relative liver weight in females (+12.4%, statistically significant), slight reduction in relative heart (-5.3%) and thymus (-24.4%) weight in females, all compared to controls

Other changes in organ weights were slightly decreased relative brain and heart weights in males. But these were within the 2-sigma scattering ranges of pooled historical organ weight data for Wistar rats and therefore not considered significant.

Summarized relevant data can be found in Attachment 6 in the attached background material. The historical organ weight data can be found in Attachment 7.
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, non-treatment-related
Description (incidence and severity):
Effects that were observed were not considered treatment related.
180 mg/m³: decreased incidence of periportal round cell infiltration in males (not dose-dependent, incidence 0/2/0/5).

Further observations included a slight hyperemia of the upper respiratory tract in few animals which is most probably due to sacrifice procedure with diethyl ether.
Statistically significant changes were found in bone marrow morphology: proerythroblasts were significantly increased in all groups of treated males. In females, an increase was seen but this was not statistically significant. Moreover, the count in controls of males and females differed by a factor of 2.5 and in peripheral blood, concentration of reticulum cells was unchanged, so that this finding can be considered incidental. Segmented neutrophils were decreased in males and females in all dose groups but no change in granulopoiesis occurred in bone marrow (band neutrophils) or peripheral blood (differential blood count). Also, the amount of segmented neutrophils in the cell population is very low (0.5-1.5%), so that counting errors have a strong impact. Due to these factors, the finding is considered to be a coincidence.
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:
General summarized results are found in Attachment 8 in the attached background material.
Key result
Dose descriptor:
NOAEC
Effect level:
5.5 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects at this dose level
Key result
Dose descriptor:
LOAEC
Effect level:
30.5 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
haematology
organ weights and organ / body weight ratios
other: effects on the liver (increased organ weight and enzyme induction)
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
30.5 mg/m³ air (analytical)
System:
hepatobiliary
Organ:
liver
Conclusions:
The study was conducted according to GLP guidelines and according to OECD Guideline 412. Since the study was conducted in 1989, there are deviations to the current guideline as described above.

Based on the effects observed on the liver, including elevated organ weights and enzyme induction, a NOAEC of 5.5 mg/m³ and a LOAEC of 30.5 mg/m³ were derived.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
5.5 mg/m³
Study duration:
subacute
Experimental exposure time per week (hours/week):
30
Species:
rat
Quality of whole database:
One reliable guideline study is available, conducted with rats, which fulfills the criteria of a key study.
System:
hepatobiliary
Organ:
liver

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Feb - Mar 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 412 (28-Day (Subacute) Inhalation Toxicity Study
Version / remarks:
adopted 2018
Deviations:
yes
Remarks:
methodological deficiencies (MMAD exceeds the currently recommended particle size, no BALF analysis or determination of lung burden was conducted, food and water consumption were not measured)
Qualifier:
according to guideline
Guideline:
OECD Guideline 412 (28-Day (Subacute) Inhalation Toxicity Study
Version / remarks:
adopted 1981
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
adopted 1984
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Winkelmann Experimental Animal Breeders, Borchen, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 2-3 months
- Weight at study initiation: 160 - 200 g
- Fasting period before study: not applicable
- Housing: in groups of five in Type III Makrolon® cages with type S 8/15 low-dust wood shavings (Ssniff Spezialdiäten GmbH, Soest, Germany)
- Diet: Altromin® 1324 Maintenance Diet for Rats and Mice (Altromin GmbH, Lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least one week

DETAILS OF FOOD AND WATER QUALITY: Tap water met drinking water standards (Drinking Water Statute of May 22, 1986; Bundesgesetzblatt Part I, page 760), feet was regularly checked for contaminants, spot checked and analyzed

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): approx. 5o
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: dust
Type of inhalation exposure:
head only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 2.37 - <= 5.7 other: µm (for details please refer to "Any information on materials and methods")
Geometric standard deviation (GSD):
1.96
Remarks on MMAD:
Results of particle size analysis are provided under "Any information on materials and methods".

Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: PVC inhalation chamber, 30 cm diameter, 28 cm height (volume approx. 20 liters)
- Method of holding animals in test chamber: Plexiglass tubes (closed, tail of the rat was outside the tube)
- Source of air and method of conditioning air: compressed air was produced with two Model SB 270/15/350D Boge compressors in parallel. Type A 110 compressed air dryer mounted behind the compressors were used for conditioning. The regulated operating pressure of the compressors was 8-10 bars (800 - 1000 kPa)
- System of generating particulates/aerosols: Wright Dust Feeder (5 and 30 mg/m³ air) and a RBG brush-type generator (180 mg/m³ air, only on the first day because of a defect thereafter) or an Exactomat 4200 (180 mg/m³ air, starting on the second day of exposure).
- Temperature, humidity, pressure in air chamber (for details on temperature and humidity please refer to "Additional information on Materials and Methods"):
- Air flow rate: continously monitored, 20-30 L/min
- Air change rate: 30 air exchanges per hour
- Method of particle size determination: aerodynamic particle sizer with a laser velocimeter (TSI APS 3300) for 5 mg/m³, 30 and 180 mg/m³: APS 3300 instrument in conjunction with two dilution stages (TSI Model 3200)
- Treatment of exhaust air: The exhaust air was treated by passing it through a cotton
wool aerosol filter. The cotton wool filters were destroyed by incineration.

TEST ATMOSPHERE
- Brief description of analytical method used: Leybold - Heraeus measuring system, temperature and humidity were determined at 10 min intervals (automatically), the data was compared to appropriate reference data. The sensors were located in the inhalation chamber cover.
- Samples taken from breathing zone: yes

VEHICLE:
- Dust is most appropriate in accordance with potential exposure route for humans. An inhalation toxicity study using an aerosol was considered inadequate because of the low solubility of the active ingredient in nontoxic vehicle substances (maximum technically producible concentration: 69 mg/m³ air).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Gravimetric filter analysis (Sartorius SM 11106 cellulose acetate filter, pore size 0.45 µm). 100, 50 and 30 liters of test atmosphere were sampled in breathing zone for 5, 30 and 180 mg/m³ air, respectively (rate: 4 L/min). If possible, 3 air samples were taken, one at the beginning of the test, one in the middle of the test and one near the end. All concentrations stated below refer to mg test substance (95.2% purity) per m³ air. Recalculation to a 100% active ingredient basis was not performed.
Duration of treatment / exposure:
28 days, 6h per exposure
Frequency of treatment:
daily, 5 times per week
Dose / conc.:
5 mg/m³ air (nominal)
Remarks:
corresponds to 5.5 mg/m³ air as analytical concentration
Dose / conc.:
30 mg/m³ air (nominal)
Remarks:
corresponds to 30.5 mg/m³ air as analytical concentration
Dose / conc.:
180 mg/m³ air (nominal)
Remarks:
corresponds to 191.2 mg/m³ air as analytical concentration
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
- Dose selection rationale: Acute inhalation toxicity studies summarised in the technical dossier under 7.2.2 "Acute toxicity: inhalation" served as range-finding studies (M-027586-01-1). LC50 values > 0.069 mg/L air and > 5.3 mg/L air were derived for aerosols and dust, respectively. Regarding orientative subacute inhalation (dust) with 20, 109 and 505 mg/m³ air, the NOEL was 20 mg/m³ air and the LC 50 > 505 mg/³. From 109 mg/m³ air, body weight was reduced slightly and liver enzyme activity was increased. Other changes were not found. Therefore, 5, 30 and 180 mg/m³ air were set as concentrations for this study.

- Fasting period before blood sampling for clinical biochemistry: only for glucose
Positive control:
not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: before exposure and after, and on exposure-free days
- parameters checked: appearance of visible mucous membranes of eyes and respiratory
tract, general state of muzzle skin and ear scoops, condition of coat, grooming activities, respiration, circulation (as far as evaluation was possible)

BODY WEIGHT: Yes
- Time schedule for examinations: prior to first exposure, then 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: No
- 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: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before first exposure and close to study termination
- Dose groups that were examined: all
- Number of animals per dose: 5 per group and sex
- parameters checked: changes in the retina, vitreous humor, lens, cornea and the outer surface of the eye (analyzed with a Heine indirect ophthalmoscope, pupil dilitation with Mydriatikum Roche® 5-10 minutes prior to examination)
And:
- Time schedule for examinations: daily
- Dose groups that were examined: all
- parameters checked: corneal and light reflexes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: Yes (not for glucose determination)
- Animals fasted: Not specified
- How many animals: all animals
- Parameters checked: Hematocrit, hemoglobin, leukocytes, erythrocytes, mean corpuscular erythrocyte volume (MCEV), mean erythrocyte hemoglobin concentration (MEHC), mean erythrocyte hemoglobin (MEH), thrombocyte count, differential blood count, reticulocytes, Heinz bodies, methemoglobin, thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at time of necropsy
- Animals fasted: no
- for glucose determination: fasted, non-anesthetized animals, in the week before necropsy
- How many animals: all animals
- Parameters checked:aspartate aminotransferase (ASAT/GOT), alanine aminotransferase (ALAT/GPT), glutamate dehydrogenase (GLDH), lactate dehyrogenase (LDH), plasma cholinesterase, alkaline phosphatase, sorbitol dehydrogenase (IDH), albumin, blood sugar, urea, bilirubin, creatinine, total protein, triglyceride, cholesterol, serum protein electrophoresis, T3 (not in all animals), T4, TBC, sodium, potassium, calcium, magnesium, phosphate, chloride, for liver: Cytochrome-P450, N-demethylase, O-demethylase, triglycerides

URINALYSIS: Yes
- Time schedule for collection of urine: overnight (from 8-16 h), one week before necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: sediment composition, pH, protein, glucose, blood, bilirubin, urobilinogen, ketone bodies

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: daily
- Dose groups that were examined: all animals
- Parameters checked: somatomotor system and behavioral pattern (including tremor, convulsions, hypersalivation, dyspnea, diarrhea, lethargy, sedation and coma), central nervous and autonomous symptoms

IMMUNOLOGY: No


BRONCHOALVEOLAR LAVAGE FLUID (BALF): No


LUNG BURDEN: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Organs that were weighed: brain, heart, testes, liver, lung, spleen, adrenals, kidneys, ovaries, pancreas, thyroid, thymus

HISTOPATHOLOGY: Yes
- Number of animals: all
- fixation: 10 % aqueous buffered formaldehyde solution
- staining: hemalum-eosin (HE), additional sections for glycogen and lipid staining were prepared from the liver, bone marrow smears were stained with May-Grünwald solution
- organs/tissues examined: aorta, eyes (including lid), vas deferens, epididymis (including accessory glands), brain (cerebellum and cerebum), skin (rhinarium - muzzle area and mamma area), harderian glands and extraorbital lacrimatory gland, urinary bladder (instillation fixation with Bouin's solution), heart, testes, pituitary gland, intestine (stomach, duodenum, jejunum, ileum, cecum, colon, rectum), bone (femur), bone marrow (femur and sternum), coagulating gland, head (nasopharynx, oropharynx, nasal and paranasal cavities), larynx, liver, lung (with main bronchi, instillation fixation), lymph nodes (mediastinal, cervical / mandibular, mesenteric), mamma, spleen, muscle (quadriceps femoral muscle), paratyroid glands, adrenals, sciatic nerve, kidneys with pelvis, esophagus, ovaries, pancreas, prostate, spinal cord (cervical, thoracic, lumbar), seminal vesicle, salivary glands (head), sternum, trachea, lacrimatory gland, thyroid, thymus, uterus (including tubes), vagina, tongue
Other examinations:
none
Statistics:
Where possible, means and standard deviation were calculated. For animal data, whenever possible, the upper and lower confidence limits at confidence levels of (1-a) = 95 % and (1-a) = 99 % were determined.

For body weight and laboratory tests, groups were compared with the Mann and Whitney rank test (U test) in the modification by Walter. Significance was set to be p < 0.05.

Organ weight was compared with ANOVA (variances between the groups were tested for homogeneity with Box's test). If differences occured, a pairwise post hoc (one and two-tailed) comparison of the groups is performed using the Games and Howell modification of the Tukey - Kramer-test was performed.

Statistical analysis of the urine was only performed for the pH (U-test).

Histological examinations were compared with the pairwise Fisher test. Necropsy findings were compared with the pairwise Fisher test with a preliminary R x C chi square test.
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):
- 180 mg/m³: significantly reduced body weight throughout the study in males (-5.8 to -8.6%) compared to controls

Summarized data can be found in Attachment 1 of the attached background material.
Food consumption and compound intake (if feeding study):
not examined
Description (incidence and severity):
not applicable
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not applicable
Ophthalmological findings:
no effects observed
Description (incidence and severity):
not applicable
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
- 180 mg/m³: HQUICK increase in females (+10%), increased blood coagulation time and elevated total serum bilirubin (females) compared to controls

Not considered toxicologically relevant were slightly decreased thrombocyte counts (-15.1%) in males compared to control animals. Isolated significantly altered values in SEGM, erythrocytes, hemoglobin, MCH and MCHC are considered as incidental findings not indicative for hematotoxiciy as those effects appeared occasionally in one sex without dose-response.

Summarized relevant data can be found in Attachment 2 in the attached background material
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- 30 mg/m³: increased ALAT (+24.7%) and APh (+20.4%) activity in females, decreased CHE activity in females (-26.1%, but not statistically significant), increased N-demethylase activity (+26.6%) in females, decreased a-globulin level in males (-7.4%) and females (-7.1%)
- 180 g/m³: increased ALAT (+70.3%), APh (+46.1%) activity in females, increased GLDH activity in males (+333.3%) and females (+731.6%), increased bilirubin (+33.3%) in females, decreased triglyceride levels in males (-48.8%) and females (-72.1%), decreased CHE activity in females (-28.2%, but not statistically significant), increased O-demthylase activity in males (+83.2%) and females (+21.5%), increased N-demthylase activity in males (+51.2%) and females (+76.5%), increased P450 content in males (+33.8%), decreased a-globulin level in males (-11.6%) and females (-8.2%)

Findings that were not considered toxicologically relevant were changes in urea content, since it is mainly dependent on food consumption and inhalation studies depend on long withdrawal from food and water. Thus, decreased plasma urea levels are a commonly occurrance. Decreased plasma protein levels in males was considered to be due to depressed hematocrit values (slight hypervolemia) determined in this sex and altered phosphorus levels in males and females were considered not biologically significant due to lack of dose-dependency.

Summarized relevant data can be found in Attachment 3, 4 and 5 in the attached background material. A summary of the results can be found in Attachment 9.
Endocrine findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
- 180 mg/m³: significantly higher pH in females compared to controls
Behaviour (functional findings):
no effects observed
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, treatment-related
Description (incidence and severity):
- 30 mg/m³: increased relative liver weight in females compared to controls (+8.8%, not statistically significant)
- 180 mg/m³: increased relative liver weight in females (+12.4%, statistically significant), slight reduction in relative heart (-5.3%) and thymus (-24.4%) weight in females, all compared to controls

Other changes in organ weights were slightly decreased relative brain and heart weights in males. But these were within the 2-sigma scattering ranges of pooled historical organ weight data for Wistar rats and therefore not considered significant.

Summarized relevant data can be found in Attachment 6 in the attached background material. The historical organ weight data can be found in Attachment 7.
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, non-treatment-related
Description (incidence and severity):
Effects that were observed were not considered treatment related.
180 mg/m³: decreased incidence of periportal round cell infiltration in males (not dose-dependent, incidence 0/2/0/5).

Further observations included a slight hyperemia of the upper respiratory tract in few animals which is most probably due to sacrifice procedure with diethyl ether.
Statistically significant changes were found in bone marrow morphology: proerythroblasts were significantly increased in all groups of treated males. In females, an increase was seen but this was not statistically significant. Moreover, the count in controls of males and females differed by a factor of 2.5 and in peripheral blood, concentration of reticulum cells was unchanged, so that this finding can be considered incidental. Segmented neutrophils were decreased in males and females in all dose groups but no change in granulopoiesis occurred in bone marrow (band neutrophils) or peripheral blood (differential blood count). Also, the amount of segmented neutrophils in the cell population is very low (0.5-1.5%), so that counting errors have a strong impact. Due to these factors, the finding is considered to be a coincidence.
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:
General summarized results are found in Attachment 8 in the attached background material.
Key result
Dose descriptor:
NOAEC
Effect level:
5.5 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects at this dose level
Key result
Dose descriptor:
LOAEC
Effect level:
30.5 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
haematology
organ weights and organ / body weight ratios
other: effects on the liver (increased organ weight and enzyme induction)
Key result
Critical effects observed:
no
Lowest effective dose / conc.:
30.5 mg/m³ air (analytical)
System:
hepatobiliary
Organ:
liver
Conclusions:
The study was conducted according to GLP guidelines and according to OECD Guideline 412. Since the study was conducted in 1989, there are deviations to the current guideline as described above.

Based on the effects observed on the liver, including elevated organ weights and enzyme induction, a NOAEC of 5.5 mg/m³ and a LOAEC of 30.5 mg/m³ were derived.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
One reliable guideline study is available, conducted with rats, which fulfills the criteria of a key study.

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29. Jun - 22. Jul 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Version / remarks:
1981
Deviations:
yes
Remarks:
methodological deficiencies (treatment time was shorter (15 days))
Qualifier:
according to guideline
Guideline:
EPA OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)
Version / remarks:
1982
Deviations:
no
GLP compliance:
yes
Limit test:
yes
Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Interfauna, UK Limited, Huntingdon, England
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 - 13 weeks
- Weight at study initiation: 2.65 - 3.43 kg (females), 2.93 - 3.09 kg (males)
- Housing: individually in Cellidor Rabbit cages with bedding (low-dust wood granulate from Ssniff Spezialdiaten GmbH, Soest)
- Diet: "ssniff K Alleindiät für Kaninchen" (Ssniff Spezialdiäten GmbH, Soest/Westfalen, Germany), given daily with automatic rabbit feeders
- Water: tap water, ad libitum
- Acclimation period: 15 days

DETAILS OF FOOD AND WATER QUALITY: The water was checked regularly, no influence on the objective of the study was indicated

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

IN-LIFE DATES: From: 29. Jun To: 22. Jul 1988
Type of coverage:
semiocclusive
Vehicle:
other: 2% (v/v) Cremophor EL in physiological saline solution
Details on exposure:
TEST SITE
- Area of exposure: Backs and flanks
- % coverage: > 10
- Type of wrap if used: the test article was covered with a muslin cloth, that was fixed with adhesive tape (Fixomullstretch: Beiersdorf AG, Hamburg)
- Time intervals for shavings or clipplings: one day before starting treatment, then twice weekly

REMOVAL OF TEST SUBSTANCE
- Washing: soap and water
- Time after start of exposure: 6 h

TEST MATERIAL
- Amount applied: 1000 mg/kg bw/day
- Concentration: 40%
- Constant volume or concentration used: yes
- For solids, paste formed: yes

VEHICLE
- Justification for use and choice of vehicle: not specified
- Amount applied: 1.5 mL/kg bw formulation agent
- Concentration (if solution): 2% (v/v) Cremophor EL in physilogical saline solution

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test article in the application formulation was confirmed analytically for 0 and 24 h, resulting in 95.4% and 95.7% of the nominal dose for 0 and 24 h, respectively.
Duration of treatment / exposure:
15 days
Frequency of treatment:
5 days/week
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5
Control animals:
yes
Details on study design:
- Dose selection rationale: The dose was set on the basis of the results of a preliminary study performed on the rabbit (study no: T4027690)
- Fasting period before blood sampling for clinical biochemistry: not reported
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily

DETAILED CLINICAL OBSERVATIONS: No data

DERMAL IRRITATION: Yes
- Time schedule for examinations: daily

BODY WEIGHT: Yes
- Time schedule for examinations: prior to start of the experiment and weekly thereafter (on Days 0, 7, 14, 22 and 23)

FOOD CONSUMPTION:
- 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: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before commencement and after the three-week treatment.
- Anaesthetic used for blood collection: no data
- Animals fasted: not reported
- How many animals: all animals
- Parameters checked: erythrocyte and leucocyte count, hemoglobin, MCV (calculation of mean corpuscular volume of individual erythrocytes), hematocrit, thrombocyte count, MCH (mean hemoglobin content of individual erythrocytes), MCHC (mean hemoglobin concentration of erythrocytes), differential blood count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: as for hematology
- Animals fasted: no data
- How many animals: all animals
- Parameters checked: Aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, urea, glucose, creatinine, bilirubin, total protein, cholesterin, inorganic phosphate, sodium, potassium, calcium, chloride;
- Time schedule for collection of liver tissue: after necropsy
- How many animals: all animals
- Parameters checked: N-demethylase, O-demethylase, cytochrome P-450, triglycerides

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Organs fixed in Bouin's fluid: treated and untreated skin, thyroids, lung, heart, liver, spleen, kidneys, adrenals, testes, epididymides, ovaries, uterus, sternum
- Organs fixed in formol calcium: liver, kidney
- absolute and relative organ weights recorded for: brain, thyroids, heart, lung, liver, kidneys, adrenals, spleen, testes, ovaries

HISTOPATHOLOGY: Yes
- Organs checked: treated and untreated skin, thyroids, lung, heart, liver, spleen, kidneys, adrenals, testes, epididymides, ovaries, uterus, sternum
Statistics:
Mean and standard deviation were calculated for food consumption, body weights, and for hematological observations and clinical chemistry. Groups were compared using the two-tailed U Test according to Mann and Whitney and Wilcoxon. Significance was defined as p < 0.05
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Soft feces was observed on Day 7 in one male animal of the control group and reduced food intake, stiff head position, transfixed bulging eyes, open mouth, salivation and wet nose in one female of the control group (caused by illness)
Dermal irritation:
no effects observed
Description (incidence and severity):
Redness and skin fold measurements did not differ for control and treated animals. Summarized data can be found in Attachment 1 and 2 in the attached background material.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Control: One female was sacrificed (Day 3) because of a fractured femur (the female was replaced)
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weights of the treated animals did not significantly differ from those of the control group.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Increased food consumption in treated males vs control animals in the first week (3.7%, statistically significant) were observed. Further, reduced food consumption in one control female on Day 14 caused by an illnes was evident.
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not applicable
Ophthalmological findings:
not examined
Description (incidence and severity):
not applicable
Haematological findings:
no effects observed
Description (incidence and severity):
not applicable
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
not applicable
Endocrine findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
not examined
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):
- 1000 mg/kg bw: absolute and relative brain weight increased in males (7.3 and 6.8%, respectively), reduced absolute ovary weight (females, 35.3%), all compared to controls

Since these finidngs were slight, they were considered incidental.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No difference was found between the control and the treatment group.
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
not applicable
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
not applicable
Other effects:
not examined
Description (incidence and severity):
not applicable
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed up to and including this dose (highest dose tested)
Key result
Critical effects observed:
no
Conclusions:
No dermal irritation or any sign of toxicity was observed in this repeated dose study over 15 days with 1000 mg/kg bw/day of the test substance applied to the skin of rabbits. Accordingly, the NOAEL is set at 1000 mg/kg bw/day. The study was conducted according to OECD Guideline 410 but the application duration was shorter than suggested (15 days instead of at least 21 days). Therefore, the study is considered reliable with restrictions. However, as no signs indicating a toxic effect were observed, the NOAEL is acceptable.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
One reliable guideline study is available, conducted with rats, which fulfills the criteria of a key study.

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29. Jun - 22. Jul 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Version / remarks:
1981
Deviations:
yes
Remarks:
methodological deficiencies (treatment time was shorter (15 days))
Qualifier:
according to guideline
Guideline:
EPA OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)
Version / remarks:
1982
Deviations:
no
GLP compliance:
yes
Limit test:
yes
Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Interfauna, UK Limited, Huntingdon, England
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 - 13 weeks
- Weight at study initiation: 2.65 - 3.43 kg (females), 2.93 - 3.09 kg (males)
- Housing: individually in Cellidor Rabbit cages with bedding (low-dust wood granulate from Ssniff Spezialdiaten GmbH, Soest)
- Diet: "ssniff K Alleindiät für Kaninchen" (Ssniff Spezialdiäten GmbH, Soest/Westfalen, Germany), given daily with automatic rabbit feeders
- Water: tap water, ad libitum
- Acclimation period: 15 days

DETAILS OF FOOD AND WATER QUALITY: The water was checked regularly, no influence on the objective of the study was indicated

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

IN-LIFE DATES: From: 29. Jun To: 22. Jul 1988
Type of coverage:
semiocclusive
Vehicle:
other: 2% (v/v) Cremophor EL in physiological saline solution
Details on exposure:
TEST SITE
- Area of exposure: Backs and flanks
- % coverage: > 10
- Type of wrap if used: the test article was covered with a muslin cloth, that was fixed with adhesive tape (Fixomullstretch: Beiersdorf AG, Hamburg)
- Time intervals for shavings or clipplings: one day before starting treatment, then twice weekly

REMOVAL OF TEST SUBSTANCE
- Washing: soap and water
- Time after start of exposure: 6 h

TEST MATERIAL
- Amount applied: 1000 mg/kg bw/day
- Concentration: 40%
- Constant volume or concentration used: yes
- For solids, paste formed: yes

VEHICLE
- Justification for use and choice of vehicle: not specified
- Amount applied: 1.5 mL/kg bw formulation agent
- Concentration (if solution): 2% (v/v) Cremophor EL in physilogical saline solution

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test article in the application formulation was confirmed analytically for 0 and 24 h, resulting in 95.4% and 95.7% of the nominal dose for 0 and 24 h, respectively.
Duration of treatment / exposure:
15 days
Frequency of treatment:
5 days/week
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5
Control animals:
yes
Details on study design:
- Dose selection rationale: The dose was set on the basis of the results of a preliminary study performed on the rabbit (study no: T4027690)
- Fasting period before blood sampling for clinical biochemistry: not reported
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily

DETAILED CLINICAL OBSERVATIONS: No data

DERMAL IRRITATION: Yes
- Time schedule for examinations: daily

BODY WEIGHT: Yes
- Time schedule for examinations: prior to start of the experiment and weekly thereafter (on Days 0, 7, 14, 22 and 23)

FOOD CONSUMPTION:
- 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: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before commencement and after the three-week treatment.
- Anaesthetic used for blood collection: no data
- Animals fasted: not reported
- How many animals: all animals
- Parameters checked: erythrocyte and leucocyte count, hemoglobin, MCV (calculation of mean corpuscular volume of individual erythrocytes), hematocrit, thrombocyte count, MCH (mean hemoglobin content of individual erythrocytes), MCHC (mean hemoglobin concentration of erythrocytes), differential blood count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: as for hematology
- Animals fasted: no data
- How many animals: all animals
- Parameters checked: Aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, urea, glucose, creatinine, bilirubin, total protein, cholesterin, inorganic phosphate, sodium, potassium, calcium, chloride;
- Time schedule for collection of liver tissue: after necropsy
- How many animals: all animals
- Parameters checked: N-demethylase, O-demethylase, cytochrome P-450, triglycerides

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Organs fixed in Bouin's fluid: treated and untreated skin, thyroids, lung, heart, liver, spleen, kidneys, adrenals, testes, epididymides, ovaries, uterus, sternum
- Organs fixed in formol calcium: liver, kidney
- absolute and relative organ weights recorded for: brain, thyroids, heart, lung, liver, kidneys, adrenals, spleen, testes, ovaries

HISTOPATHOLOGY: Yes
- Organs checked: treated and untreated skin, thyroids, lung, heart, liver, spleen, kidneys, adrenals, testes, epididymides, ovaries, uterus, sternum
Statistics:
Mean and standard deviation were calculated for food consumption, body weights, and for hematological observations and clinical chemistry. Groups were compared using the two-tailed U Test according to Mann and Whitney and Wilcoxon. Significance was defined as p < 0.05
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Soft feces was observed on Day 7 in one male animal of the control group and reduced food intake, stiff head position, transfixed bulging eyes, open mouth, salivation and wet nose in one female of the control group (caused by illness)
Dermal irritation:
no effects observed
Description (incidence and severity):
Redness and skin fold measurements did not differ for control and treated animals. Summarized data can be found in Attachment 1 and 2 in the attached background material.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Control: One female was sacrificed (Day 3) because of a fractured femur (the female was replaced)
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weights of the treated animals did not significantly differ from those of the control group.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Increased food consumption in treated males vs control animals in the first week (3.7%, statistically significant) were observed. Further, reduced food consumption in one control female on Day 14 caused by an illnes was evident.
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not applicable
Ophthalmological findings:
not examined
Description (incidence and severity):
not applicable
Haematological findings:
no effects observed
Description (incidence and severity):
not applicable
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
not applicable
Endocrine findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
not examined
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):
- 1000 mg/kg bw: absolute and relative brain weight increased in males (7.3 and 6.8%, respectively), reduced absolute ovary weight (females, 35.3%), all compared to controls

Since these finidngs were slight, they were considered incidental.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No difference was found between the control and the treatment group.
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
not applicable
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
not applicable
Other effects:
not examined
Description (incidence and severity):
not applicable
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed up to and including this dose (highest dose tested)
Key result
Critical effects observed:
no
Conclusions:
No dermal irritation or any sign of toxicity was observed in this repeated dose study over 15 days with 1000 mg/kg bw/day of the test substance applied to the skin of rabbits. Accordingly, the NOAEL is set at 1000 mg/kg bw/day. The study was conducted according to OECD Guideline 410 but the application duration was shorter than suggested (15 days instead of at least 21 days). Therefore, the study is considered reliable with restrictions. However, as no signs indicating a toxic effect were observed, the NOAEL is acceptable.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
One reliable guideline study is available, conducted with rats, which fulfills the criteria of a key study.

Mode of Action Analysis / Human Relevance Framework

No data available.

Additional information

Repeated dose toxicity 

 

 An extensive data base is available to assess the intrinsic toxicological properties of the test substance in regard to subacute, subchronic and chronic toxicity in rodents and non-rodents following oral administration. Further, subacute toxicity studies are available in rodents following inhalation and dermal application. 

Oral route 

 Subchronic toxicity studies in rodents 

 Studies performed in rats 

 M-007967-01-1 

In a 96-day oral feeding study (M-007967-01-1), Wistar rats were exposed to the test substance in concentrations of 150, 600 and 2400 ppm via the diet.Groups consisted of 10 males and 10 females, a control group received the diet without test substance. Moreover, an additional recovery control and high dose group was included. The applied concentrations were selected based on a previous study (unpublished Bayer AG report no. 17279, M- M-027593-01-1) in which 120, 600 or 3000 ppm test substance were administered to Wistar rats for 14 weeks. At 3000 ppm, weight reduction was marked, and some evidence of liver damage and enlarged testicular tubules occurred. At 600 ppm, body weight was slightly reduced (for further details, please refer to the summary provided below).

Analytical measurements confirmed stability and homogeneity of the test substance in the diet.Actual ingested doses were 14.0, 60.9 and 300.2 mg/kg bw/day in males and 20.3, 83.3 and 422.2 mg/kg bw/day in females. Cage sides were observed daily and clinical signs, body weight and food consumption were measured weekly. Ophthalmoscopic examinations were performed pre-exposure and pre-terminal (Week 14 or Week 17 for recovery animals). In Week 5 and 14 (Week 17 for recovery group), blood was taken from non-fasted animals for hematology and clinical chemistry. Urine was collected in the Week 14 (or Week 17 for recovery animals). All animals underwent gross pathological examinations. Likewise, histopathology was performed for all animals.  

 There was no mortality observed during the study and animals of the treatment groups did not show different clinical signs compared to control animals. Body weight was reduced slightly (8%) in males of the 600 ppm group and in both sexes at 2400 ppm (14-16%). This was not reversible during the recovery period. Food consumption was increased in males and females of the highest dose group (33% and 42%, relative to body weight for males and females, respectively). Reduced protein concentrations in blood were found for males in all groups (reduced from 3.1-7.6%) and in females in the highest dose group (-7.7 and -5.4% in Week 5 and 14, respectively). Other observations in hematology and clinical biochemistry in the high dose group included (but were not limited to) lower triglycerides in males after 14 (-51.9%) and 17 (-14.7%) weeks, and in females after 17 weeks (-17.6%) and longer thromboplastin times for both sexes. Alkaline phosphatase activity was increased in males in Week 5 (+14.6%) and ALAT activity in Week 14 (+24.5%). Moreover, histopathological changes were noticed in the liver of high dose male animals (round cell infiltrates, isolated cell necrosis, swollen liver cell nuclei). These changes were not found in the recovery group. Absolute organ weights of liver and kidney were reduced in both sexes at 2400 ppm. For kidney, a secondary effect to reduced body weight is considered as reason for the observed alteration, whereas a direct toxic effect on the liver cannot be excluded. Overall, the hepatic lesions determined in high dose males during histopathology including cellular necrosis, round-cell infiltration, swollen cellular nuclei, cytoplasmic lesions and changes in blood levels of cholesterol, triglyceride, protein, and albumin together with increases in blood coagulation time, elevated total serum bilirubin levels, and elevated activities of the enzymes ALT, AP, and GLDH, clearly indicate a disturbance of hepatic function due to hepatotoxicity. In regard to classification purposes, it has to be considered that histopathologically determined lesions in the liver were reversible in the recovery group and hence, classification in regard to STOT-RE is not warranted. 

 Taken together, NOAELs of 150 and 600 ppm were derived for males and females, corresponding to 14.0 and 83.3 mg/kg bw/day, respectively.Based on decreased body weights, LOAELs of 600 ppm and 2400 ppm were determined in males and females, corresponding to 60.9 and 422.2 mg/kg bw/day, respectively. In males, sings indicative for reversible liver damage and hepatic dysfunction were observed at 2400 ppm, corresponding to 300.2 mg/kg bw/day.

 Since the study was conducted in 1988, some deviations according to the current version of the OECD guideline are present, including missing organ weights for epididymides, prostate, uterus, ovaries, thymus, pituitary gland, thyroid and heart; further brain histopathology was not specified, mammary glands were not examined in histopathology, T4, T3 and TSH were not assessed and sensory reactivity was not investigated.  

 

M-027593-01-1

 Supporting evidence in regard to subchronic oral toxicity can be gained from a pilot range-finding study that was conducted to establish the dosage for a chronic toxicity study on Wistar rats (M-027593-01-1).During the study, groups of ten male and female Wistar rats were orally treated with the test substance at concentrations of 120, 600 or 3000 ppm in the diet for a period of 98 days, corresponding to 11, 56.9 and 408.9 mg/kg bw/day for males and 14.6, 77.8 and 513.2 mg/kg bw/day for females. No toxicologically significant clinical findings and no increase in mortality were observed at doses up to 3000 ppm. Food consumption was increased at the 3000 ppm, whereas water intake remained unaffected. At and above 600 ppm the body weight gain was retarded reaching statistical significance at 3000 ppm (600 ppm: males: -6 to -7 %; females: -10 %; 3000 ppm: males: -20 %; females: -15 %). No signs indicative for hematotoxicity were observed in the treatment groups at doses to 3000 ppm. Significantly elevated alkaline phosphatase and depressed glucose levels were determined in males and females of the 3000 ppm dose group and males showed also lower cholesterol levels. No treatment-related effects in urinalysis were observed. The gross pathological observations gave no indications of treatment-related organ changes. No weight differences indicating organic injury were apparent at doses up to 3000 ppm. Histopathological degenerative changes in the epithelium of the testicular tubuli were apparent in 5 out of 10 males of the 3000 ppm dose group; spermatogenesis was not negatively affected. A multifocal cell necrosis was diagnosed in the liver of one male in the 3000 ppm dose group. There were no indications of toxic effects on the other organs examined. Under the conditions described, 120 ppm was tolerated without observed effects. Therefore, the NOAEL was set to 120 ppm corresponding to 14.6 and 11.0 mg/kg bw/day for males and females, respectively, based on depressed body weight gain at 600 ppm (corresponding to 56.9 and 77.8 mg/kg bw/day in males and females, respectively). Doses of 100, 300 and 900 ppm were chosen for the chronic study on rats. 

 The study was in main accordance to OECD guideline 408 and GLP.No robust study summary was prepared regarding the overall quality of the extensive data pool of OECD-compliant sub-chronic and chronic toxicity data, especially in regard to the low number of animals included in this dose-range finding study.

 Studies performed in mice 

  M-027600-01-1 

 A dose range-finding study was conducted in a second rodent species in order to establish the dosage for a cancerogenesis study on B6C3F1 mice (M-027600-01-1).During the study, groups of mice (10 males and 10 females) were treated with 120, 600 or 3000 ppm in the diet for a period of up to 107 days. No treatment-related increased incidence of clinical symptoms was observed at doses up to 600 ppm. In the high dose group animals displayed poor general condition, rough coats and retarded weight gain. Food consumption was increased about 11 % (males) and 41 % (females). 7 males and 7 females receiving 3000 ppm died following blood withdrawal presumably due to poor general condition and pronounced weight loss. Male mice treated with 600 ppm exhibited slightly retarded weight gains. No signs of treatment-related hematotoxicity were observed in the high dose group, but indications of functional and morphological changes in the liver were determined at 3000 ppm, including significantly decreased urea and cholesterol levels (males), as well as lower alanine aminotransferase and glucose values (females). The alkaline phosphatase activities were significantly increased in males and females at 3000 ppm. Organ weight differences observed at 3000 ppm for liver, heart, spleen, kidneys, testes and adrenals were attributed to the distinct body weight decreases. No treatment-related histopathological findings were obtained. Under the conditions described, 120 ppm (corresponding to 17 mg/kg bw/day) was defined as NOAEL based on decreases in body weight gain at the next higher dose level of 600 ppm. Doses of 100, 330 and 1000 ppm were chosen for the cancerogenesis study on mice. 

 The study was conducted according to GLP and in principles similarly to OECD guideline 408.No robust study summary was prepared regarding the overall quality of the extensive data pool of OECD-and GLP-compliant sub-chronic and chronic toxicity data, especially in regard to the low number of animals included in this dose-range finding study.

 

 Chronic toxicity in rodents 

 Studies performed in rats 

 M-027741-02-1 (supplemented by M-027135-01-1) 

A 2-year combined chronic toxicity and cancerogenicity study (M-027741-02-1 and suppl. M-027135-01-1) was performed in Wistar rats according to OECD guideline 453 and GLP. The test substance was administered via diet to 50 animals per sex and dose at levels of 100, 300, 900 (main study) and 1800 ppm (supplemental study), corresponding to 5.7, 16.9, 51.3 and 102.6 mg/kg bw/day (males) and 7.6, 24.9, 73.0 and 143.7 mg/kg bw/day (females). Control animals received plain diet. Additionally, 10 animals per sex and dose were used for interim sacrifice after 1 year.The top dose group (1800 ppm) was treated in a supplementary MTD finding study (M-027135-01-1) under the same conditions as compared to the main study. 

Analytical verification of test item stability, homogeneity and concentration in the diet was performed. The test item was stable in the food for 14 days and homogeneity was confirmed, since the standard deviations of analyzed samples obtained from different areas of mixing bottles did not exceed 10%. The mean achieved analytical concentrations were within a range of 98 to 100% of respective nominal concentrations. 

 

No treatment-related effects on clinical signs, mortality, food consumption, hematology, organ weights and gross pathological findings were observed. Body weight gain was not affected at doses up to 300 ppm, but slightly lower (5% in males, 8% in females) at the 900 ppm level and significantly retarded (12% in males, 11% in females) at 1800 ppm, compared to the controls. Thus, the 1800 ppm level is regarded as maximum tolerated dose due to the significant reduction in the body weight. Also in the 1800 ppm group, significantly increased aspartate aminotransferase (both sexes) and creatine kinase levels were found at terminal sacrifice. After 12 months, significantly depressed figures for the absolute liver (14 %) and kidney (13 %) weights were observed in female rats at 900 ppm. At the end of the study (24 months), these organ weights were still significantly lower compared to control (4 and 5 % for liver and kidney, respectively), but not considered as evidence for organ damage due to the lower deviation between treated and control animals. In male rats only in the high-dose group a decrease in liver weight (13 %) at the interim sacrifice (12 months) was recorded. Alterations in liver and kidney organ weights were not attributable to liver or kidney damage, but must be seen in direct relation to the reduced body weight gain in the affected dose groups. Histopathological assessment of these organs afforded no evidence for treatment-related lesions. Mineralized particles (MPs) in the thyroid glands were observed in both sexes. This effect was dose-dependent and observed at the interim and final sacrifice. 300 ppm is considered as a threshold for this effect in male rats and 900 ppm in female rats. However, such findings occur as spontaneous phenomena in the sense of senile involution of isolated follicles in ageing rats, and thus, the observed effects are regarded as effect in the sense of premature biological ageing processes. As plasma levels of thyreotropin, triiodothyronine and thyroxine were unaffected by treatment, a toxicological effect on thyroid function is excluded. As assessed by the U.S. Environmental Protection Agency (M-677550-01-1, for details please refer to the endpoint study record in the technical dossier in chapter 7.9.3), no convincing evidence of a potential interaction with thyroid pathways exists thereby supporting that the test substance does not adversely influence thyroid function in regard to its endocrine function. Further, no interaction with the estrogen or androgen pathways was concluded. Thus, based on weight of evidence considerations, no endocrine disrupting properties were identified. 

Taking together a NOAEL of 100 ppm (equivalent to 5.7 mg/kg bw/day) was derived for male rats and a NOAEL of 300 ppm (equivalent to 24.9 mg/kg bw/day) was established for female rats based on increased incidence of colloid mineralization of the thyroid gland follicles at the next higher test concentrations (300 ppm in males and 900 ppm in females, corresponding to 16.9 and 73 mg/kg bw/day, respectively). In the absence of altered plasma hormone levels, an effect on thyroid function is excluded, as concluded previously in the biocidal assessment report (Assessment Report, 2011). No cancerogenic potential of imidacloprid may be inferred from the nature, incidence or chronology of tumours at doses up to 1800 ppm.

The study was performed under GLP condition and in accordance with OECD TG 453 (adopted 1981). Deviations to the current version of the guideline (adopted 2018) are considered minor and not to be expected to have an impact on the reliability of this study.The present study is therefore considered reliable and valid. 

 

 Studies performed in mice  

 M-026310-01-1 (supplemented by M-026038-01-1) 

Additionally, a 2-year carcinogenicity study (M-026310-01-1 and suppl. M-026038-01-1) was performed in B6C3F1 mice. The test item was administered via diet to 50 animals per sex and dose at nominal concentrations of 100, 330, 1000, and 2000 ppm, corresponding to 20.2, 65.6, 208.2, and 413.5 mg/kg bw/day (males) and 30.3, 103.6, 274.4, and 423.9 mg/kg bw/day (females). Control animals received plain diet only. Additionally, 10 animals per sex and dose were used for interim sacrifice after 1 year of dosing.The top dose group (2000 ppm) was treated in a supplementary MTD finding study (M-026038-01-1) under the same conditions as compared to the main study.  

No treatment-related effects on mortality, organ weights, gross or histopathological findings were observed. Also, clinical signs observed were considered to be non-treatment-related since incidences were similar between groups. However, at 2000 ppm a squeaking and twittering type of vocalization was perceived in male and female mice from the inception of the study onwards. Further treatment-related effects noted were a decrease of body weight and food consumption in male and female mice. Body weight gain was comparable to control mice at doses of up to and including 330 ppm (both sexes), but was slightly lower at 1000 ppm (-10% for males and -5% for females) and markedly decreased at a dose level of 2000 ppm (-29% for males and -26% for females). This effect was accompanied by a slight decrease in food consumption in the 1000 ppm group females (-10%), which was even enhanced at 2000 ppm (-35% for females and -10% for males). No effect on food consumption was observed in male mice treated with up to and including 1000 ppm test item and in female mice treated with up to and including 330 ppm test item. Hematological analysis revealed a treatment-dependent decrease of leukocyte counts in the 2000 ppm dose group (both sexes), but only in individual cases differences in e.g. erythrocyte count, hematocrit, mean cell hemoglobin or platelet count were observed between 1000 and 2000 ppm dose groups and the controls. Also in the 2000 ppm group, elevated levels of alkaline phosphatase were found in the plasma, while cholesterol levels were decreased compared to control (for further details, please refer to the robust study summary prepared in section 7.7 in the technical dossier). Hence, a NOAEL of 330 ppm (equivalent to 65.6 mg/kg bw/day in males and 103.6 mg/kg bw/day in females) was derived in B6C3F1 mice, based on the absence of test item-induced general toxicity at this dose level and decreased body weight at the next higher dose level of 1000 ppm. The nature, location incidence and latency periods of the tumours detected afforded no evidence for an oncogenic effect of imidacloprid. 

The study was performed under GLP conditions and in accordance with OECD TG 451 (adopted 1981).Deviations to the current version of the guideline (adopted 2018) are only minor and not considered to have an impact on the outcome of this study. 

 

Subacute, subchronic and chronic oral toxicity studies in non-rodents 

  Subacute toxicity 

  Studies performed in dogs 

 

 M-027014-01-1 

 Data on subacute toxicity in dogs is available based on a non-GLP compliant dose-range finding study performed according to OECD guideline 409 (1981, adapted to the objectives of a range-finding study). 

During the 28-day oral range-finding toxicity study, groups of four beagle dogs (two males and two females) were treated with 200, 1000 and 5000 ppm for 4 weeks (M-027014-01-1).This corresponds to average test article intakes of 7.3, 31.0 and 49.0 mg/kg bw/day, respectively. All animals treated at 5000 ppm died or were sacrificed in moribund conditions. Treatment-related clinical signs seen in these animals included ataxia, tremor and occasionally vomiting. A detailed discussion on the impact of tremor/trembling in regard to hazard assessment is included in the biocidal assessment report (Assessment Report, 2011) which is summarized below. There were no deaths or clinical signs at 1000 or 200 ppm. Marked reduction in food consumption accompanied by weight loss was recorded in animals treated with 5000 ppm. Slight, transient reductions in food consumption were recorded in dogs at 1000 ppm whereas those at 200 ppm were unaffected by the treatment. Body weight was not affected by the treatment at either 200 or 1000 ppm. At 5000 ppm, body weight was markedly reduced whereas it was virtually unaffected at 1000 ppm. Regarding the hearing test, ophthalmology examinations, hematology and urinalysis, no treatment-related effects were observed up to the highest dose tested. Effects in clinical chemistry were observed in the high dose group including a slight increase in urea and bilirubin, minimal increase in gamma glutamyl transferase, a marked decrease in triglycerides, and altered values of lipids, cholesterol, total protein and alkaline phosphatase. Further, a moderate decrease in alpha-1 globulin and slight decreases in T3 were observed. In total, all these changes in clinical chemistry parameters at 5000 ppm are indicative for hepatic toxicity and may be associated with the hepatic atrophy seen microscopically or may be secondary to inappetance and weight loss. At autopsy, slight increases in cytochrome P450 were observed in the samples of livers taken from males and females treated with 1000 ppm. This parameter was not measured in dogs from the high dose group. Some effects evident in the highest dose group may be regarded as secondary to loss of body weight. The morphological alterations such as hepatocellular atrophy, follicular atrophy of the thyroid glands, bone marrow atrophy, advanced involution of the thymus and the testicular tubule degeneration were considered to be probably due to the severe direct toxic effects of the test article. The low dose of 200 ppm was free of treatment-related histopathology findings. Therefore, the NOAEL was set at 200 ppm corresponding to 7.3 mg/kg bw/day (combined sex) based on liver and thyroid effects (hepatocellular hypertrophy, follicular atrophy of thyroid) in single animals at 1000 ppm. Due to the low number of animals and due to the fact that GLP- and OECD guideline studies on subchronic and chronic toxicity in the dog are available, no detailed study summary is prepared from the dose-range finder in the technical dossier. 

 

Sub-chronic toxicity 

Studies performed in dogs 

M-026540-01-1 

In the 90-day oral feeding study (M-026540-01-1), groups of four male and four female beagles were treated with 200, 600 and 1800 ppm of the test substance.200, 600 and 1800/1200 ppm corresponded to actual ingested doses of 7.8, 23.0 and 42.0 mg/kg bw/day, respectively as combined values for both sexes (recalculated based on weekly food consumption and body weight). Due to low food consumption observed in the high dose group, the highest dose was reduced from Week 4 to 1200 ppm. The dosage was initially established based on the results obtained in the subacute pilot range-finding study described above (M-027014-01-1, summarized above). The test substance was evenly blended with the food and analytical measurements confirmed stability of the test substance in the dry diet (14 days) and in moist food (24 hours). Furthermore, the test substance was homogeneously distributed in the food mix. 

Appearance and behavior of all animals was repeatedly checked each day during feeding, grooming, cleaning of the stall and during exercising.Detailed clinical observations e.g. reflex tests, body temperature and pulse rate were examined before starting the study and during the third, seventh and 13th weeks of treatment. Body weights were recorded weekly. Ophthalmoscopic examinations were performed before starting the study and during the seventh and 13th weeks of treatment. Urine and blood for clinical chemistry and hematology was collected from all animals before starting the study and during the third, seventh and 13th weeks of treatment. All animals underwent gross pathological examination and histopathology was performed for all animals. 

No mortality was observed during the study and there was no clear evidence of treatment-related clinical signs.Repeated instances of reduced food consumption were observed starting at 600 ppm which presumably resulted from the unpleasant taste of test substance. Further, lower body weight gains were observed in most animals of the high dose group during the first 3 - 6 weeks of the study. Further, from the start of exposure up to Week 5, all animals of the high dose group showed “trembling” or “slight trembling” on one or several occasions (up to six times/week). “Severe” trembling was observed during Week 1 at least once, sometimes up to five times during this time. As a consequence to reduced body weight gain and trembling, the dietary concentration was lowered from 1800 down to 1200 ppm from Week 5 on. Tremor and/or trembling were no longer observed until the end of the study when feed concentration had been lowered to 1200 ppm. Similarly, no significant differences in body weight gains compared to the controls were determined after the dose had been reduced from 1800 to 1200 ppm starting with Week 4. 

At the end of the study body weights of the treated animals were comparable to those seen in the control group.However, repeated incomplete food consumption was also observed in three animals which received 1200 ppm, and hence Pal® dog food was repeatedly blended into the food of these dogs to improve their appetite, after which complete food consumption was observed. No treatment-related changes were observed with regards to ophthalmology, hematology, clinical biochemistry, urinalysis, organ weights, gross pathology and histopathology. 

Overall, repeated instances of incomplete food consumption and consequently lower body weights were observed at 600 and 1200/1800 ppm.However, at 600 ppm, food consumption was reduced only in two females particularly during the first half of the study and mean food intake was comparable to control at 600 ppm in males and females. Further, body weight gain was only decreased in females at 600 ppm but not in males. Effects on food consumption and body weight development are considered mainly as a palatability problem and not as toxic effects.

Therefore, a NOAEL of 600 ppm corresponding to 23.0 mg/kg bw/day (both sex) was derived due to decreased body weight gain and reduced food intake at 1800/1200 ppm.

The study was performed under GLP conditions and according to OECD TG 409 (adopted 1981). Deviations to the current version (adopted 1998) are minor. Thus, the study is considered reliable and valid. 

 

 Long-term toxicity 

Studies performed in dogs 

M-027093-01-1 

In a 52-weeks oral feeding study (M-027093-01-1), male and female beagle dogs (4 per sex and dose) were exposed to the test item via the diet at dose levels of 200, 500 and 1250/2500 ppm (in the top dose group the concentration of the test item was increased from 1250 ppm to 2500 ppm from study Week 17 onwards).Control animals received plain diet. 

Analytical verification of test item stability, homogeneity and concentration in the diet was performed.The test item was stable in the food for 21 days and homogeneity was acceptable as the mean concentration (top, middle, bottom within the mixing container varied about 6%). The achieved overall mean concentrations were in a range of 95 to 101% of the respective nominal concentrations. Taking food consumption and body weight data into account the animals were exposed to 6.1, 15, 41/72 mg test item/kg bw/day for the groups 200, 500 and 1250/2500 ppm, respectively. 

No mortality or clinical signs indicative for toxicity were observed.Body weight and weight changes were unaffected by treatment. Slightly different patterns of weight gain were observed without any dose-dependency and are therefore not considered as treatment-related. Further, ophthalmological, hematological, histo- or gross pathological examinations were unremarkable. Food consumption was slightly lower (up to 5% lower in males and up to 9% lower in females) in the top dose group for one, two or three weeks of exposure following first administration of the test item and following the increase in test item intake from Week 17 onwards. Following this short period there was no treatment-related effect on food consumption in this group. There was no effect on food consumption at 200 and 500 ppm.Cholesterol levels were significantly increased in females compared to control animals in Weeks 13 and 26 and slightly higher in Week 52. Liver cytochrome P450 levels were also significantly increased in both sexes of the 1250/2500 ppm group compared to control. Further, slight increases of relative liver weights were observed. Thus, taking all these observations together, the liver is considered as target system after chronic exposure to the test substance in beagle dogs. However, based on the induction of cytochrome P450 enzymes and the slightly increased liver weights, those effects are considered as indicative for adaptation processes, especially in the absence of relevant histopathological findings, and were not considered as adverse. 

Taken together, based on the outcome of the conducted chronic t.oxicity study in dogs, a NOAEL of 500 ppm (corresponding to 15 mg/kg bw/day) for male and female beagle dogs was established based on test item-induced effects on liver parameters (liver/brain weight ratio, plasma cholesterol and cytochrome P450 induction) at 1250/2500 ppm (corresponding to 41/72 mg/kg bw/day). 

The study was performed under GLP condition and in accordance with OECD TG 452 (adopted 1981). Deviations to the current version of the guideline (adopted 2018) are considered minor and not to be expected to have an impact on reliability of this study, which is considered reliable and valid. 

 

Discussion on inconsistent results of repeated dose data in dogs 

M-029901-01-1 

As discussed previously (M-029901-01-1, 1990), a certain inconsistency in the results of the repeated dose toxicity studies performed in dogs exists in regard to a) food intake and b) the doses that produced no effect. 

Overall, in regard to food intake, a low level of acceptance at 1800 ppm was observed in the 13-week study with the aqueous food-mix (M-026540-01-1), so that the concentration had to be reduced to 1200 ppm starting at the 4th week. In contrast, in the chronic toxicity study (M-027093-01-1), doses of 1250 ppm resulted in no relevant findings which led to an increase of the exposure concentration to 2500 ppm starting in the 17th week. 

In regard to effect levels, the 13-week study indicates an apparently higher toxicity for NTN 33893 than the 52-week study. After exposure for 52 weeks, 500 ppm proved to be the no-effect concentration, whereas administration of 600 ppm in the 13-week study resulted in a distinct fall in food intake, which seems to be related to a palatability problem and periodic tremors in the animals. Since, apart from the method of feeding, the basic overall conditions of the 52-week and 13-week studies were almost comparable, the initial conclusion to be drawn is one to the effect that the apparently inconsistent results of the two studies are attributable to the method of feeding and a related problem of palatability. To evaluate the influence of the different feeding possibilities, a comparative study was performed at the Department of Toxicology, Wuppertal, Germany, in which 1200 ppm test material was supplemented to 2 female and 2 male beagle dogs each in either pelleted (similar to the 52-week study) or aqueous food-mix (similar to the 90 day study). The conditions in regard to type of food, and the mixing and provision of it were thus exactly the same as in the 52-week and 13-week studies. The comparative study supported the assumption that pelleted food was more acceptable to the animals and was almost all eaten, confirming the results of the 52-week study in regard to this parameter. The aqueous mix on the other hand was plainly refused by the animals, thus resulting in a lower food intake. This also confirmed the observations on food intake in the 13-week study. 

Overall, the inconsistency in regard to results obtained in the repeated dose toxicity studies seems to rely on the method of feeding and a subsequent palatability effect. 

 

Discussion on adversity of tremor/trembling observed in dogs 

In the assessment report provided by Germany (2011), a detailed discussion of the adversity of tremor/trembling is included. As discussed in this assessment, an inconsistency with regard to the critical finding of tremor/trembling was observed in all available dog studies. Based on this synopsis, the finding of transient trembling at 600 ppm in the 90-day study was in contradiction to the results of the other dog studies, where trembling was not reproducible at the same or even higher dose levels. It is concluded that peak doses of > 50 mg/kg bw/day were required to cause slight, transient tremor in the 28-day dog study and that, in order to elicit moderate to severe tremor in dogs, doses of approximately 80 mg/kg bw/day appear to be required. 

According to the statement on dose rationales and interpretation of results in subchronic and chronic toxicity studies in dogs (M-029901-01-1, 1990), the slight tremor occasionally observed in the 600 and 1800/1200 ppm groups in the 13-week study should probably also be looked at within the context of the repellant effect. This fine tremor is the so-called “passive tremor” observed in dogs when they are cold, afraid and anxious and is thus predominantly of a psychogenic nature. Even when this sign did not consistently appear in connection with feeding, it should be borne in mind that the animals left part of the food and were thus continually confronted in the cages with food which obviously smelt unpleasant to them. Despite identical feeding methods at 1200 ppm, it was not possible in the above comparison study to verify the findings. The reason for this could lie in the animals supplied here having a different temperament. Similarly, no tremors were observed on administering 2500 ppm in pellets in the 52-week study. Thus, the occasional slight fine tremors are most probably not related to a primary indication of intoxication but are rather related to a secondary effect. 

For these reasons, the 500 ppm value determined in the chronic feeding study on dogs is regarded as the relevant overall no observed effect level for this species (as discussed in the biocidal assessment report, 2011). 

 

Inhalation 

Subacute inhalation toxicity 

Study performed in rats 

 

 M-026004-01-1 

 A subacute inhalation toxicity study in rats is available to assess the intrinsic toxicological properties of the test substance following repeated inhalation (M-026004-01-1). The doses applied in this subacute study were selected based on the acute inhalation toxicity data which are discussed under 7.2.2 in the technical dossier (M-027586-01-1). An additional subacute inhalation toxicity study was conducted under the same study number with 10 male and 10 female rats exposed to 20, 109 and 505 mg/m³ air (dust, 5days for 6 h). The NOEC was 20 mg/m³ air and the LC 50 > 505 mg/m³. Starting at 109 mg/m³ air, body weight was reduced slightly and liver enzyme activity was increased. Other changes were not found. 

In the main study (M-026004-01-1, GLP-compliant), animals were exposed to dust at concentrations of 5, 30 and 180 mg/m³ (nominal concentration). Exposure against an aerosol was considered inadequate because of the low solubility of the active ingredient in nontoxic vehicle substances (maximum technically producible concentration as aerosol: 69 mg/m³ air). Analytical analysis of the test atmosphere gave concentrations of 5.5, 30.5 and 191.2 mg/m³. Groups consisted of 10 animals per sex and dose and exposure lasted 6h/day for 5 days per week over a period of 28 days.

The test atmosphere was generated with a Wright Dust Feeder (5 and 30 mg/m³ air) and a RBG brush-type generator (180 mg/m³ air) or an Exactomat 4200 (180 mg/m³ air, starting on the second day of exposure, since the RGB brush-type generator was defect). The MMAD for 5, 30 and 180 mg/m³ were 2.37, 4.77 and 5.70 µm, respectively. In higher concentrations, the particles agglomerated. The amount of particles below 5 µm was 95 (5 mg/m³), 53 (30 mg/m³) and 43% (180 mg/m³), respectively. The GSD were 1.54 (5 mg/m³), 1.96 (30 mg/m³) and 1.89 (180 mg/m³). Animals were checked daily for clinical signs or morbidity. Body weight was assessed weekly and ophthalmoscopic examinations were conducted before the first exposure and close to study termination. Blood was sampled at necropsy to determine hematological and clinical chemistry parameters. After necropsy, gross pathology and histopathological analysis were performed on all animals.  

All rats tolerated the treatment without test substance-induced symptoms or mortality at doses up to and including 191.2 mg/m³ air. In males, body weight was reduced throughout the study in the highest dose group (-5.8 to -8.7%). In the remaining animals, body weight development was unaffected by treatment. Clinical chemistry parameters were altered in both sexes, but more pronounced in females. These manifested in increased hepatic enzymes activity in females at 30.5 and 191.2 mg/m³ (ALAT activity up to 70.3% increased, APh activity up to 46.1% increased). Bilirubin was also increased in females (+33.3%) of the high dose group, as well as O- and N-demethylase activity in males (+83.2% and +51.2%, respectively) and females (+21.5% and +76.5%, respectively). In the middle dose group, females also showed increased N-demethylase activity (+26.6%). Further, CLH activity was decreased in females starting at 30.5 mg/m³ (-26.1% and -28.2% at 30.5 and 191.2 mg/m³, respectively), not reaching statistical significance. In the high dose group, GLDH activity was increased in males (+333.3%) and females (+731.6%) and triglyceride levels were decreased in males (-48.8%) and females (-72.1%). The serumα1-globlin fraction was reduced in both sexes at > 30.5 mg/m³ air. The urinary pH was elevated in the females at 191.2 mg/m³ air. The females exhibited an increase in the blood coagulation time. These changes are considered related to effects on the liver. The thrombocyte counts were depressed in both sexes at 191.2 mg/m³ air. Pathological examinations revealed that the relative liver weight of females in the mid and high dose group was increased (+8.8 and +12.4%, respectively), reaching statistically significance at 191.2 mg/m³. Slightly reduced relative heart and thymus weights were determined in females at 191.2 mg/m³ air. No histopathological evidence for specific adverse effects were found in any of the organs examined, including the liver.

 All other findings were considered incidental and not treatment related. These were for example slightly decreased relative brain and heart weights in males in the middle and/or high dose group (but within the 2-sigma scattering ranges of pooled historical organ weight data). Statistically significant changes were found in bone marrow morphology. Briefly, proerythroblasts were increased in all groups of treated males). In females, an increase was seen but this was not statistically significant. Moreover, the count in controls of males and females differed by a factor of 2.5 and in peripheral blood, concentration of reticulum cells was unchanged, so that this finding can be considered incidental. Segmented neutrophils were decreased in males and females in all dose groups but no change in granulopoiesis occurred in bone marrow (band neutrophils) or peripheral blood (differential blood count). In addition, the amount of segmented neutrophils in the cell population is very low (0.5-1.5%), so that counting errors has a strong impact. Due to these factors, the finding is considered a coincidence.

 Taken together, the NOAEC for the test substance after repeated inhalation for 28 days is 5.5 mg/m³ for females and males. Based on reduced body weight gain in females and effects on the liver (slight weight increase and enzyme induction) at 30.5 mg/m³ air, a LOAEC of 30.5 mg/m3was derived. 

The study was conducted according to GLP and OECD Guideline 412. Since the study was conducted in 1989, there are some deviations to the current guideline. The MMADs of the applied concentrations exceeded the currently recommended value. However, as the test substance tends to build agglomerates, and the obtained data are sufficiently conclusive to derive effect levels at which no adverse effects and adverse effects were observed, the results of the conducted study are still considered as valid. Further, no BALF analysis or determination of lung burden was conducted.

 

  Dermal route 

 Subacute dermal toxicity 

Study performed in rabbits 

 

 M-025976-01-1 

 Dermal toxicity after repeated exposure was tested in rabbits (M-025976-01-1). 5 male and female rabbits received 1000 mg/kg bw test substance over a period of 15 days. The test substance was mixed with 2% Cremophor EL in physiological saline solution to form a paste that was applied to the shorn dorsal and flank skin (covering over 10% of the rabbit skin) for 6 h/day (5 times/week). During exposure, animals were harnessed to not ingest the test item. The control group (5 male/5 females) received the vehicle only. Animals were checked daily for clinical signs, food consumption was calculated as mean intake per day and body weight was recorded weekly. At the end of the study, blood samples were taken for hematology and clinical chemistry assessment.  

Appearance, behavior, feed consumption and body weights of the treated animals were unaffected by treatment. No test substance-induced mortalities occurred (1/5 control females was sacrificed due to a fractured femur which was replaced by a healthy female). No treatment-related local skin findings were observed. The skin fold thickness of the treated animals did not differ from that of controls. In hematological or clinical chemistry, no effects occurred. Further, no treatment-related changes were observed in the examined organs. Based on the results of this study, the highest dose of 1000 mg/kg bw/day was defined as NOAEL for repeated dose toxicity following dermal application. 

The study was conducted according to GLP and OECD Guideline 410. However, due to the exposure period of only 15 days, the study is considered as reliable with some restrictions. 

 

 Overall conclusion on repeated dose toxicity 

As stated in the assessment report (2011) and summarized above, reduced body weight gain was the most sensitive parameter indicating treatment-related effects following repeated exposures after oral administration and inhalation.Moreover, the liver seems to be the main target organ after repeated administration in rats and dogs. Effects indicating adaptation processes in the hepatobiliary system were observed at lower dose levels, including induction of hepatic microsomal enzymes (elevated activities of the mixed-function oxidases, particularly cytochrome P-450). Increased liver weights were observed of limited extent without relevant histopathological findings. Thus, increased liver weights were not considered adverse. Higher doses affected hepatic function resulting in dysregulation of the lipid and protein metabolism, affecting blood levels of cholesterol, triglyceride, protein, and albumin. Further, occasional increases in blood coagulation time, elevated total serum bilirubin levels, and elevated activities of ALT, AP, and GLDH were detected in the blood which are considered as indicative for a disturbance of hepatic function. Histopathologically, liver cell necrosis was observed in high dose males in the subchronic toxicity study performed in rats (M-007967-01-1). However, no similar histopathological findings were determined in the other repeated dose toxicity studies, including longterm exposure in rats (M-027741-02-1). Moreover, reversibility of hepatic effects was established in the sub-chronic rat study as no morphological effects were visible in males at the end of the recovery period in that study (M-007967-01-1). Thus, based on the available data, the liver was identified as target organ for repeated dose toxicity based on the extensive data base in regard to short- and longterm toxicity. However, considering reversibility of liver cell necrosis and considering adaptive changes as reason for the observed hepatic effects, classification in regard to STOT-RE is not appropriate according to CLP Regulation EC 1272/2008, especially under consideration of CLP Annex I, 3.9.2.8.1. (d) and § 3.9.2.5.4. of the guidance on the application of CLP criteria (ECHA, 2017).

 

 In the combined chronic/carcinogenicity study performed in rats, effects on the thyroid were detected in form of increased incidence of mineralization in the colloid of the thyroid gland follicles observed in male (LOAEL: 17 mg/kg bw/day) and female (LOAEL: 73 mg/kg bw/day) rats. Plasma levels of thyreotropin, triiodothyronine, and thyroxine remained unchanged and thus, an effect on thyroid function can be excluded, since the increased incidence of this involution of isolated thyroid follicles is considered a treatment-related premature aging of the thyroid. However, at higher dose levels, additional effects on the thyroid (increased parafollicular hyperplasia and reduced colloid aggregation) were observed. In females of the highest dose group, absolute and relative liver weights were decreased and the body weight was markedly reduced. 

 The NOAEL for repeated oral exposure was 5.7 mg/kg bw/day in the 2-year study in rats, which was selected as starting point for DNEL derivation following the oral route.In regard to repeated dose toxicity following inhalation, a NOAEC of 5.5 mg/m³ was derived which served as starting point for DNEL derivation following inhalation. No effects indicating toxicity were observed following repeated dermal applications. Thus, a NOAEL>=1000 mg/kg bw/day was derived for repeated dose toxicity following dermal exposure. 

References not included in IUC:

Detailed information on references not included in IUC are available in the CSR and in chapter 13.

Justification for classification or non-classification

An extensive data base exists to assess repeated dose toxicity following the oral, dermal and inhalation route. Based on the available data on short- and longterm exposure, the test substance does not meet the classification criteria in regard to STOT-RE for neither route according to the CLP regulation (EC) No. 1272/2008 which is in line with the harmonized classification of the test item according to Annex VI of the CLP Regulation (EC) No. 1272/2008.