N-(2-ethylhexyl)-8,9,10-trinorborn-5-ene-2,3-dicarboximide

Endpoint summary

Description of key information

Chronic/Subchronic toxicity study by the oral route

A one-year dietary administration study of MGK 264 was performed in dogs according to EPA OPP 83 -1 (chronic toxicity). MGK® 264 was offered in the diet to beagle dogs at constant concentrations of 65, 250, and l,000 ppm for one year. A control group received ground diet on the same regimen. During the study twice- daily observations of mortality/moribundity and overt toxicity were performed. The dogs were weighed weekly and the food consumed was measured weekly. Ophthalmoscopic examinations of the eyes were performed pre-trial and again at weeks 12, 24 and 51. Clinical pathology examinations were performed pre-trial and at weeks, 12, 25 and 50. At the end of the treatment period the animals were sacrificed and necropsy examinations performed. Selected tissues were weighed and a full list of tissues processed for microscopic examination.

All dogs survived the treatment period. No adverse effects were recorded for clinical signs, body weight, food intake, clinical pathology, macroscopic findings or organ weight. Microscopic findings of cytoplasmic pigment possibly indicating biliary stasis, was noted in the livers of the 1000 ppm males and females. In addition, there was trace hepatocellular hypertrophy in 2 of the 4 males from this group. None of these changes were associated with degenerative conditions.

On the basis of the findings in the liver, the NOEL was considered to be 250 ppm which is equivalent to 7.5 mg/kg bw/day in males and 7.4 mg/kg bw/day in females based on average compound consumption. However, these changes suggested that the liver was involved in the metabolism and excretion of the test substance. Trace hepatocellular hypertrophy and cytoplasmic pigment in some animals reflected the additional work load as a consequence of ingestion of large quantities of test substance. Degenerative changes were not identified. Therefore, in the absence of degenerative changes occurring in the liver the systemic no-observed-adverse-effect-level (NOAEL) is 1000 ppm equivalent to 34.9/32.5 mg/kg bw/day in males/females.

 

Two 13-week dietary studies were performed in rat and mouse in order to assess possible toxicity of MGK 264 and select dose concentrations for the carcinogenicity studies in these species. These studies may be considered supporting studies only as a full microscopic examination of tissues was not performed. Nevertheless, liver changes either in weight, appearance or microscopically were reported and confirmed liver to be a target organ following administration of MGK 264 by the oral route.

Subchronic toxicity by dermal application

A 13-week dermal toxicity study of MGK 264 was performed in rabbits according to EPA OPP 82-3 (Subchronic dermal toxicity 90 days). Eighty New Zealand White rabbits (40 males and 40 females) were divided into four groups of 10 males and 10 females. Three groups received a solution of MGK 264 in corn oil, dermally, at dose levels of 10, 30 or 100 mg/kg/day. The remaining group received the vehicle (corn oil) alone, at the same dose volume (1 ml/kg) and served as a control. Dose levels in this study were selected based on the findings of 7 day and 14 day dose range finding studies in rabbits in which skin irritation was seen at dermal applications of 30 mg/kg bw/day or more. Animals were dosed once daily, 7 days a week for 13 weeks. The test article was left in contact with the skin for 6 hours a day under a semi-occlusive dressing. At the end of each exposure period, the backs were washed with soap and water. During the treatment period, bodyweights and food consumption were measured weekly and skin reactions and clinical observations were recorded daily. Ophthalmological examinations were carried out on all animals before treatment started and on control and high dose animals during week 13 of the study. Blood samples were taken for haematology and blood chemistry in week 13 of the study. At the end of the treatment period, all animals were killed, necropsied and the weights of the kidneys, liver and testes were recorded. A range of tissues was preserved for subsequent histopathology.

 

No treatment related effects were noted during the study. Microscopic examination identified minimal or moderate epidermal hyperplasia, with associated chronic inflammation in the dermis in many instances, present in the treated skin of the majority of control and high dose group animals. Focal scab formation and minimal hyperkeratosis were seen in a few control and treated animals. None of these findings were related to treatment. It was concluded that the administration of MGK 264 dermally to the rabbit, daily for 13 weeks, at dose levels up to 100 mg/kg/day was not associated with any overt signs of toxicity.

 

A 21-day dermal toxicity study was conducted in Crl:CD® (SD) rats to determine the potential of MGK® 264 to produce toxicity. Eighty healthy rats (40 males and 40 females) were selected for the test and equally distributed into four groups (5 males and 5 females per group). Dose levels tested were vehicle control (distilled water), 300, 600, and 1,000 mg/kg/day MGK 264.

An appropriate amount of the test substance or distilled water (control group) was applied to the skin of each rat over a 22-day (males) or 23-day (females) period, 6 hours per day under occlusion, for 5 days per week. Prior to study initiation and again on Day 20, the eyes of all rats were examined by focal illumination and indirect ophthalmoscopy. The animals were observed daily for viability, signs of gross toxicity, and behavioural changes and weekly for a battery of detailed observations. The dose site of each animal was evaluated for dermal irritation once each week. Body weights were recorded twice during the acclimation period, prior to test initiation (Day 1), weekly thereafter and at terminal sacrifice. Individual food consumption was also recorded weekly. Blood was sampled from animals during Week 3 of the study and prior to terminal sacrifice for hematology, clinical biochemistry, and serology assessments. Gross necropsies were performed on all study rats, and selected organs and tissues were evaluated histologically in the control and high dose level groups. Treated and untreated skin sections were evaluated histologically for all study rats.

There were no clinical observations, body weight, food consumption or food efficiency effects, organ weight changes, gross findings, clinical pathology, or histopathological alterations that were considered attributable to the dermal application of MGK® 264. In the treated groups, very slight to severe erythema and very slight to slight edema was observed over the course of the 21-day study. Although dermal application of MGK® 264 resulted in hyperplastic and inflammatory lesions at the treated skin site of both male and female rats at all doses studied, these findings were considered non adverse due to their non-systemic (localized) character and the apparent ability of the animals to recover from irritation upon cessation of treatment. Therefore, under the conditions of this study, a no-observed-adverse-effect level (NOAEL) for MGK® 264 following 3 weeks of dermal application for male and female rats was 1,000 mg/kg/day (the highest dose tested).

 

Subchronic toxicity by the inhalation route

A 13-week inhalation toxicity study was performed according to EPA OPP 82-4 (90-day inhalation toxicity). The toxicity of MGK 264 was assessed when administered by whole-body inhalation as a liquid aerosol to rats for 6 hours per day, 5 days per week for 13 weeks. The target concentrations in this study were 0 (control; double group), 10, 40, 135 and 400 (double group) mg/m³. The highest concentration in the study represented the maximum exposure level of MGK 264 which could be generated at the appropriate particle size in the 1000 litre chambers that were used in the study. Recovery animals were included in the control and high exposure groups and were examined after a 13-week recovery period. Exposure levels were determined gravimetrically (4 times per day) and by gas chromatography (once per day). Particle size distribution measurements of the liquid aerosol were made once per chamber/day. The animals were observed for clinical signs during each exposure and weekly for detailed physical examinations. Body weight measurements were recorded pre-test and weekly. Ophthalmoscopy examinations were performed pre-test and at the end of the study. Blood samples were withdrawn for haematological and clinical chemistry examinations before the scheduled sacrifices at the end of the treatment or recovery periods. At necropsy examination, selected organs were weighed, a full list of organs were harvested and processed for histopathological examination.

The mean MGK 264 exposure concentrations were determined to be 0, 0, 10±2 (mean± standard deviation), 43±6, 135±19, 400±28 and 407±41 mg/m³. Particle size distribution determinations indicated the test aerosol atmosphere was respirable in size to the rat.

Particle size distribution determinations showed, on average, the mass median aerodynamic diameter to be 1.1 microns with a geometric standard deviation of 2.3.

MGK 264 related changes were limited to reduced activity and excessive salivation during exposure in the high exposure groups and histopathological changes in the nasoturbinates, nasopharynx and larynx of the high exposure group. These changes which were reversible, were indicative of a localised, normal adaptive responses to an aerosol exposure and not indicative of systemic toxicity. The decreased activity was not considered to be a major detrimental response. On this basis, 135 mg/m3 was considered to be a NOAEL (no-observable-adverse-effect-level).

Summary of repeat dose toxicity

A comprehensive toxicity database is available for MGK 264 by the oral, dermal and inhalation application routes. These include a 90-day dermal toxicity study in the rabbit, a 90-day whole body inhalation toxicity study in the rat, repeat dose toxicity studies up to 1 year duration in the rat, mouse and dog and oncogenicity studies in the rat and mouse.

The target organ for MGK 264 when administered orally, is the liver. Changes observed microscopically in the liver include cytoplasmic pigment and hepatocellular hypertrophy accompanied by increases in liver weight. Similar changes have been observed in most repeat dose studies including oncogenicity and reproductive toxicity studies. It is considered that these changes suggest that the liver and bile are involved in the metabolism and excretion of MGK 264 and are supported by metabolism and excretion data which demonstrate that enterohepatic circulation plays a role in the elimination of MGK 264 (Selim, S. 1992, 1993). Therefore, it may be concluded that these changes reflect the additional work load as a consequence of ingestion of large quantities of test substance. MGK 264 can be regarded as mildly toxic to the liver at high dose levels however degenerative changes were not observed and there was no increase in liver necrosis in studies up to 2 years duration.

In the 13-week inhalation toxicity study, local point of contact toxicity was identified in the absence of systemic toxicity at the highest dose. The highest dose in the inhalation toxicity study (400 mg/m³) produced reversible microscopic changes in the nasoturbinates, nasopharynx and larynx. These were considered to be localized, normal adaptive responses to an aerosol exposure, and not indicative of systemic toxicity. In this study 135 mg/m³was considered to be the NOAEL (No-observable-adverse-effect-level).

A maximum tolerated dose of 100 mg/kg bw/day was selected for the 90-day dermal toxicity study in the rabbit based on preliminary observations of dermal irritation at application rates starting at 30 mg/kg bw for 7 days. Additionally, dermal irritation was also noted in a 21-day dermal toxicity study in the rat, in which reversible hyperplastic and inflammatory skin reactions were noted at 300 mg/kg bw. However, following application of 100 mg/kg bw/day in the 90-day dermal toxicity study, systemic toxicity was not observed and therefore this study was considered inadequate to establish a dermal endpoint. For risk assessment purposes, an endpoint for dermal risk assessment will be derived from the dietary toxicity studies in which the liver was identified as a target organ for systemic toxicity.

 Table 1 Summary of repeat dose toxicity of MGK 264

Study	Dose levels	NOAEL/NOAEC Systemic or local	Associated relevant effect
90-day inhalation toxicity rabbit (Newton, P., 1994)	0, 10, 40, 135, 400 mg/m^3	Local NOAEC: ca.135 mg/m³ air (nominal) Systemic NOAEC: 400 mg/m^3	Excessive salivation and decreased activity during the exposure period in the high exposure group; histopathology: Reversible changes related to the test material include changes in the nasoturbinates, nasopharynx and larynx indicative of a local effect of MGK 264.
90-day dermal toxicity rabbit (Lancaster, S., 1993)	10, 30, 100 mg/kg bw/day	Systemic or local NOAEL: ca.100 mg/kg bw/day (nominal)	Administration of MGK 264 dermally to the rabbit for 13 weeks was not associated with any MGK 264 related adverse effects in any of the treatment groups.
21-day dermal toxicity rabbit (Mercel, D. 2006)	300, 600, 1000 mg/kg bw/day	Systemic NOAEL:≥1000 mg/kg bw/day, Local NOAEL: < 300 mg/kg bw/day	Dermal application of MGK 264 resulted in lesions at treated skin sites at all doses studied; these findings were considered non-adverse due to non-systemic (localized) character and ability of animals to recover from irritation after treatment stopped.
1-year dietary toxicity study in dogs (Blair, M., 1991)	65, 250, 1000 ppm in the diet Average compound consumption (male/female): 2.1/2.0, 7.5/7.4, 34.9/32.5 mg/kg bw/day	Systemic NOAEL - 1000 ppm equivalent to 34.9/32.5 mg/kg bw/day in males/females	microscopic changes of brown pigment present in the liver and trace hepatocellular hypertrophy; no degenerative changes identified.
Oncogenicity study in the mouse (Blair, M. 1991)	50, 400, 800 mg/kg bw/day	Systemic NOEL: 50 mg/kg bw/day (nominal) Carcingenicity systemic NOEL: 800 mg/kg bw/day (nominal) no evidence of an oncogenic effect	Changes in the liver and gallbladder at 400 mg/kg bw/day
Oncogenicity study in the rat (Goldenthal, E.I., 1993)	50, 150, 450 mg/kg bw/day	Systemic NOEL: 50 mg/kg bw/day (nominal) Carcingenicity systemic NOEL: 450 mg/kg bw/day (nominal) - no evidence of an oncogenic effect	Body weight and weight gain decreased in animals receiving 450 mg/kg bw/day. Lower food consumption in 450 mg/kg bw/day females. Increased incidence of foci on the livers of animals treated at 450 mg/kg bw/day and an increased incidence of cysts in the liver in females from the 150 and 450 mg/kg bw/day groups. There were test article related microscopic findings in the livers of males and females from the 150 and 450 mg/kg bw/day groups and in the kidneys of 150 and 450 mg/kg bw/day females. There was no evidence of an oncogenic effect related to administration of the test article in this study at any dosage level.
2-generation reproductive toxicity study in the rat (Eiben, R., 2009)	400, 1600, 6400 ppm	Parental systemic NOAEL 400 ppm (nominal), equivalent to 33/51 mg/kg bw/day in males/females   The systemic NOAEL for reproductive parameters is 1600 ppm (127 mg/kg bw/day).	Treatment related effects on the kidney beginning at 1600 ppm (increased storage of pigment, basophilic cortical tubules, hyaline droplets in males) and liver beginning at 1600 ppm (hypertrophy and/or eosinophilic cytoplasmic change of centrilobular hepatocytes -both generations; Periportal hypertrophy increased in F0 females beginning at 1600 ppm; regarded as an adaptive response to a changed liver function and not considered as adverse).   At 6400 ppm, the maternally toxic dose level, a pup weight depression was noted in most generations (in F2b pups already before Day 14 p.p.) indicating a reprotoxic effect.
Developmental toxicity study in rats (Schardien, J., 1990)	100, 300, 1000 mg/kg bw/day	Maternal animals systemic NOEL: 300 mg/kg (nominal) Developmental systemic NOEL - 1000 mg/kg bw/day (nominal)	Maternal toxicity occurred at 1000 mg/kg bw/day level - body weight gain inhibition. No developmental toxicity was seen; no treatment related fetal malformations and variations.
Developmental toxicity study in rabbits (Schardien, J.L., 1987)	10, 30, 100 mg/kg bw/day	Maternal animals: Systemic NOAEL: 100 mg/kg bw/day (nominal) Developmental systemic NOEL: 100 mg/kg bw/day (nominal)	Treatment elicited maternally toxic effects (increased incidence of excessive salivation) at 100 mg/kg bw/day. No development toxic effects were seen at any dosage level.

 

NOAEL for systemic exposure based on changes in the liver

1. From the 1-year dietary toxicity study in dogs the systemic NOAEL was 1000 ppm equivalent to 34.9/32.5 mg/kg bw/day in males/females based on changes in the liver associated with an increased work load.

2. In the Oncogenicity study in the mouse, changes in the liver and gallbladder were present at 400 mg/kg bw/day and the Systemic NOEL was 50 mg/kg bw/day (nominal).

3. In the oncogenicity study in the rat, there were test article related microscopic findings in the livers of males and females from the 150 and 450 mg/kg bw/day groups.

4. From the 2-generation reproduction toxicity study in rats, the parental systemic NOAEL was 400 ppm (nominal), equivalent to 33/51 mg/kg bw/day in males/females. Treatment related effects on liver began at 1600 ppm (hypertrophy and/or eosinophilic cytoplasmic change of centrilobular hepatocytes in both generations whilst periportal hypertrophy was increased in F0 females beginning at 1600 ppm. It was noted that these changes were regarded as an adaptive response to a changed liver function and not considered as adverse.

 

Therefore, based on a review of the liver findings from the studies noted above, the lowest NOAEL was 32.5 mg/kg bw/day in the females from the 1 year dog study and this value may be taken as the definitive end point for the derivation of the DNEL for workers.

 

NOAEC for local effects

A 13-week inhalation toxicity study in rats identified reversible changes in the nasoturbinates, nasopharynx and larynx indicative of localized, normal adaptive responses to an aerosol exposure and not indicative of systemic toxicity of MGK 264. On this basis a local effects DNEL for workers will be also be derived based on the NOAEC of 135 mg/m³

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 1989 to December 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 83-1 (Chronic Toxicity)

GLP compliance:

yes

Specific details on test material used for the study:

MGK 264 Lot 7437, pale yellow viscous liquid was received at the experimental laboratory on 10/04/1988 in 2 drums. The test material was provded by the Sponsor. The test material was stored at rom temperature under environmentally controlled conditions. Samples of the test material were collected at 6 month intervals and sent back to the sponsor for analysis.

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

Twenty (20) male and 20 female purebred beagle dogs (approximately
3 1/2 to 4 1/2 months of age) were obtained from Ridglan
Farms, Inc., Mount Horeb, Wisconsin on September 13, 1989. A few of
the animals were slightly younger than the 4 months of age required
by the protocol at receipt. In the opinion of the Study Director
this protocol deviation did not affect the quality or the intgrity
of the study. The dogs were immunized against distemper, parvovirus,
parainfluenza, adenovirus type 2, Bordetella, leptospirosis
and rabies by the breeder prior to receipt at IRDC. Flotation tests
were conducted on stool samples obtained from each dog; the results
of the tests were negative.

At various intervals during the study, otitis was
noted for a few dogs in the control and treated groups; the appropriate
treatments were performed. Clinical pathology and ophthalmological
examinations were conducted on each dog prior to study
initiation. From these animals, sixteen males (weighing from 8.9 to
14.8 kg) and sixteen females (weighing from 9.1 to 11.8 kg) were selected
on the basis of best physical condition. The 16 dogs of each
sex were assigned at random to control or treated groups.
The dogs were individually housed in stainless steel cages
equipped with automatic watering valves and maintained in an environmentally
controlled room with a 12 hour light/12 hour dark cycle.
The temperature and humidity were continuously recorded and the recordings
were monitored for conformance with protocol specifications.
Appropriate corrective action was taken whenever readings
were outside acceptable limits. On some occasions the results of
the environmental monitoring and/or documentation of corrective
action(s) were inadvertently not recorded. In the opinion of the
Study Director, these protocol deviations did not affect the quality
or the integrity of the study. The graphs of temperature and humidity
are maintained in the study records.

Route of administration:

oral: feed

Details on route of administration:

A potential route of administration to humans is oral.
Therefore, this study employed the oral route.

Vehicle:

other: Premixed with Certified Canine Diet #5007, Purina Mills, Inc., St. Louis, Missouri

Details on oral exposure:

Route: via diet
Frequency of Administration: continuously in the diet

Preparation of Diet Mixtures: at least once weekly
Storage of Diet Mixtures: room temperature
Diet Mixtures Stable for a Period of: 21 days

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For Determination of Test Article Concentration throughout the study, 100 gram samples were taken from
the top, middle and bottom of each diet at the time test article:diet mixtures were prepared. The three samples (top, middle and bottom) were mixed to form a composite sample, which was then subdivided into three 100 gram samples. For study weeks 1-4 and every
four weeks thereafter, one set of these triplicate samples was analyzed in duplicate for determination of test article concentration
Duplicate 10g subsamples were extracted by petroleum ether using Soxhlet equipment and dose concentrations quantified by GC. The other two sets were stored frozen. At all other study weeks all three sets of samples were stored frozen.

Duration of treatment / exposure:

MGK® 264 was offered in the diet to beagle dogs at constant concentrations of 65, 250 and 1,000 ppm for
one year; a control group received ground diet on the same regimen.

Frequency of treatment:

food available ad libitum, except prior to clinical pathology testing and necropsy.

Dose / conc.:

65 ppm

Dose / conc.:

250 ppm

Dose / conc.:

1 000 ppm

No. of animals per sex per dose:

4 female/ 4 male

Control animals:

yes, plain diet

Details on study design:

MGK® 264 was offered in the diet to beagle dogs at constant concentrations of 65, 250 and 1,000 ppm for
one year; a control group received ground diet on the same regimen. Four male and four female dogs were initiated on study for each of the four groups.

Observations and examinations performed and frequency:

Mortality, moribundity and overt toxicity at least twice daily; detailed observations at least once each.
Bodyweights determined pre-test and weekly thereafter.
Food consumption determined weekly.
Ophthalmoscopic examinations carried out pretest, 12, 24 and 51 weeks of study.

On all dogs pretest and at 6, 12, 25 and 50 weeks of study:
Hematocrit; hemoglobin; erythrocyte count; mean corpuscular hemoglobin (MCH), volume (MCV), and hemoglobin concentration (MCHC); total and differential leukocyte counts; platelets; reticulocytes.
Aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, lactate dehydrogenase, leucine aminopeptidase, glucose, urea nitrogen, total bilirubin, cholesterol, albumin, globulin (calculated), albumin/globulin ratio (calculated), total prote1n,
creatinine, inorganic phosphate, creatine phosphokinase, sodium, potassium, chloride, calcium. Volume, color and appearance, pH, specific gravity, protein, glucose, ketones, urobilinogen, nitrite, bilirubin, leucocytes, occult blood, microscopic element.

Sacrifice and pathology:

Anatomic pathology on all surviving animals at study week 52. Tissues fixed in formalin (except eye, in a glutaraldehyde fixative, and testis and epididymis in Bouin's_ fixative). Bone marrow smears collected at.terminal sacrifice. Organ weights taken for all dogs sacrificed at study termination. Absolute and relative (to body and brain weights) weights of liver, kidney (2), heart, spleen, testis (2), brain, ovary (2), pituitary, thyroid/parathyroid (2), adrenal (2). Tissues examined: Adrenal (2), aorta, bone (rib), bone marrow (rib), brain (fore, mid and hind), eye including optic nerve (2), gallbladder, esophagus, stomach (glandular and nonglandular), duodenum,
jejunum, ileum, cecum, colon, rectum, ovary (2), testis with epididymis (2), heart, kidney (2), liver (3 sections), lung with mainstem bronchi (2), lymph nodes (tracheobronchial, mesenteric), mammary gland (female only), pancreas, pituitary, prostate, salivary gland
(mandibular with mandibular lymph node), sciatic nerve, skeletal muscle (thigh), skin, spinal cord (cervical, (mid)thoracic and lumbar), spleen, thymus, thyroid/parathyroid (2), trachea, urinary bladder, uterus, vagina, gross lesions, animal identification site.
Microscopic pathology examined on all dogs. Tissues sectioned at 5 microns and stained with hematoxylin and eosin.

Statistics:

Body weights, food consumption values, clinical pathology parameters and organ weights analyzed using analysis of variance and Bartlett's test. Treatment groups compared to the control group, by sex, using the
appropriate t-statistic (equal or unequal variance). Nonparametric analysis, when appropriate, by rank transformation.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No adverse effects were found during the study. In general animals receiving MGK 264 gained more weight than control animals especially in male animals.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

In general, the g/animal/day food consumption of the dogs in the treated groups was increased compared to that of the control
dogs, with the difference statistically significant in isolated instances.
However, these increases did not occur in a dose-related pattern. Similarly, the g/kg/day food consumption of the treated females was increased, although not in a dose-related fashion, during the first 6 months of the study. During the latter half of the study, the values for this parameter were comparable for the control and treated females. The q/kg/day food consumption of the treated males fluctuated in relation to the control values; no dose-related or statistically significant differences were observed.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance related organ weight variations were discerned in any of the experimental groups. A number of statistically significant variations occurred but these were considered to reflect normal biological variation because there was no dose response or correlation with any morphologic changes.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test substance related microscopic findings were limited to the liver of male and female dogs from the 1,000 ppm group. Brown pigment considered to represent biliary stasis, because of its apparent intracanalicular location and its deep, reddish brown color (which was characteristic of bile) was present for the majority of the dogs in this group. Two male dogs from the 1,000 ppm group also had
trace hepatocellular hypertrophy which was also considered test substance related.Note that these findings in the liver were not associated with degenerative change.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 ppm

Based on:

other: MGK 264 in the diet equivalent to 34.9/32.5 mg/kg bw/day in males/females

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

not specified

Lowest effective dose / conc.:

1 000 ppm

System:

hepatobiliary

Organ:

liver

Conclusions:

In summation, MGK® 264 was offered in the diet to beagle dogs at constant concentrations of 65, 250 and 1,000 ppm for one year; a control group received ground diet on the same regimen.
Four male and four female dogs were initiated on study for each of the four groups.
MGK® 264 induced microscopic changes of brown pigment in the liver and trace hepatocellular hypertrophy when offered in the diet at a concentration of 1,000 ppm to beagle dogs for one year. No other changes were observed that could be related to treatment with the test substance. The no-observed-effect-level (NOEL) dose concentration was considered to be 250 ppm in the diet which was equivalent to 7.5 mg/kg bw/day in males and 7.4 mg/kg bw/day in females based on average compound consumption.

However, these changes suggested that the liver was involved in the metabolism and excretion of the test substance. Trace hepatocellular hypertrophy and cytoplasmic pigment in some animals reflected the additional work load as a consequence of ingestion of large quantities of test substance. Degenerative changes were not identified. Therefore, in the absence of degenerative changes occurring in the liver the systemic no-observed-adverse-effect-level (NOAEL) is 1000 ppm equivalent to 34.9/32.5 mg/kg bw/day in males/females.

Executive summary:

A one year dietary administration study of MGK 264 was performed in dogs according to EPA OPP 83 -1 (chronic toxicity).

MGK® 264 was offered in the diet to beagle dogs at constant concentrations of 65, 250, and l,000 ppm for one year. A control group received ground diet on the same regimen. During the study twice daily observations of mortality/moribundity and overt toxicity were performed. The dogs were weighed weekly and the food consumed was measured weekly. Ophthalmoscopic examinations of the eyes were performed pre-trial and again at weeks 12, 24 and 51. Clinical pathology examinations were performed pre-trial and at weeks , 12, 25 and 50. At the end of the treatment period the animals were sacrificed and necropsy examinations performed. Selected tissues were weighed and a full list of tissues processed for microscopic examination.

All dogs survived the treatment period. No adverse effects were recorded for clinical signs, body weight, food intake, clinical pathology, macroscopic findings or organ weight. Microscopic findings of cytoplasmic pigment possibly indicating biliary stasis, was noted in the livers of the 1000 ppm males and females. In addition there was trace hepatocellular hypertrophy in 2 of the 4 males from this group. None of these changes were associated with degenerative conditions.

On the basis of the findings in the liver, the NOEL was considered to be 250 ppm which is equivalent to 7.5 mg/kg bw/day in males and 7.4 mg/kg bw/day in females based on average compound consumption. However, these changes suggested that the liver was involved in the metabolism and excretion of the test substance.Trace hepatocellular hypertrophy and cytoplasmic pigment in some animals reflected the additional work load as a consequence of ingestion of large quantities of test substance.Degenerative changes were not identified.Therefore, in the absence of degenerative changes occurring in the liver the systemic no-observed-adverse-effect-level (NOAEL) is 1000 ppm equivalent to 34.9/32.5 mg/kg bw/day in males/females.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

17 Feb 1986 to 20 May 1986. Reported 04 Feb 1987 (report amended 24 Oct 1991)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Range finding study prior to initiation of oncology study

Qualifier:

no guideline followed

Principles of method if other than guideline:

13 Week Dietary Range-Finding Study in Mice with MGK 264

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

MGK 264
N-Octyl bicycloheptene dicarboximide 98%
INERT INGREDIENTS: 2%
LOT 3843

Species:

mouse

Strain:

CD-1

Details on species / strain selection:

The mouse is a standard laboratory animal used· to evaluate the chronic toxicity of chemicals. Considerable control data have been accumulated .
Eighty-two (82) male and 82 female Charles River CD-1 mice (35 days old) were received from the Charles River Breeding Laboratories, Inc., Portage, Michigan, on February 5, 1986. Prior to receipt, a viral screen was conducted by the breeder on the colony of mice from which the acquired mice originated.

Sex:

male/female

Details on test animals or test system and environmental conditions:

The mice were individually housed in wire-mesh cages in a temperature (73°F ± 0.7°F; mean ± standard deviation), humidity (48% ± 5.1%; mean ± standard deviation) and light (12-hour light/12-hour dark) controlled room. Neither temperature nor humidity values exceeded the protocol specified range of 73 ± 4°F and 50 ± 20% during the study.
Water and diet (Certified Rodent Chow #5002, Ralston Purina) were available ad libitum. Certification analysis of each lot of diet was performed by the supplier. The IRDC water supply was analyzed on a quarterly basis for the presence of heavy metals, pesticides and coliforms. Water and diet analyses applicable to the study are retained in the Archives of IRDC.
Each mouse was identified by cage, group and individually by toe clip. Toe clips were verified after the initial clipping, at study initiation, at each cage change and prior to necropsy.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

Beginning on February 17, 1986, the test article:diet mixtures were offered at concentrations such that doses of 125, 250, 500, 1000 and 2000 mg/kg/day of the test article would be consumed. Control mice were offered untreated ground diet as received from the supplier.
Beginning study week 7, the 125 mg/kg/day dosage level was increased to 4000 mg/kg/day in an attempt to induce test substance related change in body weight or food consumption.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples extracted from Rodent Chow using Soxhlet apparatus and petroleum ether.

Extract was then analysed by GC FID using the below conditions:
a) Tracor 560 gas chromatograph or equivalent, equipped with FID detector.
b) Column: 6' x 4 mm glass column packed with 5% OV-101 on 80/100 mesh Gas Chrom Q.
c) Temperatures
i. Column: 155°c
ii. Injection port: 250°c
iii. Detector: 300°c
d) G&s flow:. N2• 30 ml/min; air, 1.6 SCFH; H2, 35 ml/min
e) Detector sensitivity: 5 x 10-11

Mean ± S.D. of the test article content in 10 stratified samples/diet (mixing time of 10 minutes) were 102 ±. 2.7, 98 ± 3.6 and 96 ± 1.7% of target levels for the 125, 1000 and 4000 mg/kg/day groups, respectively.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Daily in diet

Dose / conc.:

0 mg/kg diet

Remarks:

Control

Dose / conc.:

125 mg/kg diet

Remarks:

Dosage level increased to 4000 mg/kg at week 7

Dose / conc.:

250 mg/kg diet

Dose / conc.:

500 mg/kg diet

Dose / conc.:

1 000 mg/kg diet

Dose / conc.:

2 000 mg/kg diet

Dose / conc.:

4 000 mg/kg diet

Remarks:

Dosed at 4000 mg/kg/day from week 7 onwards, prior to this, the animals were dosed at 125 mg/kg/day

No. of animals per sex per dose:

10 animals per sex per dose.
NOTE: From week 7 onwards 125 mg/kg/day animals (group 2) were dosed with 4000 mg/kg/day in an attempt to induce a test article related change in body weight and food consumption.

Control animals:

yes, concurrent no treatment

yes, plain diet

Details on study design:

Test article was offered orally in the diet co Charles River CD-1 mice at dosage levels of 0, 125/4000, 250, 500, 1000 and 2000 mg/kg/day for 13 weeks. Summaries of methods, results and conclusions are presented below.

Computerized random selection in a block design based on body weights. Animals with large absolute differences from the quarantine population body weight mean eliminated prior to randomization and homogeneity of group body weight variances used as the criteria for acceptance.

Housed individually in wire-mesh cages in an environmentally controlled room with fluorescent lighting providing illumination 12 hours per day. Food and water available ad libitum.

Positive control:

N/A

Observations and examinations performed and frequency:

Mortality, morbidity and signs of overt toxicity three times daily, Monday through Friday, and twice daily on weekends. Detailed observations and palpation for masses at least once weekly.

Body weights pretest and weekly thereafter. Food consumption and compound consumption weekly.

Sacrifice and pathology:

Macroscopic Pathology:
On all mice. Tissues fixed in formalin (except eyes, in a glutaraldehyde fixative). Bone marrow smears collected at terminal sacrifice.
Organ Weights:
On all mice at terminal sacrifice. Absolute and relative (to brain and to body weights) weights of adrenal (2), brain (and brain stem), heart, kidney (2), liver, spleen. Paired organs weighed separately.

Microscopic examination of the liver for all dosage groups was performed.

Other examinations:

The mice were observed for signs of overt toxicity at the times of the moribundity/mortality checks. Detailed observations of appearance and condition, behaviour and activity, excretory functions, respiration, orifices, eyes and palpation for masses were conducted at least once each week.

Statistics:

STATISTICS:
Body weights, food consumption and absolute and relative (to body and brain weights) organ weights analyzed using Bartlett's test and analysis of variance. Treatment groups compared to the control group, by sex, using the appropriate t-statistic (equal or unequal variance) and Dunnett's
multiple comparison tables.
All statistical tests were two-tailed, with p<0.05 and p<0.01 used as levels of significance.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Decreased defecation was evident for each mouse at the 2000 mg/kg/day level throughout the study, and dark yellow urine was seen for two males at this dose level. These two observations were also apparent for all or most mice in the 125/4000 mg/kg/day group after the dose level was increased at week 7. Firm areas in the abdomen, tremors, yellow material on the anogenital region, reduced motor activity, labored breathing and coolness to the touch were also observed for males and females at the 125/4000 mg/kg/day level following the dose Level increase. One male in the 2000 mg/kg/day group also had firm areas in the abdomen. Mice in the other treated groups were comparable to the control mice in appearance and behaviour.

Mortality:

mortality observed, treatment-related

Description (incidence):

Two deaths occurred in the 125/4000 mg/kg/day male group during weeks 8 and 9 (after the dose level increase), and one female from the same group was sacrificed extremis during week 11 after being observed in a moribund condition. No mortalities were noted in the control or other treated groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight values for the 125/4000 mg/kg/day group were comparable to the control group during weeks 1-6. When the dosage level increased at week 7 in this group, mean values for the males decreased 15% from week 6 and mean values for the females decreased 10% from the previous week. Body weight means began increasing for this group beginning week 9, and by week 13 mean values for females were comparable to those for the control group and values for the males were only 8% lower than controls. Mean body weight values for the males at the 2000 mg/kg/day level were slightly lower than the control values (6-8%) throughout the study, but no differences were seen for the females at this level. Mean body weights in all other treated groups were comparable to the control values.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food consumption values (g/animal/day) for the 125/4000 mg/kg/day group were comparable to those of the control group through week 6. Consumption decreased markedly during week 7 when the dosage level was increased to 4000 mg/kg/day; mean values were 29.6 and 23.3% (male and female, respectively) lower than control values at this interval. However, consumption increased in the following weeks, and by week 10, mean values were again comparable to control values. The 250 mg/kg/day group values were comparable to the control values throughout the study. The 500, 1000 and 2000 mg/kg/day groups mean values were slightly lower than control but the differences are less then 10%.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Mean absolute liver weight, mean liver weight relative to body weight, and mean liver weight relative to brain weight were statistically significantly increased in the male mice given 500, 1000, 2000 and 4000 mg/kg/day. Mean absolute liver weight and mean liver weight relative to body weight were also statistically significantly increased in the 250 mg/kg/day male group. In female mice, similar findings were noted for liver organ weights in 1000, 2000 and 4000 mg/kg/day groups. This increase in liver weight was considered both test article and dose related. Male mean absolute spleen weight, mean spleen weight relative to body weight, and mean spleen weight relative to brain weight were increased at the 2000 and 4000 mg/kg/day dosage levels. Changes in the female groups were occasional and inconsistent.

Several other absolute and relative organ weights achieved statistical significance but these variations were not considered to be related to test article administration.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

During the macroscopic examination, hepatomegaly and hepatic discoloration were seen with high incidence in the 2000 and 4000 mg/kg/day groups of both sexes. Discoloration only was observed in the 1000 mg/kg/day male group. Macroscopic findings in the remaining test article treated groups were considered unremarkable.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The 500 and 250 mg/kg/day dose levels were considered no effect levels with respect to test article related microscopic liver changes although one female mouse (13673) from the 250 mg/kg/day level had mild bile stasis, portal bile duct proliferation and portal mononuclear cell infiltrate. This mouse may have been a highly sensitive individual. Test article related macroscopic changes were observed in the liver of most male and female mice from the 4000, 2000 and 1000 mg/kg/day groups. These changes consisted of bile stasis, portal bile duct proliferation, portal mononuclear cell infiltration, cholangiofibrosis and hepatocellular hypertrophy. These lesions tended to be somewhat less severe in females. There was little dose response with respect to incidence and severity between the 4000 and 2000 mg/kg/day groups, but there was a good dose response between the 1000 and 2000 mg/kg/day levels.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOEL

Effect level:

ca. 500 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Lack of microscopic liver changes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg diet

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

In conclusion, based on the complete results of this Range-Finding Toxicity Study, the dose level of 500 mg/kg/day is recommended as the maximum dose for the proposed carcinogenicity study.

Executive summary:

MGK264 Insecticide Synergist was administered orally in the diet to Charles River CD9-l mice at dosage levels of 125, 250, 500, 1000 and 2000 mg/kg/day. Ten male and 10 female mice were initiated at each dose level. A further group served as the control group and was provided laboratory diet as received from the supplier. The objective of this study was to obtain data to establish dosage levels for an 18 month dietary oncogenicity study in mice.

During the study the animals were observed daily for morbidity, mortality and overt toxicity. Body weights and food consumption were measured weekly. The animals were sacrificed after 13 weeks tratment and a necropsy examination performed. Selected organs were weighed and a full list of tissues harvested and preserved for possible microscopic examination. A microscopic examination of liver was performed.

Effective the start of study week 7, the dose Level of .group 2 was increased from 125 mg/kg/day to 4000 mg/kg/day in an attempt to induce a test article related change in body weight and food consumption. Two mice died in the 125/4000 mg/kg/day male group during weeks 8 and 9 after the dose Level was increased, and one female mouse from the same dose level was sacrificed in extremis during week 11. No mortalities were seen in the control or other treated groups during the course of the study.

 

Decreased defecation was evident for each mouse at the 2000 mg/kg/day level throughout the study, and dark yellow urine was seen for two males at this dose level. These two observations were also apparent for all or most mice in the 125/4000 mg/kg/day group after the dose level was increased at week 7. Firm areas in the abdomen, tremors, yellow material on the anogenital region, reduced motor activity, labored breathing and coolness to the touch were also observed for males and females at the 125/4000 mg/kg/day level following the dose level increase. One male in the 2000 mg/kg/day group also had firm areas in the abdomen. Mice in the other treated groups were comparable to the control mice in appearance and behavior.

 

Mean body weight values for the 125/4000 mg/kg/day group were comparable to the control values during weeks 1-6. When the dosage level was increased to 4000 mg/kg/day beginning study week 7, body weights dropped sharply in the males in weeks 7 and 8 and then gradually recovered during the remainder of the study to be slightly less than that of the control group. Body weight reductions in the 4000 mg/kg/ day female group were not so great as the males and recovery was evident by week 11.

 

Mean body weight values for the males at the 2000 mg/kg/day level were slightly lower than that of the controls throughout the study. Body weight values in all the other male and female groups administered test article were considered comparable to those of the control group.

 

Mean food consumption mg/animal/day) decreased markedly in the 4000 mg/kg/day males during weeks 7 and 8. Thereafter, food consumption recovered quickly to normal values. A similar pattern was observed for the 4000 mg/kg/day female group. The decrease inbodyweights in the new high dose group was a result of· a temporary decrease in food consumption. Food consumption values quickly returned to normal, but body weight values were slower to recover. Mean food consumption values were considered unremarkable in all the remaining groups.

 

During the macroscopic examination, hepatomegaly and hepatic discoloration were seen with high incidence in the 2000 and 4000 mg/kg/day groups of both sexes. Discoloration only was observed in the 1000 mg/kg/day male group. Macroscopic findings in the remaining test article treated groups were considered unremarkable. Mean absolute liver weight, mean liver weight relative to body weight, and mean liver weight relative to brain weight were statistically significantly increased in the male mice given 500, 1000, 2000 and 4000 mg/kg/day. Mean absolute liver weight and mean liver weight relative to body weight were also statistically significantly increased in the 250 mg/kg/day male group. In female mice, similar findings were noted for liver organ weights in 1000, 2000 and 4000 mg/kg/day groups. This increase in liver weight was considered both test article and dose related.

 

Male mean absolute spleen weight, mean spleen weight relative to body weight, and mean spleen weight relative to brain weight were increased at the 2000 and 4000 mg/kg/day dosage levels. Changes in the female groups were occasional and inconsistent.

 

Several other absolute and relative organ weights achieved statistical significance, but these variations were not considered to be related to test article administration.

 

Based on macroscopic findings, liver was examined microscopically. The 500 and 250mg/kg/daydose levels were considered no effect levels with respect to test article related microscopic liver changes although one female mouse (13673) from the 250mg/kg/daylevel had mild bile stasis, portal bile duct proliferation and portal mononuclear cell infiltrate. This mouse may have been a highly sensitive individual. Test article related macroscopic changes were observed in the liver of most male and female mice from the 4000, 2000 and 1000 mg/kg/day groups. These changes consisted of bile stasis, portal bile duct proliferation, portal mononuclear cell infiltration, cholangiofibrosis and hepatocellular hypertrophy. These lesions tended to be somewhat less severe in females. There was little dose response with respect to incidence and severity between the 4000 and 2000 mg/kg/day groups, but there was a good dose response between the 1000 and 2000 mg/kg/day levels.

 

In conclusion, based on the complete results of this Range-Finding Toxicity Study, the dose level of 500 mg/kg/day is recommended as the maximum dose for the proposed carcinogenicity study.

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

May 1988 - August 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

no guideline followed

Principles of method if other than guideline:

13 Week Dietary Range-Finding Study in Rats with MGK 264

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

MGK 264
N-Octyl bicycloheptene dicarboximide 98%
INERT INGREDIENTS: 2%
LOT 7437

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Sixty (60) males (weighing from 146 to 169 g) and 60 females
(weighing from 124 to 141 g) were randomly selected and assigned, as
described previously, to the following control or treated dosage
levels:
Dosage.Level, Number of Animals
mg/kg/day
0 (Control) 10 Male 10 Female
125 10 Male 10 Female
250 10 Male 10 Female
500 10 Male 10 Female
1,000 10 Male 10 Female
2,000 10 Male 10 Female

Diet (Certified Rodent Chow #5002, Ralston Purina Company, St. Louis, Missouri) and water were available ad libitum, except prior to clinical laboratory tests when food only was removed.
Animals were obtained from Charles River Laboratories, Inc., Portage, Michignn on October 10, 1988. The rats were observed during a 10 day acclimation period for any clinical signs of disease and all animals were given a detailed physical examination. For the first 3 days of the conditioning period, animals were housed 3 per cage to allow for acclimation to the automatic watering system.
Each rat was identified by cage, group and individually by Monel metal ear tag. Ear tags were verified after initial tagging,
before and after blood sample collections, at each cage change and prior to necropsy.

Route of administration:

oral: feed

Details on route of administration:

A potential route of administration to humans is oral.
Therefore, this study employed the oral route.

Vehicle:

other: Premixed with Certified Canine Diet #5002, Purina Mills, Inc., St. Louis, Missouri

Details on oral exposure:

Route: via diet
Frequency of Administration: continuously in the diet

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Ten subsamples taken from trial batches of test diets
prepared at levels of 1,000 and 40,820 ppm from a 1,000 g premix
contained 80 to 99 percent and 94 to 102 percent of the respective
target concentrations. 'l'he mean percent of claim values/diet were
95 and 98 percent and the corresponding relative standard deviations
of the 10 replicates/diet were <6 percent. It was concluded that
the test diets were homogeneous.
Additional homogeneity tests were run on January 20,
1989 using a 500 g premix for the purpose of validating the mixing
procedure that was utilized throughout the study. Results were comparable
to the previous data.

Duration of treatment / exposure:

MGK 264 was administered in the diet with concentrations adjusted to give target dose levels of 125 to 2000 mg/kg bw/day for 13 weeks. A control group was offered the basal diet. On humane grounds the 2,000 mg/kg/day was discontinued on study day 23 (males) and study day 41 (females).

Frequency of treatment:

food continuously available ad libitum, except prior to clinical pathology testing and necropsy.

Dose / conc.:

0 other: Control

Dose / conc.:

125 mg/kg bw/day (nominal)

Remarks:

135 mg/kg bw/day (actual dose)

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

270 mg/kg bw/day (actual)

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

543 mg/kg bw/day (actual)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

1080 mg/kg bw/day (actual)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

1890 mg/kg bw/day (actual)

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, plain diet

Details on study design:

MGK 264 was offered orally in the diet at target dosage levels of 125, 250, 500, 1,000 and 2,000 mg/kg/day for 13 weeks. A control group was offered the basal diet. Ten male and ten female rats were initiated on study in each of the six groups.

Mortality, moribundity and overt toxicity observations were performed at least twice daily. Cage side observations for appearance, behaviour and condition were performed daily. Body weight was determined pre-test and weekly and food consumed by each animal was measured weekly. Clinical pathology examinations (haematology and biochemistry) were performed at the end of the dosing period in week 13. All surviving animals in the control, 125, 250, 500 and 1000 mg/kg bw/day groups were sacrificed at the end of the tratment period examined for gross abnormalities and protocol listed tissues harvested for microscopic examination. Selected organs were weighed.

Three males and six females died or were sacrificed in extremis at the 2,000 mg/kg/day level. Deaths were considered the consequence of very low food consumption and associated low body weights. On humane grounds male rats at the 2,000 mg/kg/day level were removed from test material administration and allowed access to basal diet for the remainder of the study beginning on study day 23. Females at this level were placed on recovery on study day 41.



No animals died during the recovery period. Body weight values of both sexes in this group increased substantially during the recovery period. No animals died or were sacrificed in extremis in the other groups.

Observations and examinations performed and frequency:

Mortality, moribundity and overt toxicity at least twice daily; cageside observations for appearance, behavior and condition conducted daily.
Body weights determined pretest and weekly; food consumption determined weekly.

Sacrifice and pathology:

Macroscopic Pathology: All surviving animals at study termination.On all animals. Tissues fixed in formalin (except eyes in a glutaraldehyde fixative). Bone marrow smears collected at the terminal sacrifice.
Organ weights - On rats sacrificed at terminal sacrifice.
Absolute and relative (to body and brain weights) weights of adrenal (2), brain (and brain stem), heart, kidney (2), liver, testis (2), ovary (2), spleen.
Tissues preserved - Adrenal (2), aorta, bone (femur), bone marrow (femur), brain (fore, mid, hind), eye including optic nerve (2), esophagus, stomach (glandular and nonglandular), duodenum, jejunum, ileum, cecum, colon, rectum, ovary (2), testis
with epididymis (2), heart, kidney (2), lung with mainstem bronchi (2), liver (2 lobes), lymph nodes (mediastinal, mesenteric, mammary region (females only), pancreas, pituitary, prostate and seminal vesicle (2), salivary gland (mandibular with mandibular lymph
node), sciatic nerve, skin, spinal cord (cervical, thoracic and lumbar), spleen, "thymic region, thyroid/parathyroid complex, trachea, urinary bladder, uterus, vagina, all gross lesions, skeletal muscle (thigh).
Microscopicc examination was not performed.

Statistics:

Body weights, food consumption values, clinical pathology parameters and organ weight values analyzed using analysis
of variance and Bartlett's test. Treatment groups compared to the control group, by sex, using the appropriate
t-statistic (equal or unequal variance). Nonparametric analysis, when appropriate, by rank transformation.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Signs related to a moribund state were seen in the 2,000 mg/kg/day group. Animals in all the other dosage groups had no changes in physical appearance or behavior that would differentiate them from the control group.

Mortality:

mortality observed, treatment-related

Description (incidence):

Three males and six females died or were sacrificed in extremis at the 2,000 mg/kg/day level. Deaths were considered the consequence of very low food consumption and associated low body weights. On humane grounds male rats at the 2,000 mg/kg/day level were removed from test material administration and allowed access to basal diet for the remainder of the study beginning on study day 23.
Females at this level were placed on recovery on study day 41. No animals died during the recovery period. Body weight values of both
sexes in this group increased substantially during the recovery period.
No animals died or were sacrificed in extremis in the other groups. Animals in all the other dosage groups had no changes in physical appearance or behaviour that would differentiate them from the control animals.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Prior to being placed on recovery the mean body weight for males and females at the 2,000 mg/kg/day level was about 55% and 30% respectively below that of animals in the control group. Toxicologically significant weight differences (i.e. more than 10%) were detected for both sexes in the 1,000 mg/kg/day group. Body weight values for the remaining groups were comparable to those of the control group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

food consumption values (g/animal/day) were reduced to a toxicologically significant extent for male rats in the 2,000 mg/kg/day group and for female rats in the 500, 1,000 and 2,000 mg/kg/day groups. Food consumption values for the remaining groups on study were similar to those of the control groups.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Description (incidence and severity):

Absolute and relative liver weights were significantly increased in males and females of the 250, 500 and 1,000 mg/kg/day groups.
Liver weights in the 2,000 mg/kg/day males and females were either not significantly different from control animals (males) or showed lower magnitude increases over control animals (females).than did those of animals in the 250, 500 and 1,000 mg/kg/day groups.
Additionally, absolute brain weight was lower in 2,000 mg/kg/day females. Males in the corresponding dose group showed no significant difference from control animals. The toxicologic significance of these changes is unknown. However, increased liver weights are not unusual in animals ingesting large quantities of chemicals. This phenomenom has been referred to as "work hypertrophy" and is the consequence of the additional work required to metabolize and excrete such chemicals.
Various other relative organ weights were significantly different from control animals in several test groups (male and female). However, none of the changes occurred in any dose related pattern. These changes are considered incidental.

Reduced body weights were noted for both sexes in this 1,000 mg/kg/day group. Liver organ weight values were consistently
increased for both sexes in the 250, 500 and 1,000 mg/kg/day dosage groups. Such changes may not be toxic in nature but merely adaptation to additional metabolic workload.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Various red focal, red discoloration or hemorrhagic lesions were observed macroscopically in a variety of tissues in 6 of 6 females
and 1 of 3 males of the 2,000 mg/kg/day group that died or were sacrificed prior to study termination. Affected sites included the
body cavities (abdominal, cranial, thoracic), kidney, liver, skeletal muscle, skin, sternum and thymus in females and epididymis, kidney and skin in males. Similar lesions were not observed in other test or control groups or in terminally sacrificed animals of the 2,000 mg/kg/day (i.e. after recovery). The toxicologic significance of these lesions is unknown. All other observed macroscopic lesions were considered spontaneous in nature and incidental.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

Based on this study the no toxic effect dose level of MGK 264 was considered to be 500 mg/kg/day for both male and female rats.

Key result

Dose descriptor:

NOEL

Effect level:

ca. 500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

food consumption and compound intake

organ weights and organ / body weight ratios

Conclusions:

MGK 264 has been offered orally in the diet at dosage levels of 125, 250, 500 and 1,000 mg/kg/day for 13 weeks.
2,000 mg/kg/day dosage level males and females were dosed for 23 and 41 days, respectively, then placed on recovery status. A control group was offered the basal diet.
Based on this study the no toxic effect dose level of MGK 264 was considered to be 500 mg/kg/day for both male and female rats.

Executive summary:

Groups of 10 male and 10 female rats were administered MKG 264 mixed with the diet at target dosage levels of 0 (control group receiving basal diet only), 125, 250, 500, 1000 and 2000 mg/kg bw/day for 13 weeks. The purpose of the study was to evaluate the subchronic toxicity of MGK 264 in a dietary dose-range finding study in rats and thereby obtain data to establish dosage levels for a 2 year oncogencity study in rats.

During the study the animals were observed daily for morbidity, mortality and overt toxicity. Body weights and food consumption were measured weekly. At the end of the treatment period blood samples were taken for haematological and biochemical examination. The animals were sacrificed after 13 weeks tratment and a necropsy examination performed. Selected organs were weighed and a full list of tissues harvested and preserved for possible microscopic examination. A microscopic examination of the tissues was not performed.

Treatment of the 2000 mg/kg bw/day group was discontinued on study day 23 (males) and 41 (females) on welfae grounds. The animals did not consume sufficient food to sustain life which may have been due to the palatability of the MGK 264 dietary mix. Rats at this dose level gained body weight when allowed access to basal diet. Rats given dose levels up to l,000 mg/kg/day for 13 weeks survived the study without any changes in physical appearance or behavior. Reduced body weights were noted for both sexes in this 1,000 mg/kg/day group. Liver organ weight values were consistently increased for both sexes in the 250, 500 and 1,000 mg/kg/day dosage groups. Such changes may not be toxic in nature but merely adaptation to additional metabolic workload. No liver weight changes were observed in the 2,000 mg/kg/day group after a period of recovery.

Based on this study the no toxic effect dose level of MGK 264 was considered to be 500 mg/kg/day for both male and female rats.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

32.5 mg/kg bw/day

Study duration:

chronic

Species:

dog

Quality of whole database:

Database considered realiable. Justification - data is based on a GLP guideline study

System:

hepatobiliary

Organ:

liver

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March - September 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 82-4 (90-Day Inhalation Toxicity)

GLP compliance:

yes

Specific details on test material used for the study:

Test Material: MGK® 264
Supplier: Mclaughlin Gormley King Company
8810 Tenth Avenue North
Minneapolis, Minnesota 55427
Date Received: 30 September 1991 and 21 January 1993
Lot No.: 3843
Active Ingredient: 98% MGK® 264 Insecticice Synergist
Description: Liquid
Expiration Date: None. Test material Was shown to be stable for the duration of the study.

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Sprague-Dawley - derived (CD®) [CD® - Crl: (CD®)BR]
Supplier: Charles River Breeding Laboratories, Inc.Kingston, New York 124E4

Sex:

male/female

Details on test animals or test system and environmental conditions:

age at receipt - 6 weeks
age at initiation - 8 weeks

Non exposure periods: Animals were doubly housed in suspended stainless steel wire mesh cages during the first week of the acclimation period and individually house thereafter
Food was ad libitum; Purina Ceritified Rodent Chow® #5002 meal, Purina Mil's Inc., St. Louis,MO. Fresh food presenied as required.
water was ad libitum; by automated watering system (Elizabethtown Water Campany).
Approximately 12 hour · light/dark cycle (7 AM to 7 PM) via automatic timer. Temperature and relative humidity were monitored and recorded twice daily and
maintained, to the maximum extent possible, within the range presented below.
Temp: Desired: 20-24°C
Actual : 22 ± 1 °C (X ± S. D.)
Humidity: Desired: 40-70%
Actual: 54 ± 9% (X ± S.D.)

During Exposure: Animals were individually housed in stainless steel, wire mesh cages within a 1L glass and stainless steel exposure chamber. no water/food
Temp: Desired: 20-24°C
Actual : 24 ± 1 °C (X ± S. D.)
Humidity: Desired: 40-60%
Actual: 50 ± 1% (X ± S.D.)

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

1.1 µm

Geometric standard deviation (GSD):

2.3

Remarks on MMAD:

It was concluded that the liquid aerosol was highly respirable to the rat with a similar MGK 264 particle size amongst all the test material exposed groups.

Details on inhalation exposure:

Appropriate amounts of MGK® 264 were placed into a 3-neck flask resting in a heating mantle regulated by a variable autotransformer. House-line air was delivered through a regulator and backpressure gauge via 1/4" plastic tubing to a plastic T-tube where it split the airflow to the generation and dilution systems. For the generation system the air was directed from the T - tube, to a Nupro® metering valve, Dwyer® flowmeter and Matheson® backpressure gauge. The air was then carried to a 1 or 2-barrel Laskin nebulizer contained inside a 3-neck flask. For the dilution system, air was directed d to a Nupro® metering valve and Dwyer® flowmeter where it converged at a glass T-Tube with the generation air to generate the test atmosphere. The resultant liquid aerosol was directed into the chamber which housed the animals. The animals remained in the chamber for 30 minutes following the exposure to allow the chamber to clear, using room air at the same airflow rate used during exposure.

Analytical verification of doses or concentrations:

yes

Remarks:

Gravimentrical determinations were made 4 times per chamber per day and gas chromatatography was performed once per chamber per day.

Details on analytical verification of doses or concentrations:

The nominal concentration was determined by weighing the generation apparatus containing the test material before and after the exposure and dividing the difference in these weights by the total volume of air delivered during exposure (volumetric flow rate times total exposure time).

Duration of treatment / exposure:

6 hours per day
Exposure levels were determined gravimetrically (four times per chamber per day) and by Gas Chromatography (GC) (one time per chamber per day). Particle size distribution measurements of the liquid aerosol were made once per chamber/day using a Delron DCI-6 cascade impactor.

Frequency of treatment:

5 days per week, for 13 weeks

Dose / conc.:

0 mg/m³ air (nominal)

Remarks:

Group 1 and 2

Dose / conc.:

10 mg/m³ air (nominal)

Remarks:

Group 3

Dose / conc.:

40 mg/m³ air (nominal)

Remarks:

Group 4

Dose / conc.:

135 mg/m³ air (nominal)

Remarks:

Group 5

Dose / conc.:

400 mg/m³ air (nominal)

Remarks:

Group 6

Dose / conc.:

400 mg/m³ air (nominal)

Remarks:

Group 7

No. of animals per sex per dose:

15/sex/group

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

Physical observations for abnormal signs were performed once during each exposure for all animals; detailed physical examinations were conducted on all animals once pretest and weekly thereafter. Ophthalmoscopic examinations were performed on all animals pretest and once prior to scheduled sacrifice. Body weight measurements were recorded twice pretest, weekly thereafter and just prior to scheduled sacrifice. Food consumption measurements were conducted once pretest and weekly thereafter. Blood samples for analysis of hematology and clinical chemistry parameters were withdrawn just prior to the scheduled sacrifices. Following 13 weeks of exposure, 7 (groups I and VI) or 8 (Groups II and VII) animals/sex/group were sacrificed. Recovery animals were allowed a 13 week recovery period prior to sacrifice. Selected organs were weighed and organ/body and organ/brain weight ratios calculated for all animals. Complete macroscopic postmortem examination of selected tissues were conducted on all animals.

Sacrifice and pathology:

Following 13 weeks of exposure, 7 (groups I and VI) or 8 (Groups II and VII) animals/sex/group were sacrificed. Recovery animals were allowed a 13 week recovery period prior to sacrifice.

Statistics:

Body weight, change in body weight from Week 0, food consumption, haematology and clinical chemistry parameters, organ weights, organ/body and organ/brain weight ratios were analyzed. Mean values of the two control groups (I and II) and mean values of the two high dose groups (VI and VII) were compared in order to show comparable results regarding test material effects. When combined, the mean values of all exposure groups were compared to control at each time interval.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In-Chamber Observations: The only clearly test material related observation recorded during the exposures was decreased activity. This was seen in almost all animals in the high exposure groups over the entire study.

Weekly Detailed Observations: Weekly detailed observations noted dose-related incidences of red facial stains, nasal discharge and excessive salivation in both sexes. Excessive salivation occurred almost exclusively at the high exposure level and primarily during the first two weeks of the study. The red facial stains and nasal discharge were present over the entire exposure period. Red nasal discharge or staining is a common finding in inhalation studies. The animals were essentially all within normal limits during the recovery period.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One control group male was found dead on Day 46 of the study. One high exposure level male was sacrificed moribund during the recovery period. Neither of these deaths is considered to be test material related.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic morphologic abnormalities considered to be related to the whole-body exposure to MGK® 264 were seen in the nasoturbinal tissues, nasopharynx and in the larynx of both males and females. In the nasoturbinates, these changes were seen and included an increased severity of hypertrophy/hyperplasia of goblet cells in the respiratory epithelium of males and a slight increase in the severity of intracytoplasmic eosinophilic material in the epithelium covering the respiratory and/or olfactory mucosa in males and females. In the nasopharynx, an increased incidence of hypertrophy/hyperplasia of the goblet cells in the epithelium lining the nasopharynx was seen in the males and females of the high exposure group. In the larynx, an increased severity of subacute (chronic active)/chronic inflammation was seen in the high exposure group. Squamous/squamoid metaplasia/hyperplasia of the pseudostratified columnar epithelium, frequently keratinized, of the larynx was seen in all treated groups (almost all animals) but not in any control animals. Hyperplasia and hyperkeratosis of the stratified squamous epithelium was seen in groups receiving exposures of 135 or 400 mg/m3. These changes were considered to be a direct local effect of the test material. At the end of the month post exposure recovery period, all of these findings had for all practical purposes, completely reversed.

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 135 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

400 mg/m³ air (nominal)

System:

respiratory system: upper respiratory tract

Organ:

larynx

other: nasoturbinates and nasopharynx

Tabulated data are presented in the final report which is attached to this end point summary.

Conclusions:

Exposures to MGK® 264 as a liquid aerosol for 6 hours/day, 5 days/week for 13 weeks at levels of 10, 40, 135 and 400 mg/m3, produced test material related increases in red facial stains and nasal discharge in all test material exposure groups and excessive salivation in the high exposure group. Decreased activity was seen in the high exposure group during the exposures.
Body weight gain, food consumption, ophthalmology, hematology and clinical chemistry parameters, organ weights and organ/body weight ratios were not affected by these exposures.
Microscopic findings related to the test material included reversible changes in the nasoturbinates, nasopharynx and larynx.
All of these microscopic results were considered to be localized, normal adaptive responses to an aerosol exposure, and not indicative of systemic toxicity. Therefore, 135 mg/m3 was considered to be a NOAEL No-observable-adverse-effect-level).

Executive summary:

The toxicity of MGK 264 was assessed when administered by whole-body inhalation as a liquid aerosol to rats for 6 hours per day, 5 days per week for 13 weeks. The target concentrations in this study weere 0 (control; double group), 10, 40, 135 and 400 (double group) mg/m3. The highest concentration in the study represented the maximum exposure level of MGK 264 which could be generated at the appropriate particle size in the 1000 litre chambers that were used in the study. Recovery animals were included in the control and high exposure groups and were examined after a 13 week recovery period.

Exposure levels were determined gravimetrically (4 times per day) and by gas chromatography (once per day). Particle size distribution measurements of the liquid aerosol were made once per chamber/day.

The animals were observed for clinical signs during each exposure and weekly for detailed physical examinations. Body weight measurements were recorded pre-test and weekly. Ophthalmoscopy examinations were performed pre-test and at the end of the study. Blood samples wre withdrawn for haematological and clinical chemistry examinations before the scheduled sacrifices at the end of the treatment or recovery periods. At necropsy examination, selected organs were weighed, a full list of organs were harvested and processed for histopathological examination.

The mean active ingredient exposure concentrations were determined to be 0, 0, 10±2 (mean± standard deviation), 43±6, 135±19, 400±28 and 407±41 mg/m3 for

Groups I to VII, respectively. Particle size distribution determinations indicated the test aerosol atmosphere was respirable in size to the rat.

Particle size distribution determinations showed, on average, the mass median aerodynamic diameter to be 1.1 microns with a geometric standard deviation of

2.3.

MGK 264 related changes were limited to reduced activity and excessive salivation during exposure in the high exposure groups and histopathological changes in the nasoturbinates, nasopharynx and larynx of the high exposure group. These changes which were reversible, were indicative of a localised, normal adaptive responses to an aerosol exposure and not indicative of systemic toxicity. The decreased activity was not considered to be a major detrimental response. On this basis, 135 mg/m3 was considered to be a NOAEL (no-observable-adverse-effect-level).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March - September 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 82-4 (90-Day Inhalation Toxicity)

GLP compliance:

yes

Specific details on test material used for the study:

Test Material: MGK® 264
Supplier: Mclaughlin Gormley King Company
8810 Tenth Avenue North
Minneapolis, Minnesota 55427
Date Received: 30 September 1991 and 21 January 1993
Lot No.: 3843
Active Ingredient: 98% MGK® 264 Insecticice Synergist
Description: Liquid
Expiration Date: None. Test material Was shown to be stable for the duration of the study.

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Sprague-Dawley - derived (CD®) [CD® - Crl: (CD®)BR]
Supplier: Charles River Breeding Laboratories, Inc.Kingston, New York 124E4

Sex:

male/female

Details on test animals or test system and environmental conditions:

age at receipt - 6 weeks
age at initiation - 8 weeks

Non exposure periods: Animals were doubly housed in suspended stainless steel wire mesh cages during the first week of the acclimation period and individually house thereafter
Food was ad libitum; Purina Ceritified Rodent Chow® #5002 meal, Purina Mil's Inc., St. Louis,MO. Fresh food presenied as required.
water was ad libitum; by automated watering system (Elizabethtown Water Campany).
Approximately 12 hour · light/dark cycle (7 AM to 7 PM) via automatic timer. Temperature and relative humidity were monitored and recorded twice daily and
maintained, to the maximum extent possible, within the range presented below.
Temp: Desired: 20-24°C
Actual : 22 ± 1 °C (X ± S. D.)
Humidity: Desired: 40-70%
Actual: 54 ± 9% (X ± S.D.)

During Exposure: Animals were individually housed in stainless steel, wire mesh cages within a 1L glass and stainless steel exposure chamber. no water/food
Temp: Desired: 20-24°C
Actual : 24 ± 1 °C (X ± S. D.)
Humidity: Desired: 40-60%
Actual: 50 ± 1% (X ± S.D.)

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

1.1 µm

Geometric standard deviation (GSD):

2.3

Remarks on MMAD:

It was concluded that the liquid aerosol was highly respirable to the rat with a similar MGK 264 particle size amongst all the test material exposed groups.

Details on inhalation exposure:

Appropriate amounts of MGK® 264 were placed into a 3-neck flask resting in a heating mantle regulated by a variable autotransformer. House-line air was delivered through a regulator and backpressure gauge via 1/4" plastic tubing to a plastic T-tube where it split the airflow to the generation and dilution systems. For the generation system the air was directed from the T - tube, to a Nupro® metering valve, Dwyer® flowmeter and Matheson® backpressure gauge. The air was then carried to a 1 or 2-barrel Laskin nebulizer contained inside a 3-neck flask. For the dilution system, air was directed d to a Nupro® metering valve and Dwyer® flowmeter where it converged at a glass T-Tube with the generation air to generate the test atmosphere. The resultant liquid aerosol was directed into the chamber which housed the animals. The animals remained in the chamber for 30 minutes following the exposure to allow the chamber to clear, using room air at the same airflow rate used during exposure.

Analytical verification of doses or concentrations:

yes

Remarks:

Gravimentrical determinations were made 4 times per chamber per day and gas chromatatography was performed once per chamber per day.

Details on analytical verification of doses or concentrations:

The nominal concentration was determined by weighing the generation apparatus containing the test material before and after the exposure and dividing the difference in these weights by the total volume of air delivered during exposure (volumetric flow rate times total exposure time).

Duration of treatment / exposure:

6 hours per day
Exposure levels were determined gravimetrically (four times per chamber per day) and by Gas Chromatography (GC) (one time per chamber per day). Particle size distribution measurements of the liquid aerosol were made once per chamber/day using a Delron DCI-6 cascade impactor.

Frequency of treatment:

5 days per week, for 13 weeks

Dose / conc.:

0 mg/m³ air (nominal)

Remarks:

Group 1 and 2

Dose / conc.:

10 mg/m³ air (nominal)

Remarks:

Group 3

Dose / conc.:

40 mg/m³ air (nominal)

Remarks:

Group 4

Dose / conc.:

135 mg/m³ air (nominal)

Remarks:

Group 5

Dose / conc.:

400 mg/m³ air (nominal)

Remarks:

Group 6

Dose / conc.:

400 mg/m³ air (nominal)

Remarks:

Group 7

No. of animals per sex per dose:

15/sex/group

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

Physical observations for abnormal signs were performed once during each exposure for all animals; detailed physical examinations were conducted on all animals once pretest and weekly thereafter. Ophthalmoscopic examinations were performed on all animals pretest and once prior to scheduled sacrifice. Body weight measurements were recorded twice pretest, weekly thereafter and just prior to scheduled sacrifice. Food consumption measurements were conducted once pretest and weekly thereafter. Blood samples for analysis of hematology and clinical chemistry parameters were withdrawn just prior to the scheduled sacrifices. Following 13 weeks of exposure, 7 (groups I and VI) or 8 (Groups II and VII) animals/sex/group were sacrificed. Recovery animals were allowed a 13 week recovery period prior to sacrifice. Selected organs were weighed and organ/body and organ/brain weight ratios calculated for all animals. Complete macroscopic postmortem examination of selected tissues were conducted on all animals.

Sacrifice and pathology:

Following 13 weeks of exposure, 7 (groups I and VI) or 8 (Groups II and VII) animals/sex/group were sacrificed. Recovery animals were allowed a 13 week recovery period prior to sacrifice.

Statistics:

Body weight, change in body weight from Week 0, food consumption, haematology and clinical chemistry parameters, organ weights, organ/body and organ/brain weight ratios were analyzed. Mean values of the two control groups (I and II) and mean values of the two high dose groups (VI and VII) were compared in order to show comparable results regarding test material effects. When combined, the mean values of all exposure groups were compared to control at each time interval.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In-Chamber Observations: The only clearly test material related observation recorded during the exposures was decreased activity. This was seen in almost all animals in the high exposure groups over the entire study.

Weekly Detailed Observations: Weekly detailed observations noted dose-related incidences of red facial stains, nasal discharge and excessive salivation in both sexes. Excessive salivation occurred almost exclusively at the high exposure level and primarily during the first two weeks of the study. The red facial stains and nasal discharge were present over the entire exposure period. Red nasal discharge or staining is a common finding in inhalation studies. The animals were essentially all within normal limits during the recovery period.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One control group male was found dead on Day 46 of the study. One high exposure level male was sacrificed moribund during the recovery period. Neither of these deaths is considered to be test material related.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic morphologic abnormalities considered to be related to the whole-body exposure to MGK® 264 were seen in the nasoturbinal tissues, nasopharynx and in the larynx of both males and females. In the nasoturbinates, these changes were seen and included an increased severity of hypertrophy/hyperplasia of goblet cells in the respiratory epithelium of males and a slight increase in the severity of intracytoplasmic eosinophilic material in the epithelium covering the respiratory and/or olfactory mucosa in males and females. In the nasopharynx, an increased incidence of hypertrophy/hyperplasia of the goblet cells in the epithelium lining the nasopharynx was seen in the males and females of the high exposure group. In the larynx, an increased severity of subacute (chronic active)/chronic inflammation was seen in the high exposure group. Squamous/squamoid metaplasia/hyperplasia of the pseudostratified columnar epithelium, frequently keratinized, of the larynx was seen in all treated groups (almost all animals) but not in any control animals. Hyperplasia and hyperkeratosis of the stratified squamous epithelium was seen in groups receiving exposures of 135 or 400 mg/m3. These changes were considered to be a direct local effect of the test material. At the end of the month post exposure recovery period, all of these findings had for all practical purposes, completely reversed.

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 135 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

400 mg/m³ air (nominal)

System:

respiratory system: upper respiratory tract

Organ:

larynx

other: nasoturbinates and nasopharynx

Tabulated data are presented in the final report which is attached to this end point summary.

Conclusions:

Exposures to MGK® 264 as a liquid aerosol for 6 hours/day, 5 days/week for 13 weeks at levels of 10, 40, 135 and 400 mg/m3, produced test material related increases in red facial stains and nasal discharge in all test material exposure groups and excessive salivation in the high exposure group. Decreased activity was seen in the high exposure group during the exposures.
Body weight gain, food consumption, ophthalmology, hematology and clinical chemistry parameters, organ weights and organ/body weight ratios were not affected by these exposures.
Microscopic findings related to the test material included reversible changes in the nasoturbinates, nasopharynx and larynx.
All of these microscopic results were considered to be localized, normal adaptive responses to an aerosol exposure, and not indicative of systemic toxicity. Therefore, 135 mg/m3 was considered to be a NOAEL No-observable-adverse-effect-level).

Executive summary:

The toxicity of MGK 264 was assessed when administered by whole-body inhalation as a liquid aerosol to rats for 6 hours per day, 5 days per week for 13 weeks. The target concentrations in this study weere 0 (control; double group), 10, 40, 135 and 400 (double group) mg/m3. The highest concentration in the study represented the maximum exposure level of MGK 264 which could be generated at the appropriate particle size in the 1000 litre chambers that were used in the study. Recovery animals were included in the control and high exposure groups and were examined after a 13 week recovery period.

Exposure levels were determined gravimetrically (4 times per day) and by gas chromatography (once per day). Particle size distribution measurements of the liquid aerosol were made once per chamber/day.

The animals were observed for clinical signs during each exposure and weekly for detailed physical examinations. Body weight measurements were recorded pre-test and weekly. Ophthalmoscopy examinations were performed pre-test and at the end of the study. Blood samples wre withdrawn for haematological and clinical chemistry examinations before the scheduled sacrifices at the end of the treatment or recovery periods. At necropsy examination, selected organs were weighed, a full list of organs were harvested and processed for histopathological examination.

The mean active ingredient exposure concentrations were determined to be 0, 0, 10±2 (mean± standard deviation), 43±6, 135±19, 400±28 and 407±41 mg/m3 for

Groups I to VII, respectively. Particle size distribution determinations indicated the test aerosol atmosphere was respirable in size to the rat.

Particle size distribution determinations showed, on average, the mass median aerodynamic diameter to be 1.1 microns with a geometric standard deviation of

2.3.

MGK 264 related changes were limited to reduced activity and excessive salivation during exposure in the high exposure groups and histopathological changes in the nasoturbinates, nasopharynx and larynx of the high exposure group. These changes which were reversible, were indicative of a localised, normal adaptive responses to an aerosol exposure and not indicative of systemic toxicity. The decreased activity was not considered to be a major detrimental response. On this basis, 135 mg/m3 was considered to be a NOAEL (no-observable-adverse-effect-level).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

135 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

Database considered realiable. Justification - data is based on a GLP guideline study

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 1992 - March 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 82-3 (Subchronic Dermal Toxicity 90 Days)

GLP compliance:

yes

Specific details on test material used for the study:

The test article; a pale yellow liquid, used for this study came from a 17kg (gross weight) sample received at Toxicol on 16th September 1991. It was supplied in a grey metal drum as MGK 264 Synergist and labelled as follows : Lot 3843; No 1 of 1; G 38.19; T 4.5; N 33.69
When not in use the test article was stored at room temperature in the dark .

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

Forty-five male and 45 female New Zealand 2.5 to 3.1 kg in weight on arrival, were were supplied by Interfauna U.K. Ltd.,
England. White rabbits, 11-17 weeks old and delivered on 6th March 1992. All animals were found to be healthy on arrival. During the acclimatisation period 2 animals were lethargic and were euthanased. The animals were acclimatised for 18 days instead of the 7-14 days required by the protocol. The surviving animals were re-examined, confirmed to be suitable for experimental use and 80 were selected for the study. Predose ophthalmoscopy detected an ocular defect in one male animal. This animal was rejected from the study and replaced with a male, from the same batch. At the start of treatment the males weighed 3.0 - 3.6kg and the females weighed 2.9 - 3.6kg.

The animals were individually housed in grid-bottomed metal cages (Biotech/Stephen Clark Fabrications) suspended over cardboard lined excreta trays. The experimental rooms (Bl and 2) were illuminated by fluorescent lighting controlled automatically to give a cycle of 12 hours light (0600 to 1800 hours), and 12 hours dark. They were air conditioned and recorded temperature and relative humidity were in the ranges 16 - 25°C and 41 - 72% respectively. The temperature in room Bl rose above the protocol specified upper limit (21°C) by 1°C on five occasions, and by 2°C on three occasions, (see deviations, page 7). The high relative humidity of 72% was recorded on two occasions in this room. In room B2, the temperature rose above the protocol specified upper limit by 1°C on sixteen occasions, by 2°C on three occasions, by 3°C on one occasion and by 4°C on one occasion.

All animals were offered a pelleted, antibiotic-free, rabbit diet (SQC Stanrab (P) Special Diets Services Limited, Witham, England). Mains water was provided by an automatic watering system. With the exception of periods of deprivation associated with laboratory investigations (see section 4.3) both were freely available throughout the study.

Type of coverage:

semiocclusive

Vehicle:

corn oil

Details on exposure:

Twenty-four hours before the start of treatment, the application sites were clipped free of fur. During the treatment period the application sites were clipped as often as necessary to maintain them free of fur.
The test article was applied dermally to a clipped area (about 10 x 10 cm) of the upper back. Animals were dosed once daily, 7 days a week for 13 weeks The application site was then covered with a semi-occlusive dressing for a period of 6 hours. On day 82 only, the test article applied to animal 52 (group 2F) was left in contact with the back for approximately 24 hours because of a technical oversight. When this was discovered on day 83, the back was immediately washed. The animal was allowed a period of rest and was then dosed. The dressing consisted of a piece of gauze placed over the application site and a elastic stockinette tubular bandage (12cm diameter, supplied by IMS Limited, Cheshire, U.K.). Holes were then made in the tubular bandage for the legs, to prevent the dressing from slipping. At the end of each exposure period, the application site was thoroughly washed with warm.water and a mild soap ('Simple', ex. Smith and Nephew Ltd., Birmingham, England) to remove as much residual test article as possible and the skin was then blotted dry with a paper towel. Elizabethan collars were fitted for the duration of each 6-hour exposure period, to prevent oral ingestion of the test article.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

As a check on the accuracy of preparation, samples of each formulation (including the control formulation) prepared on one day in each of weeks 1 and 13 were analysed for test article at Toxicol Lahoratories. (For procedural details and results refer to Appendix 13 in the final report attached to this end point study record).

Duration of treatment / exposure:

The test article was applied dermally to a clipped area (about 10 x 10 cm) of the upper back. Animals were dosed once daily, 7 days a week for 13 weeks. Elizabethan collars were fitted for the duration of each 6-hour exposure period, to prevent oral ingestion of the test article.

Frequency of treatment:

Animals were dosed once daily, 7 days a week for 13 weeks

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

controls - recieived the vehicle (corn oil).

No. of animals per sex per dose:

10 male / 10 female per dose. A constant dose volume of 1 ml/kg bodyweight was used.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected in light of previous 7 and 14 day range finding studies.
In the 7 day preliminary range-finding study with MGK 264, doses of neat material as high as 1000 mg/kg/day were used and were associated with severe skin reactions.
For a subsequent 14 day preliminary range-finding study MGK 264 was diluted in corn oil and doses up to l100 mg/kg/day were used. Due to the degree of skin irritation noted at 30 and 100 mg/kg/day, in this study and since the definitive GLP study was to be of 90 days duration, it was considered that if such irritation was allowed to continue, possibly becoming more pronounced, any debility could be due to the irritation and not due to systemic toxicity. Therefore, it was decided that a dose level of MGK 264 in corn oil not exceeding 100 mg/kg/day should be used in order to avoid severe chronic skin reactions of animals on the study, especially as 7 days/week dosing was planned.

Observations and examinations performed and frequency:

During the treatment period, bodyweights and food consumption were measured weekly and skin reactions and clinical observations were recorded daily.
Ophthalmological examinations were carried out on all animals before treatment started and on control and high dose animals during week 13 of the study. Blood samples were taken for haematology and blood chemistry in week 13 of the study.

Sacrifice and pathology:

At the end of the treatment period, all animals were killed, necropsied and the weights of the kidneys, liver and testes were recorded. A range of tissues was preserved for subsequent histopathology .

Statistics:

Bodyweight, haematological and organ weight data were evaluated by analysis of variance (ANOVA) and, if a between groups difference significant at the 5% level occurred, by pairwise t-tests between the control and treatment groups.
Blood chemistry data were analysed by Kruskal-Wallis ANOVA and, if a between group difference significant at the 5% level occurred, significant differences between the control and treatment group were determined by Multiple Comparison test.

Clinical signs:

no effects observed

Dermal irritation:

effects observed, non-treatment-related

Description (incidence and severity):

Observations recorded during the study included: pustule formation, desquamation, eschar formation, wrinkling of the skin, erythema and oedema.
Erythema was seen to cover 10 - 100% of the appliration site in group 1, 20-100% in groups 2 and 4 and 10-100% in group 3. Oedema covered 10-100% of the application site in group 1, 20-100% in group 2, 10-100% in group 3 and 40-100% in group 4. Skin reaction scores for hnth erythema and oedema ranged from 0 (no erythema or oedema) to 3 (moderate to severe erythma or moderate oedema) in control and treated animals. Occasional scores of 4 (severe erythema or oedema) were seen in group 3 and 4 males.
Varying degrees of pustule formation, desquamation and eschar formation were seen in animals from all groups.
It was concluded that the skin reactions observed were due mainly to the semi-occlusive dressing and corn oil vehicle and were not related to treatment with MGK 264 Synergist.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related microscopic changes .
Minimal or moderate epidermal hyperplasia, with associated chronic inflammation in the dermis in many instances, was present in the treated skin of the majority of control and high dose group animals. Focal scab formation and minimal hyperkeratosis were seen in a few control and treated animals. Superficial haemorrhage was noted in one group 3 male and one group 3 female.
All other minor changes observed showed no group relationship and were recognised as that which can occur spontaneously in the laboratory rabbit.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Administration of MGK 264 dermally to the rabbit for 13 weeks was not associated with any MGK 264 related adverse effects in any of the treament groups.

Key result

Critical effects observed:

no

Conclusions:

The administration of MGK 264 dermally to the rabit, daily for 13 weeks, at dose levels up to 100 mg/kg/day was not associated with any overt signs of toxicity .

Executive summary:

Eighty New Zealand White rabbits (40 males and 40 females) were divided into four groups of 10 males and 10 females. Three groups received a solution of MGK 264 in corn oil, dermally, at dose levels of 10, 30 or 100 mg/kg/day. The remaining group received the vehicle (corn oil) alone, at the same dose volume (1 ml/kg) and served as a control. Dose levels in this study were selected based on the findings of 7 day and 14 day dose range finding studies in rabbits in which skin irritation was seen at dermal applications of 30 mg/kg bw/day or more.

Animals were dosed once daily, 7 days a week for 13 weeks. The test article was left in contact with the skin for 6 hours a day under a semi-occlusive dressing. At the end of each exposure period, the backs were washed with soap and water.

During the treatment period, bodyweights and food consumption were measured weekly and skin reactions and clinical observations were recorded daily.

Ophthalmological examinations were carried out on all animals before treatment started and on control and high dose animals during week 13 of the study. Blood samples were taken for haematology and blood chemistry in week 13 of the study.

At the end of the treatment period, all animals were killed, necropsied and the weights of the kidneys, liver and testes were recorded. A range of tissues was preserved for subsequent histopathology.

No treatment related effects were noted during the study. Microscopic examination identified minimal or moderate epidermal hyperplasia, with associated chronic inflammation in the dermis in many instances, present in the treated skin of the majority of control and high dose group animals. Focal scab formation and minimal hyperkeratosis were seen in a few control and treated animals. None of these findings were related to treatment.

It was concluded that the administration of MGK 264 dermally to the rabbit, daily for 13 weeks, at dose levels up to 100 mg/kg/day was not associated with any overt signs of toxicity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

Database considered realiable. Justification - data is based on a GLP guideline study