clothianidin (ISO); (E)-1-(2-chloro-1,3-thiazol-5-ylmethyl)-3-methyl-2-nitroguanidine

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May - Aug 1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

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:

housing conditions were slightly different to the guideline, no detailed clinical observations (sensory reactivity), blood clotting time, weight of prostate and pituitary gland not provided, justification for choice of vehicle not given

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

assumed to be 1981

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

433-460-1

EC Name:

-

Cas Number:

210880-92-5

Molecular formula:

C6H8ClN5O2S

IUPAC Name:

(E)-N'-[(2-chloro-1,3-thiazol-5-yl)methyl]-N-methyl-N''-nitroguanidine

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The rat was chosen since it is the recommended rodent species in OECD TG 408.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Michigan, USA
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: 258 - 304 g for males and 176 - 211 g for females
- Housing: individually housed in stainless steel wire-mesh cages with deotized cage board in the bedding tray
- Diet: Purina Mills Rodent Lab Chow 5001-4, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 2 weeks

DETAILS OF FOOD AND WATER QUALITY:
Feed, water, and corn oil (used in diet preparation to facilitate mixing of the test substance in the feed) were periodically sampled and analyzed for a variety of potential impurities. The results of these analyses, which have been archived, were unremarkable.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26
- Humidity (%): 40 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: acetone/corn oil mixture

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Replacement admixtures for each treatment group were prepared weekly
- Mixing appropriate amounts with (Type of food): acetone/corn oil mixture was used as a vehicle to suspend the test substance prior to mixing in the basal diet
- Storage temperature of food: stored under refrigerator conditions

VEHICLE
- Purity: Corn oil was periodically sampled and analyzed for a variety of potential impurities. The results of these analyses, which have been archived, were unremarkable.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration:
The concentration of the test substance in the various test diets was analytically verified 4 separate times during the in-life phase of this study (weeks 1, 5, 9, and 14). The mean analytical concentrations for each dose group were 138, 423, and 2945 ppm, with all values remaining within approximately 15% of the corresponding nominal concentrations of 150, 500, and 3000 ppm, respectively.

Homogeneity:
Feed was analyzed for uniformity of distribution/homogeneity of the test substance in rat diet. Therefore, three samples were taken from each of three distinct layers (top, middle and buttom) in a mixing bowl containing a nominal concentration of either 50 or 5000 ppm. The mean concentration was determined to be 46.5 ppm and 4341 ppm, respectively. Based on the results, it was judged that the test substance was homogeneously distributed in the feed over a concentration range of 50 - 5000 ppm (with a coefficient of variation below to 10%).

Stability:
A time and temperature analysis of feed containing nominal concentrations of 50 and 5000 ppm of the test substance was conducted. Based on the criteria of at least 80% recovery of the initial chemical concentration, the test substance mixed in rodent ration was judged to be stable following room temperature storage for a minimum of 7 days and refrigerator storage for a minimum 28 days, respectively, over a concentration range of 50 - 5000 ppm.

Duration of treatment / exposure:

approximately 14 weeks

Frequency of treatment:

continuously in the feed

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

150 ppm

Remarks:

corresponding to 9 mg/kg bw/day (males) and 10.9 mg/kg bw/day (females)

Doses / concentrations 2

Dose / conc.:

500 ppm

Remarks:

corresponding to 27.9 mg/kg bw/day (males) and 34.0 mg/kg bw/day (females)

Doses / concentrations 3

Dose / conc.:

3 000 ppm

Remarks:

corresponding to 202 mg/kg bw/day (males) and 254.2 mg/kg bw/day (females)

No. of animals per sex per dose:

Main groups: 15
Recovery groups: 15 (control and high-dose only)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses selection was based on a previous 13-week oral feeding study with the test substance.
- Fasting period before blood sampling for clinical biochemistry: yes, overnight
- Post-exposure recovery period in satellite groups: 7 weeks

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations checked: moribundity and mortality

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: once each week on all animals

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: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before and after exposure duration
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to their respective termination
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters checked: platelet count (PLTS), leucocyte count (WBC), erythrocyte count (RBC), hemoglobin (Hgb, Hg), hematocrit (HCT, PCV), mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), leukocyte differential counts, erythrocyte morphology, prothrombin time (PT), reticulocyte (Retic) and heinz body (HZ) counts

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to their respective termination
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters checked: sodium (Na, Na+), potassium (K, K+), chloride (Cl), magnesium (MG), urea nitrogen (UN), glucose-fasting (Glue), creatinine (Creat, Crea), uric acid (Uric-A), triglyceride (Trig), cholesterol (Choi), creatine kinase (CK, CPK), aspartate aminotransferase (AST, GOT, SGOT), alanine aminotransferase (ALT, GPT, SGPT), gamma-glutamyl transpeptidase (GGT, GGTP), alkaline phosphatase (ALP), total bilirubin (T-Bili), direct bilirubin (D-Bili), total protein (T-Prot, Prot), albumin (ALB, ALBU), phosphorus (Phos, PO4), calcium (Calc, Ca), and globulin (Glob), Thyroxine (T4), Triiodothyronine (T3), and Thyroid stimulating hormone (TSH)
Enzyme activities not specified in the guideline: cytochrome P-450 (Cyto P-450), N-demethylase (N-Demeth), O-demethylase (O-Demeth), Ethoxyresorufin O-deethylase (EROD), and Pentoxyresorufin O-dealkylase (PROD) which are all hepatic enzymes and lactate dehydrogenase (LD, LDH, LDH-L) which indicates tissue damage

PLASMA/SERUM HORMONES/LIPIDS: refer to the list above under clinical chemistry

URINALYSIS: Yes
- Time schedule for collection of urine: the week prior to blood collection
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked: pH (pH), urine protein (Pro), urine glucose (Glu), ketones (Ket), urine bilirubin (Bil), urine blood (Bid), urobilinogen (Uro), Leukocytes (U-Leu), and nitrite (Nit), urine volume (uVOL), urine appearance, urine clarity (Clarity), urine color (Color), specific gravity (Sp.Gr.), and microscopic observation of solids (Micro)

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER:
- Estrous cycle staging: The estrous cycle of each female was determined by examining daily vaginal smears and charted over a 3-week period prior to their respective termination. If possible, animals were selected for terminal sacrifice that were determined to be in the same estrous cycle stage in order to alleviating any possible effects of non-synchronous estrous cycle stage on various measured parameters (uterus weight, follicular population, etc.).

Sacrifice and pathology:

All animals were sacrificed by exsanguination and/or CO2 asphyxiation and subject to a postmortem examination for documenting and saving all gross lesions, weighing designated organs, and collecting representative tissue specimens for histopathologic evaluation. All tissues from control and high dose (3000 ppm) animals, as well as gross lesions from all animals were examined under a light microscope.

GROSS PATHOLOGY: Yes
The following organs were weighed: adrenal glands, brain, epididymides, heart, kidneys, liver, lungs, ovaries, spleen, testicles, thymus, thyroid, and uterus

HISTOPATHOLOGY: Yes
Histopathologic evaluation included the following organs/tissues: integumentary- skin, mammary gland; musculoskeletal- bone (femur, rib/cc jet, sternum), joint (fem/tib), muscle (protocol), skull; respiratory- lungs, larynx, trachea, nasopharynx; cardiovascular- heart, aorta; hematopoietic- spleen, bone marrow, lymph node (cervical), lymph node (mesenteric), thymus; digestive- liver, cecum, colon, esophagus, pancreas, rectum, salivary gland, small intestine (duodenum, ileum, jejunum), stomach; urogenital- kidneys, ovaries, testicles, cervix, clitoral gland, epididymis, preputial gland, prostate, seminal vesicles, urinary bladder, uterus, vagina; endocrine- adrenal glands, parathyroid, pituitary, thyroid; nervous- brain (cerebellum, cerebrum-midbrain, medulla/pons), optic nerve, sciatic nerve, spinal cord (cervical, lumbar, thoracic); special senses- exorbital lacrimal gland, eyes, harderian glands, Zymbal's gland; gross lesions, physical identifier, and associated tissues

Statistics:

Continuous data that were examined statistically were evaluated initially for equality or homogeneity of variance using Bartlett's test (Snedecor and Cochran, 1967). Group means were further analyzed by a one-way variance analysis (ANOVA) (Snedecor and Cochran, 1967) followed by Dunnett's test (Dunnett, 1955, 1964). In the event of unequal variances, and at the discretion of the study director, data were subject to non-parametric procedures consisting of a Kruskal-Wallis ANOVA (Hollander and Wolfe, 1973) followed by the Mann-Whitney-U test for between-group comparisons. Frequency data were initially examined for trends; data suggestive of a potential effect were then statistically evaluated using the chi-square, Fisher exact, or chi-square and Fisher exact tests. On a case by case basis, data were subject to additional statistical procedures other than those mentioned above. For the Bartlett test, a probability (p) value < 0.001 was considered significant; for all other statistical tests, differences with p values < 0.05 were considered statistically significant.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs attributable to exposure to the test substance were observed.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Survival of the treated animals was comparable to control animals. One male rat given 500 ppm was found dead on Day 17. The cause of death was undetermined.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight gain was comparable to the control in both sexes at doses up to and including 500 ppm. Animals given 3000 ppm showed a reduced body weight gain for both sexes of approximately 11 and 12% in the male and female, respectively.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption and utilization was similar to the control at all doses tested.

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):

Pre- and post-exposure ophthalmological examinations provided no evidence that the test substance induced ophthalmic toxicity in either sex at any dose tested.

Haematological findings:

no effects observed

Description (incidence and severity):

Evaluation of the data from blood collected approximately 14 weeks into the study provided no indication that the test article induced change in either sex at any dose tested.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At the highest dose tested, increased hepatic enzyme activities were measured. There was statistically increased induction of N-demethylase and O-demethylase enzyme activity in the liver tissue of the 3000 ppm males. Slight, non-statistical increase in cytochrome P-450 in 3000 ppm males and females associated with increased N-demethylase in 3000 ppm females suggest subtle response to compound exposure.
In general, these changes in enzyme activity may reflect an adaptive response by the liver to an increased need to facilitate the metabolism and excretion of an exogenously administered test substance. Morphological evidence of a direct toxicological effect of the test item on the liver was not observed.

Endocrine findings:

not examined

Description (incidence and severity):

not applicable

Urinalysis findings:

no effects observed

Description (incidence and severity):

Evaluation of urine chemistries from samples collected approximately 14 weeks into the study provided no indication that the test substance induced change in either sex at any dose tested.

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):

Relative organ weight increases were seen for brain, kidney, and testis of 3000 ppm males and for brain and heart of 3000 ppm females at the 13-week termination. Relative organ weight increases were also present in 3000 ppm recovery females for brain, lung, spleen, and ovaries. These variations were considered secondary due to alterations in body weight gain observed in both sexes at the highest dose tested. This conclusion was also supported by the lack of microscopic evidence of direct toxicological effect of the test item on any tissue examined in this study.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related gross lesions at necropsy.

Neuropathological findings:

not examined

Description (incidence and severity):

not applicable

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant increased incidence of pigmentation which was associated with exposure to the test item was observed in the spleen of 3000 ppm males (13/15) compared to the control animals (6/15). However, this microscopic lesion was not present at the recovery groups termination. The increased incidence of pigmentation was suggested to be deposition of hemosiderin, an iron-containing pigment derived from the hemoglobin of disintegrating red blood cells.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

not applicable

Other effects:

no effects observed

Description (incidence and severity):

Estrous cycle staging:
There was no effect of treatment on the duration and stages of the estrous cycle in females at any dose level, and no effects on ovarian follicular populations.

Details on results:

Recovery group:
By conclusion of the recovery period, reversibility was suggested in every parameter that had been influenced; many parameters had fully reverted to control levels.

Effect levels

1

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: corresponding to 27.9 mg/kg bw/day for males and 34.0 mg/kg bw/day for females

2

Key result

Dose descriptor:

LOAEL

Effect level:

3 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

histopathology: non-neoplastic

Remarks on result:

other: corresponding to 202.0 mg/kg bw/day for males and 254.2 mg/kg bw/day for females

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The study was performed under GLP conditions and similar to OECD TG 408. Deviations from the current OECD guideline (2018) were related to detailed clinical observations, specific blood parameters and specific organ weights. However, the study is considered reliable and valid. Under the conditions of this study, the NOAEL for subchronic toxicity was considered to be 500 ppm, corresponding to 27.9 mg/kg bw/day for males and 34.0 mg/kg bw/day for females based on the occurrence of reduced body weight gain in both sexes, and hepatic microsomal enzyme induction in both sexes at 3000 ppm and pigmentation of the spleen in males only at 3000 ppm.