CHAPTER 7 Insecticides that Interfere with Insect Growth and Development TIMOTHY C MARRS Edentox Associates, Pinehurst, Four Elms Road, Edenbridge, Kent, UK. Email: [email protected]7.1 Introduction The attraction of insecticides that interfere with insect growth and development is that they have no direct target organ or system in mammals analogous to those that they target in insects, so that their toxicity tends to be unrelated to their insecticidal action, conferring considerable specificity towards killing insects and not mammals. This has an important consequence, in that the mammalian toxicity is generally rather non-specific and relatively high exposures are required to produce adverse effects. However, the compounds are not generally effective against adult insects. Some authorities classify all this group of compounds as insect growth regulators (IGRs), whereas others use the term IGR as a synonym for juvenile hormone (JH) analogues. To some extent the ecdysone agonists (see below) are exceptions to the rule that interference with mammalian metabolism is unlikely, in that ecdysone is a steroid and structurally related to mammalian steroid hormones. Despite the structural dissimilarity of the three main groups of insecticides discussed in this chapter, curiously, many have effects on the mammalian haematological sys- tem. A widely accepted scheme for mechanism grouping has been proposed by the Insecticide Resistance Action Committee (IRAC); 1 this is used in this chapter with some modifications (see Table 7.1). Issues in Toxicology No. 12 Mammalian Toxicology of Insecticides Edited by Timothy C. Marrs r The Royal Society of Chemistry 2012 Published by the Royal Society of Chemistry, www.rsc.org 221 Downloaded by University of Illinois - Urbana on 11 March 2013 Published on 19 January 2012 on http://pubs.rsc.org | doi:10.1039/9781849733007-00221
Transcript
CHAPTER 7
Insecticides that Interfere withInsect Growth and Development
The attraction of insecticides that interfere with insect growth and developmentis that they have no direct target organ or system in mammals analogous tothose that they target in insects, so that their toxicity tends to be unrelated totheir insecticidal action, conferring considerable specificity towards killinginsects and not mammals. This has an important consequence, in that themammalian toxicity is generally rather non-specific and relatively highexposures are required to produce adverse effects. However, the compounds arenot generally effective against adult insects. Some authorities classify all thisgroup of compounds as insect growth regulators (IGRs), whereas others use theterm IGR as a synonym for juvenile hormone (JH) analogues.To some extent the ecdysone agonists (see below) are exceptions to the rule
that interference with mammalian metabolism is unlikely, in that ecdysone is asteroid and structurally related to mammalian steroid hormones. Despite thestructural dissimilarity of the three main groups of insecticides discussed in thischapter, curiously, many have effects on the mammalian haematological sys-tem. A widely accepted scheme for mechanism grouping has been proposed bythe Insecticide Resistance Action Committee (IRAC);1 this is used in thischapter with some modifications (see Table 7.1).
Issues in Toxicology No. 12
Mammalian Toxicology of Insecticides
Edited by Timothy C. Marrs
r The Royal Society of Chemistry 2012
Published by the Royal Society of Chemistry, www.rsc.org
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7.2 Insect Growth Regulators
JHs are a group of substances that regulate many aspects of insect physiology.JHs are acyclic sesquiterpenoids that control and regulate development andreproduction.2 They are secreted by the corpora allata, which are a pair ofendocrine glands behind the insect brain.
7.2.1 Actions of Insect Growth Regulators
The effect of IGRs (JH analogues) is to prevent reproduction by preventingmetamorphosis of insect larvae into viable adults, when such insecticides areapplied to the larvae.3 The analogues used as insecticides are heterogeneous instructure: some are esters of long-chain fatty acids (methoprene and hydro-prene) and these clearly act as JH analogues.1 Cyromazine, which IRAC1
separately classifies as a moulting disruptor, whose target protein is unknownor uncharacterized, is a triazine. Dicyclanil is a cyclopropylaminopyrimidine.Fenoxycarb is a carbamate whose target protein is unclear.1 Although com-pounds similar to insect JHs exist in crustaceans, the juvenile hormone systemis not present in mammals, and IGRs are generally of low acute mammaliantoxicity (oral LD50 c.5 g kg�1).
7.2.2 Methoprene
Methoprene is the ISO name for 1-methylethyl (E,E)-11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate (IUPAC). The structural formula is:
Methoprene is a racemic mixture of the R and S enantiomers in a 1:1 ratio,activity as a JH analogue being restricted to the S enantiomer.4 Some studieshave been carried out using S-methoprene while others have used the racemicmixture.
Table 7.1 Insecticides that interfere with insect growth and development.
7.2.2.1 Absorption, Distribution, Metabolism and Excretion
Studies of the absorption, distribution, metabolism and excretion ofmethoprene after single doses have been undertaken in mice, rats, guinea-pigs,cows and chickens. Generally methoprene was rapidly absorbed and theradiolabel is excreted in the urine, faeces and expired air, mostly within 5 days.Methoprene was extensively metabolized by mammals and in guinea-pigs muchof the label in urine was present as glucuronic acid conjugates.5 In rats,concentrations of methoprene in fat were observed to decline very slowly.
7.2.2.2 Acute Toxicity, Irritancy and Sensitization
Methoprene and S-methoprene are of low mammalian toxicity;5–7 the oral ratLD50 both of the racemate and S-methoprene is greater than 5 g kg�1 bw.5
Methoprene was not irritating to the eye in two studies of the racematein rabbits, while slight irritancy was observed in a third study usingS-methoprene. Significant irritancy was not observed to the skin of rabbits,with racemic methoprene or S-methoprene. In a study in guinea-pigs, not tomodern standards, the racemate appeared to have no sensitizing properties butin a study with a formulation containing 20% S-methoprene, skin sensitizationwas seen.
7.2.2.3 Repeated Dose Studies
In repeated dose studies, increased relative liver weight were frequently seen,not always accompanied by histopathological changes, but methoprene causedchanges such as bile duct proliferation in the livers of rats during a 2-year studyat doses of about 220mg kg�1 bw day�1.5,7 In a 90-day study in dogs, usingdietary concentrations giving intakes of up to 120mg kg�1 bw day�1 ofracemate, no deaths occurred, nor were there adverse effects on behaviour,bodyweight, food consumption or haematological endpoints. Serum alkalinephosphatase activity was increased in both sexes at the highest dose. At thatconcentration, at termination, the relative liver weight was increased in bothsexes, but this finding was not accompanied by any histopathologicalabnormality.
7.2.2.4 Carcinogenicity and Mutagenicity
Methoprene was carcinogenic in neither rats nor mice, but bile duct pro-liferation in the livers of rats was seen during a 2-year study (see above).Methoprene did not appear to be mutagenic.5 The range of tests was limited:Ames tests were carried out both on racemate and S-methoprene, while a testfor chromosomal aberrations in Chinese hamster ovary (CHO) cells was per-formed on the racemate and tests for mitotic recombination, gene conversionand reverse mutation in Saccharomyces cerevisiae D7 were undertaken usingS-methoprene.
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There was no evidence of reproductive or developmental toxicity at dosesbelow those causing maternal toxicity, except in a mouse study of develop-mental toxicity, where the dams were allowed to litter and suckle their pups:effects on organ weights were observed in pups at the top dose. A study onendocrine activity in mice was reviewed by the JMPR,5 in which immaturefemale mice (19–21 days of age) received racemic methoprene subcutaneouslyat doses of 0.015 or 0.15mg kg�1 bw day�1 for 3 days. No increase in theuterine weight relative to bodyweight ratio was seen. When methoprene wasgiven subcutaneously to castrated male rats at doses of 0.37 or 3.7mg kg�1 bwday�1 for 7 days, no increase was seen in the seminal vesicle, ventral prostate orlevator ani weights relative to bodyweight. In adrenalectomized male rats,21–23 days old, subcutaneous injection of methoprene at 0.9 or 9mg kg�1 bwday�1 for 6 days did not affect the thymus weight relative to bodyweight ratio.It was concluded that these studies suggested that methoprene had nooestrogenic, androgenic, anabolic or glucocorticoid activity.
7.2.2.6 Reference Dose
The JMPR5 established an acceptable daily intake (ADI) of 0.09mg kg�1 bw:this allowed for the purity (c.70%) of the racemate tested in the relevant study,which was the 90-day study in dogs. A safety factor of 100 was allocated. Theeffects observed at the lowest observed effect level (LOAEL) were increasedliver weight and an increase in alkaline phosphatase activity. An acute referencedose (ARfD) was considered unnecessary.
7.2.3 Hydroprene
Hydroprene is the ISO name for ethyl (E,E)-(RS)-3,7,11-trimethyldodeca-2,4-dienoate (IUPAC). The structural formula is:
Hydroprene is similar in structure to methoprene. Like methoprene, it is aracemic mixture of the R and S enantiomers and activity as a JH analogue islimited to the S enantiomer. Both the racemate and S-hydroprene have a LD50
greater than 5 g kg�1 bw in the rat. The toxicology, which was reviewed by theUK Advisory Committee on Pesticides (ACP),8,9 is broadly similar to that ofmethoprene and will therefore be discussed in less detail. In a 4-week study inthe rat, using the racemate, the main finding was increased liver weights(absolute and relative). In a 13-week rat study using S-hydroprene, there weredisturbances in haematological and clinical chemistry parameters, includingprothrombin time (dose-related decrease in males) (such changes were not seenwith methoprene—see section 7.2.2.3). Increased liver, kidney and ovarianweights were observed at necropsy. Histopathogically, there was increasedhomogeneity of liver cytoplasm at high doses and in females, vacuolatedinterstitial cells in the ovaries. In a long-term/carcinogenicity study in rats usingS-hydroprene, findings were similar and S-hydroprene was not carcinogenic.Neither hydroprene nor S-hydroprene appear to be mutagenic. The racematewas not fetotoxic or teratogenic in the rabbit nor was S-hydroprene in therat. In a multigeneration study in rats, using S-hydroprene, there was someevidence of an effect on seminiferous tubules in males, associated with an effecton fertility; it was unclear whether these effects were test-material related.Hydroprene (racemate) had no oestrogenic activity in mice and no androgenic,anabolic or glucocorticoid activity in rats.8,9 The ACP did not identify an ADI,as the evaluation was not for the use of hydroprene as a plant protectionproduct, but the lowest NOAEL was 4.62mg kg�1 bw day�1 in the rat long-term study on S-hydroprene based on ovarian lipid accumulation and regres-sion of corpora lutea. However, the Australian ADI was 0.5mg kg�1 bw.10
7.2.4 Cyromazine
Cyromazine is the ISO name and international non-proprietary name (INN)for N-cyclopropyl-1,3,5-triazine-2,4,6-triamine. The structural formula is:
Cyromazine is used inter alia as a sheep ectoparasiticide. The absorption,distribution, metabolism, excretion and toxicology has been reviewed twice bythe JMPR.11,12
7.2.4.1 Absorption, Distribution, Metabolism and Excretion
In most species (rat, sheep, goat, monkey), the majority is excreted within 24 h,as unchanged cyromazine. A higher proportion seems to be metabolized in the
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goat. Melamine (a bladder carcinogen) was a metabolite of cyromazine inmammals.11,12
7.2.4.2 Acute Toxicity, Irritancy and Sensitization
Cyromazine is of low acute toxicity (rat oral LD50 c.3–4g kg�1 bw). In rabbits,cyromazine was not an irritant to the skin or eyes, nor does this insecticide havesensitizing potential in the guinea-pig.11,12
7.2.4.3 Repeated Dose Studies
In short-term feeding studies in rats and dogs and in long-term studies in miceand rats, effects on bodyweight were seen. Red blood cell counts andhaemoglobin levels were reduced in dogs at high dietary concentrations.11 A1-year dog study using diets containing cyromazine at concentrations givingintakes of up to 110mg kg�1 bw day�1 was also undertaken.12 Similarhaematological effects (slight hypochromic and microcytic anaemia), were seenas in the earlier dog studies. Increased heart and liver weights (both absoluteand relative) were seen at high doses. The effects on the blood were accom-panied by hypercellularity of the bone marrow. In addition there werehistopathological effects on the myocardium (chronic myocarditis) and kidney(focal chronic tubular epithelial regeneration) at high doses. The NOAEL of5.7mg kg�1 bw day�1 proved to be the critical NOAEL for establishing theJMPR ADI. This NOAEL was based upon haematological effects in males atthe next highest dietary concentration.
7.2.4.4 Carcinogenicity and Mutagenicity
In long-term studies in mice and rats, dietary concentrations were used thatgave intakes of up to 476mg kg�1 bw day�1 in the former and 210mg kg�1 bwday�1 in the latter. There was a slight increase in the number of females withmammary gland adenocarcinomas in the highest dose group in both species,but there was no clear dose relationship. The JMPR11,12 considered thatcyromazine was neither carcinogenic nor genotoxic. The NOAEL for the long-term rat study, which formed the basis of the 1990 JMPR ADI11 (now super-seded) was a dietary concentration giving an intake equal to 1.8mg kg�1 bwday�1, based on the bodyweight changes noted in females seen in the mid dose(the 2006 JMPR12 considered the NOAEL for the rat study to be 15mg kg�1
bw day�1).
7.2.4.5 Reproductive and Developmental Toxicity
In a rat multigeneration study, cyromazine did not affect fertility, but there wasincreased perinatal pup mortality and reduced pup weight, at maternally toxicdoses. A study of developmental in rats showed fetal toxicity at the highest dose
(600mg kg�1 bw day�1) and maternal toxicity at the next lower dose:teratogenic effects were absent. Several studies in rabbits were evaluated by theJMPR: abnormalities, including cyclopia with multiple head anomalies in twofetuses, were observed in one study, but there was no dose response and similarfindings were not present in other rabbit studies. The JMPR did not considercyromazine to be teratogenic.11,12
7.2.4.6 Reference Doses
The JMPR established an ADI of 0.06mg kg�1 bw based on a NOAEL of5.7mg kg�1 bw day�1 for haematological effects in the 1-year study of toxicityin dogs, seen at 23mg kg�1 bw day�1 in males. A safety factor of 100 was used.An ARfD of 0.1mg kg�1 bw was based on the NOAEL of 10mg kg�1 bwday�1. This was based on bodyweight loss and decreased food consumptionobserved soon after the start of dosing at 25mg kg�1 bw day�1 in dams instudies of developmental toxicity in rabbits treated by gavage, a safety factor of100 being used.12
7.2.5 Dicyclanil
Dicyclanil is the ISO name and the INN for 4,6-diamino-2-cyclopropylami-nopyrimidine-5-carbonitrile (IUPAC). The structural formula is:
Dicyclanil is an insecticide also used on sheep as an ectoparasiticide.
7.2.5.1 Absorption, Distribution, Metabolism and Excretion
The absorption, distribution, metabolism, excretion and toxicology ofdicyclanil has been reviewed by the Joint Expert Committee on FoodAdditives (JECFA).13 When orally administered to rats for 7 days, label fromradiolabelled dicyclanil was rapidly excreted (>90% within 24 h of thelast dose) predominantly in the urine. Most of the label was present as meta-bolites. Biotransformation of dicyclanil in rats and sheep was limited to thecyclopropyl ring.
227Insecticides that Interfere with Insect Growth and Development
7.2.5.2 Acute Toxicity, Irritancy and Sensitization
The toxicology of dicyclanil has been reviewed, also by JECFA.13 Dicyclanil ismoderately hazardous by mouth (rat oral LD50 c.500mg kg�1 bw), i.e. it issomewhat more acutely toxic than many of the other insecticides discussed inthis chapter. In a skin irritancy test in rabbits, very slight erythema wasobserved, while in an eye irritancy test, also in rabbits, slight chemosis of theconjunctiva was observed in two out of three animals 1 h after instillation, butcomplete recovery was seen within 24 h. Dicyclanil was not a sensitizer inguinea-pigs.
7.2.5.3 Repeated Dose Studies
In a 28-day study in the rat, dietary concentrations were used whichachieved intakes of up to 160mg kg�1 bw. Piloerection was seen in both sexes atthe top dose in the latter part of the study. Dose-dependent reductions in foodconsumption, bodyweight gain, and terminal bodyweight were noted in alltreated groups. Increased incidences of erythrocytic anisocytosis and poly-chromasia were observed in males at the highest dose. Clinical chemistrydisturbances at the highest dietary concentration were also seen in both sexes,notably raised blood urea and elevations in aminotransferase activities. Atnecropsy, reductions in the absolute and relative weights of the prostate inmales at the mid and high doses were observed. Histopathologically, reducedspermatogenesis and accumulation of cellular debris in the epididymal duct inmales at the high dose were noted. In a 3-month study in rats, including a4-week recovery period, intakes of up to 34mg kg�1 bw day�1 were achieved.Adverse clinical signs were not seen. Small reductions in bodyweight gain andfood consumption were observed in animals of both sexes at high doses:bodyweights of animals at the high dose were comparable to those of thecontrols at the end of the recovery period. Higher organ:bodyweight ratioswere observed for a number of organs (kidneys, brain, and testis in males at thehigh dose and for liver and brain in females at the highest dose) but these werereversible during the 4-week recovery period. No treatment-related adverseophthalmological, haematological or histopathological findings were seen. In a3-month study in dogs, intakes of up to 42mg kg�1 bw day�1 were achieved.Clinical signs of neurotoxity (ataxia, raised tails and tremor) were noted atweeks 9–11 at the high dose. Vomiting was also seen as was, in the males,decreased food consumption and bodyweight gain. Changes in clinicalchemistry (increased plasma cholesterol and phospholipid concentrations athigher doses) and haematology (reduced haemoglobin and haematocrit withmicrocytosis and erythrocytic hypochromasia at the top dose) were observed.Liver weights (absolute and relative) were increased at high doses in both sexes.The absolute and relative weights of the testes were decreased at the top dose.The main histopathological findings were minimal to moderate focal ormultifocal subcapsular inflammation with fibrosis in the livers of some animalsof both sexes at the highest dose. Enlarged oedematous hepatocytes were
observed in some females at in the test groups. The testes in 3/4 males showedminimal tubular atrophy at the high dose and there was marked reduction inspermatogenesis in all males of that group. A 1-year dietary study of dicyclanilwas undertaken in dogs with a 4 week recovery period, with 4 test groups anddaily intakes of up to 23mg kg�1 bw day�1. One top-dose male was killed inextremis (vomiting and apathy) and one top-dose female died. Vomiting wasobserved in females at that dose. Adverse haematological effects were not seen.Throughout the treatment period, plasma cholesterol concentrations wereincreased in animals at the highest dose rate (in females not statistically sig-nificantly). This finding provided the basis for the NOAEL of the study, whichwas 0.71mg kg�1 bw day�1.
7.2.5.4 Carcinogenicity and Mutagenicity
Long-term studies were carried out in mice and rats, the former with intakes ofup to 210mg kg�1 bw day�1 and the latter up to 26mg kg�1 bw day�1. In themice, there were clinical signs of toxicity and deaths in the high-dose groups, forwhich reason survivors were killed at weeks 58–59. There were findings atautopsy in a number of organs, the most important being the liver. Macro-scopically, there were enlarged livers with masses and/or nodules of the liver atthe higher doses. Microscopically, these findings were associated with Kupffercell pigmentation, hepatocellular necrosis, increased in the number of hepato-cellular mitotic figures and/or multinucleated hepatocytes. The incidence ofhepatocellular adenomas was higher in females at the two highest doses than incontrols. Additionally, the incidence of hepatocellular carcinomas wasincreased in females at the highest dose. Pigmentation of the olfactory epi-thelium was observed at higher doses. Moto et al.14,15 have studied theaetiology of the liver tumours seen in mice exposed to dicyclanil; they con-cluded that inhibition of apoptosis and DNA damage due to oxidative stressmay be involved in the mechanism of hepatocarcinogenesis (see also Umemuraet al.16). Moreover, the effect appears to be thresholded.17 In the rat long-termstudy, there were effects on bodyweight, haematological and clinical chemistryendpoints; as in mice, pigmentation of the olfactory epithelium on both sexeswas observed. Dicyclanil was not tumorigenic in the rat. Dicyclanil was notmutagenic in a variety of tests (including one test in vivo) and JECFA13 con-sidered that dicyclanil was neither genotoxic nor a carcinogenic risk to humans(see also Moto et al.).18,19
7.2.5.5 Reproductive and Developmental Toxicity
In a multigeneration study in rats, dicyclanil was not a reproductive toxin at thehighest dietary concentration tested, while the NOAEL for pup toxicity(reduced bodyweight gain) was above the NOAEL for parental toxicity.Developmental toxicity studies were carried out in rats and rabbits, in theformer using gavage doses of up to 75mg kg�1 bw day�1, and in the latter doses
229Insecticides that Interfere with Insect Growth and Development
of up to 30mg kg�1 bw day�1. In rats, fetotoxicity was seen at doses abovethose that were maternally toxic (reduced fetal weight, increased renal pelvicdilatation, and increased skeletal anomalies and variations consistent with aslight delay in skeletal maturation). In the rabbit, dicyclanil was not teratogenicand was only fetotoxic at doses above those that were maternally toxic.Findings in the fetuses were consistent with delayed ossification (reduced fetalweight and increased skeletal variations).
7.2.5.6 Other Studies
Additional studies have been carried out on the pharmacological properties ofdicyclanil both in vitro and in vivo. Dicyclanil at high concentrations hadantagonistic effects on smooth muscle contractions induced by agonists. Asingle oral dose of 100mg kg�1 bw (the highest dose tested) slightly inhibitedboth exploratory activity and the startle response in mice. Moreover, there werereductions in static activity and motor coordination. The same dose in ratsaffected the heart rate, and tidal and minute lung volume. Locomotor activitywas also affected.
7.2.5.7 Reference Dose
JECFA13 considered an ADI of 0.007mg kg�1 bw appropriate. This was basedon the NOAEL of 0.71mg kg�1 bw day�1 in a 1-year study of toxicity in dogsconcentrations and a safety factor of 100.
7.2.6 Fenoxycarb
Fenoxycarb if the ISO name for ethyl 2-(4-phenoxyphenoxy)ethylcarbamate(IUPAC). The structural formula is:
7.2.6.1 Action
In structure, fenoxycarb is a carbamate, without acetylcholinesterase-inhibitingproperties and it exerts most of its insecticidal activity as a JH analogue. Theabsorption, distribution, metabolism and excretion and toxicology of fenox-ycarb has been reviewed by a number of regulatory authorities, for example theUSEPA and the UK PSD (now the Chemical Regulations Directorate,CRD).20
7.2.6.2 Absorption, Distribution, Metabolism and Excretion
In a study in rats using radiolabelled fenoxycarb, the majority of the label wasexcreted in 24 h mostly in the faeces. A study in bile duct cannulated ratsshowed that there was considerable excretion in the bile. In both single andrepeated dose studies, concentration of the label in the liver was notable.Metabolism in the rat was complex involving loss of the ethylcarbamate sidechain and hydroxylation. Sulfate conjugates were prominent in the urine.20
7.2.6.3 Acute Toxicity, Irritancy and Sensitization
Fenoxycarb was of low acute mammalian toxicity when administered to rats bymouth, via the skin or by inhalation.20 The rat oral LD50 was greater than 10 gkg�1 bw. Fenoxycarb was not irritant to rabbit skin but is a mild eye irritant.Fenoxycarb was nor a sensitizer in the guinea-pig.
7.2.6.4 Repeated Dose Studies
In a 28-day rat study using gavage, using doses of up to 1000mg kg�1 bwday�1, there were no deaths or treatment-related clinical signs, nor weredisturbances in bodyweight or food consumption seen. Slight but significanteffects were seen at the highest dose on haematological parameters (decreasederythrocyte count, haemoglobin and haematocrit); in the same group increasesin the alkaline phosphatase activity and calcium and albumin concentrationswere seen. The most important finding was an increase in relative and absoluteliver weights in all treated groups with a dose relationship in both sexes.Microscopically, there was hypertrophy and eosinophilia of hepatocytes, con-fined to the highest-dose group. Another microscopic finding was follicularhyperplasia of the thyroid, also confined to the highest-dose group. On electronmicroscopic examination, there was peroxisome proliferation in the livers of thehigh dose group, more marked in males. In a 13-week dietary study, with a4-week reversibility group and using doses of up to 800mg kg�1 bw day�1,there were clinical signs (alopecia with some evidence of dose response, anddiuresis and shaggy fur) at the top dose. Bodyweight and weight gain wasreduced in both sexes at the top dose. Haematological disturbances were seen atthe top dose and in some cases in the lower-dose treatment groups in females,the changes comprising decreased red blood cell counts, haemoglobin levelsand packed cell volume. Anisoscytosis and poikilocytosis were also seen.Clinical chemistry changes at the high- and/or mid-dose group were seen: theseincluded increased alanine aminotransferase, glutamate dehydrogenase, andalkaline phosphatase activities. Bilirubin levels were increased at the top twodoses in both sexes. Total protein was increased in the top-dose males and thetop two doses of females and the albumin:globulin ratio was increased in thesegroups. There was a dose-related increase in relative and absolute liver weights(all dose groups, both sexes). Increased thyroid weight was also observed athigh doses. Histopathological changes observed included hepatocyte
231Insecticides that Interfere with Insect Growth and Development
hypertrophy (mainly centrilobular), changes in the thyroids of females (smallactive follicles) and renal tubular changes. Most of these findings appeared tobe reversible, but there was not a clear NOAEL for the study. Another ratstudy was conducted at lower doses (highest dose was 203mg kg�1 bw day�1),but otherwise using a similar design with a reversibility group. No clinical signswere seen at any dose. Bodyweight gain was decreased at the highest dose inboth sexes (greater in females) and this was reversible only in the males.Top-dose females had reduced food consumption. No effects were seen onophthalmoscopy or on haematological parameters. Changes in clinicalchemistry parameters, generally confined to the top dose group were seen inaminotransferases (males) and alkaline phosphatase (both sexes). The top-dosemales and females had increased albumin:globulin ratios. Reduced bilirubinconcentrations were seen in the top-dose females. Absolute and relative liverweights were increased in both sexes at the top two doses. Absolute and relativethyroid weights were increased in females. Hepatocellular hypertrophy, largelycentrilobular, was seen at the higher doses in females and the top dose in males.Hypertrophy of the thyroid follicular epithelium was seen in females at the toptwo doses and also in males at the top dose. A 13-week mouse study wasundertaken, using dietary concentrations giving doses of up to 900mg kg�1 bwday�1. There were no adverse clinical signs or effects on food consumption,bodyweight, weight gain or food consumption. In males there were decreases inwhite blood cell counts and in females of thrombocytes. Dose-dependentchanges in plasma proteins were seen in females. Significant increases inliver weights were seen in the top dose in both sexes. Periportal fatty changewas seen in the liver, most notably in males. A capsule study in dogs wasundertaken using doses of up to 260mg kg�1 bw day�1. Treatment-relatedclinical signs were not seen, but bodyweight gain was reduced in the high-dosegroup. No haematological effects were observed but there was an increase inrelative liver weight at the high dose. No treatment-related histopathologicaleffects were seen.
7.2.6.5 Carcinogenicity and Mutagenicity
Long-term/carcinogenicity studies in the mouse and rat were reviewed byACP.20 In the rat study, dietary concentrations which gave intakes up to about104mg kg�1 bw day�1 were used. There was a slight reduction in bodyweight atthe top dose in males and in bodyweight at termination in females. Effectsobserved were broadly consistent with short-term rat studies, buthaematological effects were minor. In males at the top dose and at the interimsacrifice, there was slight centrilobular hypertrophy of the liver and fibrosis wasalso observed. At termination, there were no test-material related effects ontumour incidence, but at the top dose effects on the liver were seen, includingcentrilobular hypertrophy and sometimes focal necrosis. Two mouse studieswere available for review: the first was carried at dietary concentrations givingintakes of up to 56mg kg�1 bw day�1. The effects observed were mainly on the
liver but fenoxycarb also increased the incidence of alveolar/bronchiolar andHarderian gland tumours. These findings were reviewed and while the presenceof Harderian gland tumours was considered to be incidental, the lung tumourswere considered to be significant. In the second study, which was carried out athigher dietary concentrations, an increase in lung tumours was also observed.Increased liver weights were seen as was enlargement and extramedullaryhaematopoiesis in the spleen. Fenoxycarb was non-mutagenic both in vivo andin vitro.
7.2.6.6 Reproductive and Developmental Toxicity
A rat multigeneration study was carried at dietary concentrations producingintakes of up to approximately 135mg kg�1 bw day�1. There were effects onliver weight in F1 parental females and F1 pups. Effects were seen on durationof gestation and pup weight. A NOAEL was not achieved for this study, but aderived no effect level (NOEL) was used to calculate the ADI. In a study ofdevelopmental toxicity in the rat, developmental toxicity, including ter-atogenicity was not seen even at the highest dose (500mg kg�1 bw day�1). In astudy in rabbits of developmental toxicity, there was some evidence of ter-atogenicity at the highest dose (300mg kg�1 bw day�1). In a follow-up studyusing doses up to 200mg kg�1 bw day�1 such findings were not seen, but it wasconcluded that it would be prudent to regard 200mg kg�1 bw day�1 as aNOAEL for these effects. In a study of reproductive toxicity in sheep, fenox-ycarb was administered at doses of 0, 0.69 or 1.38mg kg�1 per day in gelatinecapsules; nothing adverse was observed in the dosed ewes or rams or in theiroffspring up to 28 days.21
7.2.6.7 Neurotoxicity
Fenoxycarb is not thought to be an anticholinesterase, but Smulders and col-leagues22 observed that fenoxycarb inhibited rat neuronal a4b4 nicotinicreceptors, expressed in Xenopus laevis oocytes.
7.2.6.8 Reference Dose
The ACP proposed an ADI based on a derived NOAEL (considered to be3mg kg�1 bw day�1) from the multigeneration study. The ADI was 0.03mgkg�1 bw day�1.20
7.3 Chitin Synthesis Inhibitors
Chitin is a modified polysaccharide which contains nitrogen and is found inmany organisms. It is the main component of the exoskeletons of arthropods,including insects and crustaceans. Insects grow by repeatedly moulting andcreating a new exoskeleton, and this requires the synthesis of chitin. Chitin
233Insecticides that Interfere with Insect Growth and Development
synthesis inhibitors prevent this process. The action of chitin synthesisinhibitors such as diflubenzuron is very specific, in that the compounds do notappear to inhibit hexosamine transferases, which are responsible for connectivetissue glycosaminoglycan formation in mammals.23 The chitin synthesisinhibitors, which are benzoylphenylureas, are generally of low mammaliantoxicity. Many of these compounds have haematological effects in mammals,probably because of metabolism to substituted anilines.Many of the chitin synthesis inhibitors have uses in veterinary medicine as
well as in plant protection.
7.3.1 Diflubenzuron
Diflubenzuron is the ISO name for 1-(4-chlorophenyl)-3-(2,6-difluorobenzoyl)urea (IUPAC). The structural formula is:
7.3.1.1 Absorption, Distribution, Metabolism and Excretion
The absorption, distribution, metabolism and excretion were reviewed by theJMPR.5,23 Diflubenzuron administered orally was absorbed to a limited extentin mice, rats and cats, the rate of absorption being rapid. The extent ofabsorption decreased with increasing dose. The highest tissue concentrations oflabel in the rat were found initially in liver and fat. Diflubenzuron was rapidlyexcreted, most of the dose being excreted within 24 h. Absorbed diflubenzuronwas excreted primarily in urine, with involvement of biliary excretion andenterohepatic circulation. Diflubenzuron (unchanged) was the only componentexcreted to a significant extent in the faeces. A number of metabolites werefound in the urine. The primary metabolic steps were hydroxylation of theanilino ring, ureido bridge cleavage and sulfate conjugation.
7.3.1.2 Acute Toxicity, Irritancy and Sensitization
The acute toxicity of diflubenzuron is very low, the LD50 in mice and rats bygavage was greater than 4600mg kg�1 bw.5 Diflubenzuron is not a skin irritantor a skin sensitizer. It is marginally irritant to the eyes of rabbits.24
As with many other insecticides discussed in this chapter, a notable tox-icological effect of diflubenzuron in experimental animals is to cause changes inhaematological parameters, such as methaemoglobinaemia, sulfhaemoglobi-naemia and reticulocytosis, together with a fall in the red blood cell count andincreased Heinz body formation. This may result from the metabolism of thecompound to inter alia 4-chloroaniline.23 Such changes were seen in rats, in a7/13-week dietary study, but the JMPR5 considered the findings not to bebiologically significant, as they were sporadic. Increases in spleen and liverweight were seen. Histopathologically, there was, haemosiderosis and conges-tion of the spleen, erythroid hyperplasia of the bone marrow in all treatedgroups, and haemosiderosis in the liver. In a 13-week dietary study in the dog,achieved intakes were up to 6.4mg kg�1 bw day�1. Changes similar to thoseseen in rats were observed (reduction in haemoglobin concentration anderythrocyte count and an increase in methaemoglobin and free haemoglobinconcentrations). In a study using dosing by capsule for a year, with doses of upto 250mg kg�1 bw day�1, there were signs of haemolytic anaemia, destructionof erythrocytes and compensatory regeneration of erythrocytes. Increases inmethaemoglobin and sulfhaemoglobin concentrations were evident at somedoses and the haematological effects appeared dose related. At autopsy,increased pigmentation of Kupffer cells and macrophages in the liver, wasobserved.
7.3.1.4 Carcinogenicity and Mutagenicity
In a long-term study in mice, diflubenzuron was given in the diet at con-centrations producing intakes of up to 960mg kg�1 bw day�1. Increases insulfhaemoglobin concentrations were noted and Heinz bodies were seen.Increased serum alkaline phosphatase and aspartate aminotransferase activitieswere also observed. There were elevations in liver and spleen weights, andhistopathological changes were see in both organs. In the liver, these changesincluded hepatocyte enlargement and vacuolation and the presence of pig-mented Kupffer cells. In the spleen evidence of extramedullary haematopoiesiswas seen. No increase in tumour incidence was associated with administrationof diflubenzuron. Two long-term studies in the rat were seen by the JMPR,5
only one of which demonstrated an overall NOAEL. In the first study, dietaryconcentrations used were calculated to produce daily doses of up to 8mg kg�1
bw. Increased methaemoglobin concentrations were seen. The study wascharacterized by poor survival and the range of tissues examined was limited.In the second rat study, much higher dietary concentrations were used andintakes up to 612mg kg�1 bw day�1 were achieved. There were alterations inerythrocyte parameters (reductions in red blood cell count and haemoglobin,and increased methaemoglobin and reticulocyte count). Bone-marrow hyper-plasia indicated a response to haemolysis. Splenomegaly was seen accompaniedhistopathologically by pigmented macrophages in the spleen and liver and
235Insecticides that Interfere with Insect Growth and Development
erythroid hyperplasia of the bone marrow. No NOAEL was identified asincreased concentrations of methaemoglobin and sulfhaemoglobin were seen atthe lowest dietary concentration (equal to an intake of 7.1mg kg�1 bw day�1).In neither rat study was there evidence of carcinogenicity. Interestingly, thecompound is reported to have antitumour activity in mice,25 but it stimulateshaematopoiesis in mice.26 Diflubenzuron was not genotoxic in vitro, in a varietyof systems and in the presence or absence of metabolic activation. The pesticidewas not genotoxic in vivo.
7.3.1.5 Reproductive and Developmental Toxicity
In a two-generation study in rats seen by the JMPR,5 the most notable effectwas a reduction in pup bodyweight gain during lactation (F1 pups only).Specific reproductive toxicology was not observed, even at the highest dietaryconcentration, which produced intakes of diflubenzuron of up to 4300mg kg�1
bw day�1. Haematological effects were observed in the parents at all doses.Developmental toxicity studies in rats and rabbits were available to the JMPR.5
In both species the NOAELs for maternal toxicity, embryotoxicity, fetaltoxicity and teratogenicity were considered to be the highest dose tested(1000mg kg�1 bw day�1 for both species).
7.3.1.6 Reference Dose
The JMPR established an ADI of 0.02mg kg�1 bw.27 This was based on theNOAEL for haematological effects in the 2-year rat studies and the 1-yearstudy in dogs and was later reaffirmed. An ARfD was deemed unwarrented.5
7.3.2 Flufenoxuron
Flufenoxuron is the ISO name for 1-[4-(2-chloro-a,a,a-trifluoro-p-tolyloxy)-2-fluorophenyl]-3-(2,6-difluorobenzoyl)urea (IUPAC). The structural formula is:
7.3.2.1 Absorption, Distribution, Metabolism and Excretion
The metabolism and toxicology of the compound has been reviewed.28–30 TheUnited States Environmental Protection Agency (EPA)29 considered the major
metabolic route in rats to be hydrolysis to benzoic acid, a substituted anilinederivative and a phenylurea metabolite. The major metabolite in the urinewas benzoic acid. In rats at low doses the material was well absorbed but not athigh doses, i.e. saturation occurred.
7.3.2.2 Acute Toxicity, Irritancy, and Sensitization
The rat oral LD50 is greater than 3 g kg�1 bw. The material is not a skin irritantin the rabbit and is only transiently and minimally irritant to the rabbit eye.EFSA considered the active substance to be non-sensitizing to guinea-pigskin.30
7.3.2.3 Repeated Dose Studies
The toxicology of flufenoxuron is similar to that of diflubenzuron and conse-quently it will be discussed in less detail. As with other pesticides of the group,the major effects in repeated dose studies are on the haematological system.These include decreases in red blood cell count, haemoglobin and haematocritand the presence of methaemoglobin and sulfhaemoglobin, dogs being moresusceptible than the rat.
7.3.2.4 Carcinogenicity and Mutagenicity
In one (out of two) mouse oncogenicity studies vascular tumours (haem-angiosarcomas in the liver and spleen) were observed. This was considered to bea high dose effect and the EPA29 considered flufenoxuron not likely to becarcinogenic to humans. Flufenoxuron was negative in a variety of genoxicitystudies.
7.3.2.5 Reproductive and Developmental Toxicity
In a study of reproductive toxicity in the rat, parental toxicity was not seen andoffspring toxicity consisted of reduced pup weight during lactation (theNOAEL for this effect was critical to the evaluation). Parental mating per-formance and fertility was unaffected. In a study of developmental toxicity inthe rat, adverse effects were seen neither in the dams nor in the offspring. Inrabbits, maternal toxicity was not seen, but there were effects on fetal growth atthe highest dose.29
7.3.2.6 Neurotoxicity
There was no evidence of neurotoxicity in a 28-day rat neurotoxicity study.29
237Insecticides that Interfere with Insect Growth and Development
There have been few case reports of human poisonings, although lactic acidosiswas reported in one individual, who appeared to have ingested about 100 mL ofa preparation containing flufenoxuron and other components including sur-factants and solvents.31
7.3.2.8 Reference Dose
The EPA established a chronic reference dose (ADI) of 0.0375mg kg�1 bwday�1. This was based upon the NOAEL of 3.75mg kg�1 bw day�1 in the two-generation study of reproductive toxicity in the rat and a uncertainty factorof 100.29
7.3.3 Lufenuron
Lufenuron is the ISO name for (RS)-1-[2,5-dichloro-4-(1,1,2,3,3,3-hexa-fluoropropoxy)phenyl]-3-(2,6-difluorobenzoyl)urea. The structural formula is:
7.3.3.1 Absorption, Distribution, Metabolism and Excretion
The absorption, distribution and metabolism and toxicity of lufenuron hasbeen reviewed.32 Several studies of absorption, distribution and metabolismwere available. The major route of excretion after gavage in rats with radi-olabelled lufenuron was the faeces, particularly at high doses, and unchangedlufenuron was the only major component in the faeces. Elimination in the urinewas very low at all doses. Following intravenous administration, the majorroute of elimination was again the faeces, but excretion in the first 24 h wasmuch lower than after oral administration suggesting that, after oral admin-istration, much of the lufenuron was simply not absorbed. The rate of elim-ination of the material which was absorbed or administered intravenously wasslow. The highest tissue residue levels were in fat.
7.3.3.2 Acute Toxicity, Irritancy and Sensitization
The acute oral toxicity in mice and rats was greater than 2000mg kg�1 bw.The material was non-irritant to the skin and eyes. However, lufenuron was askin sensitizer in guinea-pigs.32
Compared to other insecticides of its group, studies on lufenuron show a paucityof haematological signs but evidence of neurotoxicity (albeit at high doses). In90-day study in rats, seizures were seen at very high doses (intakes ofapproximately 1 g kg�1 bw day�1). Slightly increased haematocrits and pro-thrombin times were seen in females. Bodyweights were lower in males at highdoses compared to controls. Seizures were also observed in range finding studiesin mice and this effect appeared to be dose-related. In a 90-day study of thetoxicity of lufenuron admixed into the diet of beagles, maximum intakesachieved were approximately 2 g kg�1 bw day�1. Adverse clinical effects werenot seen. A small decrease was seen in the highest-group males in red blood cellcount and haematocrit: some changes in clinical biochemistry (decrease inpotassium and phosphorus and increases in alkaline phosphatase and choles-terol) also occurred. Increased liver weight was observed at higher doses, andthis was unaccompanied by histopathological changes. In a 1-year toxicity studyin dogs, similar dietary concentrations were used. Neuromuscular signs andconvulsions were observed at the higher doses starting at least 20 weeks afterinception of the study. Increased adrenal weights were observed at higher doses.There was also a slight increase in thyroid weight (at higher doses), with folli-cular dilatation (at all doses) and an increase in liver weight (at higher doses)accompanied by histopathological alterations (hepatocyte hypertrophy andincreased intracellular pigmentation). Changes were also seen in the adrenalcortex and lung at the higher doses. The rapporteur member state consideredthat no NOAEL could be derived for the study. A further dog study over 12months was carried out at lower dietary concentrations (highest intakes were31.8mg kg�1 bw day�1). One early death was seen at the highest dose and threein that dose group were killed after convulsions. Clinical signs seen at the highestdietary concentrations included convulsions, which were preceded by reducedactivity, tremor and ataxia. Lower bodyweight was seen at the highest dietaryconcentration. There was an increase in liver weights at the higher doses and inadrenal weights in the high-dose males. Hypertrophy of hepatocytes with pig-mentation of hepatocytes was seen at the higher doses. At the highest dose therewere changes in the lymphoreticular tissue of the gastrointestinal tract (deple-tion of lymphocytes in Peyer’s patches) and lungs (foamy histiocytes).
7.3.3.4 Carcinogenicity and Mutagenicity
In a long-term study in mice, the highest intakes achieved were approximately63 mg kg�1 bw day�1. Convulsions were seen at the higher doses, and at thehighest dose the study was terminated early. Moreover, at the next to highestdose, there was excess mortality. At that dose, fatty liver was observed. In thefemales necrotic changes were seen in livers at the highest dose. Excess lungadenomas were seen. The relevance of these tumours was considered equivocal.Histopathological evidence of chronic inflammation of the prostate was seen atthe highest dose. In a long-term oral toxicity study in the rat, dietary
239Insecticides that Interfere with Insect Growth and Development
concentrations were used that produced daily intakes of up to 114mg kg�1 bw.There were convulsions at high doses. Because of the clear evidence of toxicity,the animals of both sexes at the highest dose were killed at week 14. Someminor haematological changes were observed in these animals, but not theothers in the study. There was no evidence of tumorigenesis. At higher dosesthere were pulmonary alveolar foam cells, ulcerative and/or inflammatorychanges in the forestomach and focal inflammatory changes in the caecum andcolon. Fatty change in the livers and urinary tract inflammation were seen infemales. In a number of tests in vitro and in vitro there was no evidence ofgenotoxicity.
7.3.3.5 Reproductive and Developmental Toxicity
In a multigeneration study in the rat, the highest dietary concentration was250 ppm, where intakes were approximately 20mg kg�1 bw day�1, exceptduring lactation where intakes were higher. There was little evidence ofreproductive toxicity: at the high dose, appearance of the righting reflex wasdelayed in both F1 and F2 pups. Bodyweights of the F1 adults (both sexes) wereincreased by comparison with the controls. There was no histopathologicalevidence of effects related to treatment. In a rat study of reproductive toxicity, thehighest dose being 1000mg kg�1 bw day�1, there was slight maternal toxicity atthat dose (reductions in bodyweight and food consumption). No embryofetaltoxicity was seen and there was no evidence of teratogenicity. A reproductivetoxicity study was carried out in rabbits, using the same doses as in the rats.Maternal toxicity was not observed, There were no effects of treatment onfertility. Embryofetal toxicity and teratogencity was not observed at any dose.
7.3.3.6 Neurotoxicity
The neurotoxicity of lufenuron was investigated in male rats at four doses, withcontrols, in a 4-month feeding study (the section in the draft assessment reportis incorrectly headed ‘Delayed neurotoxicity’, unlikely to be an issue withlufenuron). Daily test-material intakes achieved ranged up to 27mg kg�1 bw.At the higher doses there was a 2-month recovery period. At the highest dosethere was an episode of fasciculation in one rat and convulsions in another.Specific neurological examinations (motor activity, startle habituation, mazelearning) were carried out, and the results did not differ between doses. Lufe-nuron, at the highest dose facilitated convulsions induced by pentalene tetra-zole. No effects were seen of lufenuron in the central or peripheral nervoussystem on microscopy.
7.3.3.7 Endocrine Effects
In a study of the endocrine system of the rat, an increase in adrenocorticotropichormone (ACTH) levels was seen at the highest dose: this was considered to be
a stress response. Elevated prolactin and FSH levels were not considered to bebiologically significant. No effects were observed on the female genital systemor oestrous cycle length.
7.3.3.8 Reference Dose
EFSA32 proposed an ADI of 0.015mg kg�1 bw day�1 based on the 1-year dogstudy NOAEL of 1.5mg kg�1 bw day�1 with a safety factor of 100.
7.3.4 Hexaflumuron
Hexaflumuron is the ISO name for 1-[3,5-dichloro-4-(1,1,2,2-tetra-fluoroethoxy)phenyl]-3-(2,6-difluorobenzoyl)urea (IUPAC). The structuralformula is:
Hexaflumuron is a chitin synthesis inhibitor used predominantly as a ter-miticide, as are many other insecticides of this group.33 It is of very low acutemammalian toxicity. As it is usually used in bait stations, exposure of the publicis unlikely. The mammalian organ-specific toxicity is not dissimilar to that ofdiflubenzuron, with effects on the liver and haematological system.34
7.4 Ecdysone Agonists
Ecdysone is the precursor of 20-hydroxyecdysone, the major insect moultinghormone, which acts at the ecdysone receptor, a ligand-activated transcriptionfactor. The ecdysone moulting system is a general one in arthropods, henceactivity of the ecdysone agonists may be expected in other arthropod speciessuch as crabs. Unlike the other insect hormonal systems discussed above, wherethe hormones are structurally unrelated to known mammalian hormones,ecdysone resembles hormones important in mammalian physiology. Bothecdysone and 20-hydroxy ecdysone are steroids and, unsurprisingly, arepharmacologically active in mammals (see review by Lafont and Dinan).35
The main insecticides in this group are tebufenozide and methoxyfenozide.They mimic the action of ecdysone and bind to the ecdysone receptor, which
241Insecticides that Interfere with Insect Growth and Development
brings about an incomplete and premature unsuccessful moult, leading even-tually to death.
7.4.1 Tebufenozide
Tebufenozide is the ISO name for N-tert-butyl-N0-(4-ethylbenzoyl)-3,5-dime-thylbenzohydrazide (IUPAC). The structural formula is:
Tebufenozide is predominantly used to control lepidopteran pests in crops,including fruit and vegetables. Tebufenozide is fairly specific for lepidopteraninsects.36,37
7.4.1.1 Absorption, Distribution, Metabolism and Excretion
The absorption, distribution and metabolism of tebufenozide has beenreviewed.38,39 In rats given a single dose by gavage of labelled tebufenozide, theprofiles of excretion of radiolabel by males and females were similar.Absorption and excretion of label were rapid, with more than 70% of theadministered dose eliminated within 48 h. The faeces was the main route ofexcretion, only minor amounts being excreted in the urine and trace amounts inexpired air. Little radiolabel was retained in organs or tissues by 7 days afterdosing. In a similar study in bile duct cannulated rats, most of the radiolabel(67–70%) was unabsorbed and was eliminated in the faeces. The major com-ponent in the faeces was unchanged tebufenozide, together with numerousmetabolites. Parent tebufenozide was not found in the urine, but there weremetabolites, as seen in the faeces. The extent of metabolism of tebufenozide washighly dose-dependent, being lower at higher doses. The major route ofmetabolism of tebufenozide appeared to be oxidation of benzylic carbons. Inbile duct cannulated rats, parent tebufenozide was not seen in the bile.
7.4.1.2 Acute Toxicity, Irritancy and Sensitization
The toxicology of tebufenozide has been reviewed by the JMPR.38–40 Tebufe-nozide is of low oral acute toxicity (lethal dose >5.0 g kg�1 bw in the rat).Dermal administration resulted only in transient and mild effects (erythema) atthe site of application to rabbits. Tebufenozide was minimally irritating to theeyes of male rabbits and was not a skin sensitizer in guinea-pigs. A single-dose
oral study was carried out in dogs to evaluate the NOAEL for haematotoxicity;this was used to set an ARfD (see below).
7.4.1.3 Repeated Dose Studies
In repeated dose studies, the primary target of tebufenozide is the red bloodcell, resulting in increased erythrocytic turnover and a compensatory haema-topoiesis. In short-term (2-week) toxicity studies in mice, using diets containingtebufenozide, giving intakes of up to 1100mg kg�1 bw day�1, increased liverweights were observed. In a 13-week study in mice using diets containingtebufenozide giving intakes of up to 4200mg kg�1 bw day�1 reduced meanbodyweight gain was seen in males. At higher dietary concentrationshaemolytic changes and a reduced mean myeloid:erythroid ratio in bonemarrow were observed and there were dose-related increases in the weights ofthe spleen and liver. Histopathologically there was an increased frequency and/or severity of pigment deposition in the liver, spleen and kidney tubules,accompanied by increased extramedullary haematopoiesis in the spleen. In a 2-week study in rats, tebufenozide was admixed into the diet to provide intakes ofup to 780mg kg�1 bw day�1. The only adverse effects seen were increased liverweights. In a 13-week rat study, using higher dietary concentration (maximumintake 1600mg kg�1 bw day�1), there were significant decreases in overallbodyweight gain and mean food consumption. As with mice, slight haemolyticanaemia, increased bone-marrow erythropoiesis and increased deposition ofpigment in the spleen were observed. At the highest dose, additional effectsincluded overt haemolytic changes, slightly and tubular ‘nephrosis’ of thekidney in four males. Several dietary studies in dogs were carried out. In a 2-week study maximum intakes achieved were 290mg kg�1 bw day�1, in the 6-week study 42mg kg�1 bw day�1 and in the 90-day study, 200mg kg�1 bwday�1. In the 52-week study, the highest intake achieved was 56mg kg�1 bwday�1. Again the main effects seen were haematological: mild haemolyticanaemia (significantly reduced erythrocyte count, haemoglobin, andhaematocrit) was observed. The 4-week study incorporated a recovery periodafter cessation of exposure and total recovery had occurred four weeks aftercessation of treatment. In the 90-day study, Heinz bodies were observedtogether with elevated total bilirubin levels. Furthermore, there were increasedabsolute and relative spleen weights. Histopathologically, there was anincreased incidence of haemosiderin deposition in the Kupffer cells of the liverand increased haematopoiesis and sinusoidal engorgement in the spleen.At the highest dietary concentration, significant haemolytic changes and
increased bone-marrow erythropoiesis (reduced mean myeloid:erythroid ratio)were seen at weeks 6 and 13 of treatment. In the 52-week study, slight butconsistent haemolytic changes and a slightly elevated total plasma bilirubinlevel were seen. The mean absolute and relative spleen weights of females andthe mean relative liver weight of males were increased, and there was anincreased incidence of pigment deposition in the Kupffer cells of the liver.
243Insecticides that Interfere with Insect Growth and Development
Increased haematopoiesis in the spleen and splenic sinusoidal engorgement,and bone-marrow hyperplasia were also observed. The NOAEL for this study(1.8mg kg�1 bw day�1) provided one of the bases for the ADI.
7.4.1.4 Carcinogenicity and Mutagenicity
A study of long-term (18-month) toxicity was undertaken in mice, whichincluded an interim sacrifice at 52 weeks. Maximum intakes achieved were190mg kg�1 bw day�1. Survival rates were affected at the higher doses, butotherwise, the main effects seen were in the haematological system. There wasmild haemolytic anaemia, accompanied by a small but significant increase inthe blood level of methaemoglobinaemia and increased prevalence of poly-chromasia and echinocytosis in erythrocytes. Increases in splenic pigmentdeposition were observed. Extramedullary haematopoiesis in the spleen infemales was noted. There was no evidence of carcinogenicity. Additionally,there was a rat 2-year study, with an interim sacrifice at about 1 year. Tebu-fenozide was admixed in the diet to produce intakes of test material up to120mg kg�1 bw day�1. At high doses, there were effects on bodyweightand weight gain. Signs of mild haemolytic anaemia were seen during the first52 weeks. Minor increases in the incidence and/or severity of pigment deposi-tion (haemosiderin) were seen in the spleens of animals of both sexes at boththe interim sacrifice and at termination. There was no evidence of carcino-genicity. In a battery of genotoxicity tests, mostly in vitro but including atest for chromosomal aberrations in rats in vivo, tebufenozide was non-genotoxic.
7.4.1.5 Reproductive and Developmental Toxicity
In two rat studies of reproductive toxicity, the main findings werehaematological pigment deposition in the spleen and extramedullary haema-topoiesis). In the earlier study, intakes achieved were up to 170mg kg�1 bwday�1. Effects were seen at high doses on parental weight gain. The number ofimplantation sites in F1 females was decreased and gestational length in F1
(but not F0) dams was increased at the highest dose. The NOAEL was 0.8mgkg�1 bw day�1, providing one of the bases for the ADI. The findings weresimilar in the later study, but reproductive toxicity was not observed (this studyinvolved dietary concentrations giving intakes up to 140mg kg�1 bw day�1).There was evidence of pup toxicity: significant reductions in mean bodyweightgain were seen in F1 and F2 pups at the highest dose between lactation days14 and 21. In a rat study of developmental toxicity, initial slight, transientreductions in mean bodyweight gain and food consumption was observed in thedams. Embryotoxicity, fetotoxicity and teratogenicity were not observed. In arabbit study of developmental toxicity, maternal toxicity, embryofetotoxicityand teratogenicity were not seen. In both rats and rabbits, the highest dose usedwas 1000mg kg�1 bw day�1.
In a neurotoxicity test in rats, single doses of up to 2000mg kg�1 bw wereused. The study included a functional observational battery (FOB) testing andmotor activity assessment before treatment and 1–3 h and 7 and 14 days afterdosing. No effects referable to treatment were seen. At the end of the 14-dayobservation detailed neuropathological examination was carried out: treat-ment-related neuropathological changes were not found at any dose.
7.4.1.7 Reference Doses
The JMPR38 set the ADI at 0.02mg kg�1 bw based on the NOAEL from the1-year dog study and one of the rat studies of reproduction, with a safety factorof 100. The JMPR39 established an ARfD of 0.9mg kg�1 bw based on theNOAEL of 89.4mg kg�1 bw (the highest dose tested) in a single-dose oral studyof haematotoxicity in dogs and a safety factor of 100.
7.4.2 Methoxyfenozide
Methoxyfenozide is the ISO name for N-tert-butyl-N0-(3-methoxy-o-toluoyl)-3,5-xylohydrazide (IUPAC). The structural formula is:
The action of methoxyfenozide in insects is similar to that of tebufenozide.The toxicology of the two compounds is also very similar.
7.4.2.1 Absorption, Distribution, Metabolism and Excretion
The absorption, distribution and metabolism and excretion of methox-yfenozide was reviewed by the JMPR.39 When given orally to rats [14C]-methoxyfenozide is absorbed rapidly, 58–77% of the administered dose beingexcreted within 24 h (mostly in the faeces). The peak plasma and blood con-centrations of radioactivity (Cmax) were attained approximately 15–30 min afterdosing. Elimination of radiolabel from the plasma showed a biphasic pattern.Concentrations of radioactivity at Cmax were highest in the liver, with con-centrations in the adrenals and in the spleen also being higher than that inwhole blood. Using methoxyfenozide radiolabelled at several sites, more than30 metabolites of methoxyfenozide were identified in rat urine, faeces and bile,demethylation, glucuronidation and hydroxylation being the main metabolic
245Insecticides that Interfere with Insect Growth and Development
processes. Methoxyfenozide was poorly absorbed (o4%) in the rat after der-mal exposure.
7.4.2.2 Acute Toxicity, Irritancy and Sensitization
The toxicity of methoxyfenozide was reviewed by the JMPR.39 Methoxy-fenozide was of low acute toxicity when administered by the oral route to ratsand mice (LD50> 5 g kg�1 bw) and was of low toxicity in rats by the dermaland inhalation routes. Methoxyfenozide was not irritant to the skin of rabbitsand produced only minimal, irritancy to the rabbit eye. Methoxyfenozide wasnot a sensitizer in guinea-pigs.
7.4.2.3 Repeated Dose Studies
In a 3-month study in mice, methoxyfenozide was given in diets at con-centrations producing intakes up to 1742mg kg�1 bw day�1. Deaths were notseen nor were there treatment-related clinical signs of toxicity. Bodyweight gainafter 4, 8 and 13 weeks was consistently reduced in both sexes at the highestdose. Substantial substance-related haematological effects were not seen,although a small increase in mean cell volume and mean cell haemoglobinconcentration for females was seen at the highest dose. No important changesin any clinical chemistry parameter were seen. Clear effects were not noted onorgan weights at any dose in either sex. A small increase in relative liver weight,probably secondary to reduced bodyweight was observed without histo-pathological correlates and there was no other evidence of hepatic toxicity inthe study. No test-material related gross or microscopic pathological changefindings was observed in any tissue. A 90-day dietary study of methoxyfenozidewas undertaken in the rat, using 5 dietary concentrations and controls. Intakesachieved ranged up to 1531mg kg�1 bw day�1. No clinical signs of toxicitywere noted and there was little effect on bodyweight, bodyweight gain, foodconsumption or ophthalmoscopic examination. Haematological findings indi-cative of mild anaemia were noted at the highest dose (decreased erythrocytecount and haemoglobin in females). Relative liver weights were increased inboth sexes, and there was evidence of slight to moderate periportal hepatocytehypertrophy in both sexes at the top two doses.A total of five dog studies were reviewed by the JMPR.39 They were two 2-
week studies (highest intakes observed being 1225 and 1186mg kg�1 bw day�1),a 90-day study, a 1-year study and a study of reversibility of blood effects. Inthe 2-week studies, the main changes of interest were haematological (decreasederythrocyte counts and haemoglobin concentration, presence of Heinz bodiesand an increase in methaemoglobin). Howell–Jolly bodies were also seen. Therewas an increase in plasma total bilirubin concentrations. Reticulocytosis wasseen in one study, especially in females. Absolute and relative splenic weightswere increased at high dietary concentrations. Minimal haemosiderin accu-mulation in Kupffer cells was observed in liver in one study (livers were not
examined histopathologically in the other). In a 13-week dietary study in dogs,intakes achieved were up to 209mg kg�1 bw day�1. All dogs survived to ter-mination and test-material-related clinical signs were not noted. Dogs from thelowest dose test group were dosed for a further 2 weeks at that dose and thenfor 6 weeks at a very high dietary concentration (intake about 460mg kg�1 bwday�1): there were no concurrent controls for that dose, which made inter-pretation difficult. Bodyweight and weight gain of females at the highest dosefor which there were concurrent controls were decreased, but the significance ofthis was unclear. No effects were seen on food consumption by either sex duringthe study. Consistent effects on haematological parameters were not seenduring the main 90-day study. There was no clear evidence of methaemoglobin-aemia; moreover, there were no haematological changes in animals in the groupin which the intake had been increased to approximately 600mg kg�1 bw day�1.There were no clear effects on clinical chemistry measurements. Ophthalmo-scopic findings were similar in all groups. There were small rises in relative, butnot absolute liver weights at some doses in both sexes compared with those of theconcurrent controls, but as there was no clear dose–response relationshipthe significance of this is dubious. Absolute kidney weights were slightlydecreased inmales at all test doses, and this effect appeared to be dose-related.Notest-material related morphological findings were recorded post mortem at theend of the main study or at the end of exposure to 460mg kg�1 bw day�1.A 1-year dietary study in beagles was also undertaken, using dietary con-
centrations that gave intakes of up to 1199mg kg�1 bw day�1. Mortality wasnot observed, nor were there clinical signs of toxicity. Bodyweight gain wasreduced in males at the top dose in the early part of the study, while in females,bodyweight gain was reduced at the top dose during the later part of the study.Food consumption was similar in all groups. There was some evidence ofmethaemoglobinaemia at the top dose in both sexes compared with controlsand the top-dose group pretreatment. It should be noted that these measure-ments were on fasting blood samples and therefore may well have under-estimated peak methaemoglobin values. Decreases were seen in erythrocytecounts at the top two dietary concentrations and nucleated erythrocytes weredetected in both sexes at the top dose. Platelet numbers were increased in bothsexes at the top two doses. There were no effects on leukocytes. Total bilirubinin both blood and urine was increased in both sexes at the top two doses.Ophthalmological examinations were all normal. The NOAEL was equal to9.8mg kg�1 bw day�1, based on changes in the blood and liver.A study to investigate the reversibility of haematological effects seen in the
1-year dog study was undertaken. There were two groups, controls and a testgroup similar to the high dose group in the 1-year study, the animals (all male)being dosed for 4 weeks and retained for 4 weeks untreated. Blood sampleswere collected from all dogs for haematology analysis before the start of thestudy, after 4 weeks of treatment and after 2 and 4 weeks of the period duringwhich the dogs were not treated. There were no mortalities nor were adverseclinical signs observed. Methoxyfenozide produced a range of haematologicaleffects after the 4-week period of dosing with methoxyfenozide, such as reduced
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haemoglobin and red blood cell count and elevated methaemoglobin. Therewas substantial evidence of recovery during the reversibility period.
7.4.2.4 Carcinogenicity and Mutagenicity
The JMPR39 reviewed long-term studies in mice and rats. Dietary concentra-tions used in mice produced intakes of up to 1354mg kg�1 bw day�1. Therewere no effects on survival and treatment-related clinical signs of toxicity werenot seen. Minor increases in the prevalence of extramedullary haematopoiesisin the spleen were observed at the highest dose in both sexes. The incidence ofbronchiolo-alveolar adenoma or carcinoma was increased in females at the toptwo doses; however, the increase was small and was not considered to betreatment-related, as the prevalence was within the historical control range.The NOAEL for carcinogenicity and for other effects was the highest dietaryconcentration. In rats, the dietary concentrations used gave intakes of up to1248mg kg�1 day�1. There was some evidence of reduced survival in thehighest dietary concentration group, but survival was poor in all groups, whichlimited the power of the study. No test-material related clinical signs of toxicitywere seen. Decreased bodyweight gain was seen at the highest-dose group infemales towards the end of the study. Minor haematological changes wereobserved at the top two dietary concentrations (reduced red blood cell countsand haemoglobin and a small degree of methaemoglobinaemia). At the top twodietary concentrations, there was an increase in g-glutamyltransferase activity.Increased liver weights (absolute and relative) were seen in both sexes at the toptwo dietary concentrations, and increased adrenal weights in females at thehighest dietary concentration. Histopathological changes were seen in liver(periportal hepatocellular hypertrophy) at the top two dietary concentrations inboth sexes and kidney (glomerular nephropathy) at the highest dietary con-centration in females only. Follicular hyperplasia of the thyroid was observedat the top two dietary concentrations in males and the top dietary concentra-tion in females. Changes were observed at the highest dose in females in theforestomach. There was a small increase in the incidence of hepatocellularadenoma in females at the highest dose, although the prevalence was within thehistorical control range. There were two instances of statistically significantincreases in tumour incidence (C-cell adenoma of the thyroid in males andmammary gland tumours in females), but in neither case was there any doserelationship. The study authors considered there to be no clear oncogenicresponse. The overall NOAEL was equal to 10mg kg�1 bw day�1. This was onthe basis of haematological changes and histopathological changes in the liverand thyroid at the next highest dietary concentration. Methoxyfenozide wasnon-genotoxic both in vitro and in vivo.
7.4.2.5 Reproductive and Developmental Toxicity
The JMPR39 reviewed a two-generation study, which included sperm tests,oestrous cycling and developmental milestones. There were no deaths or
clinical signs of toxicity in the parents or pups during the lactation period.Bodyweight gain was decreased at the highest dose in both generations ofparental males but not females. Test-material-related effects on oestrouscycling, sperm parameters, mating performance, litter size and pup bodyweightand viability were not observed. There was an increased in the number of F1
stillbirths but this was not seen in the F2 offspring. A significant but small delayin vaginal opening was noted in F1 female pups, but this was not significant inthe F2 generation. No effect was observed on reproductive success. There wereno test-material-related effects on reproductive organs. In parental rats at thehighest dose, there was an increase in absolute and relative liver weights,accompanied by hepatocellular hypertrophy, with pigmentation of Kupffercells. Minimal effects on the liver at the mid dietary concentration were notconsidered to be an adverse effect. The NOAEL for toxicity was 143mg kg�1
bw day�1. This was on the basis of liver toxicity. The NOAEL for reproductivetoxicity was 1474mg kg�1 bw day�1, the highest dose tested. The NOAEL forpup toxicity was 143mg kg�1 bw day�1 because of minor delays in vaginalopening. In a study of developmental toxicity, mated female rats were givenmethoxyfenozide (purity 99.2%) at a doses of up to 1000mg kg�1 bw day�1 ondays 6–15 of gestation. Dosing was by gavage, dams being sacrificed at day 20 ofgestation. No deaths occurred and clinical signs of toxicity were not seen. Therewere no treatment-related effects on the dams’ bodyweights, weight gain or foodconsumption. There was no evidence of developmental toxicity or of treatmentrelated-effects on the fetuses. There was no evidence of teratogenicity. TheNOAELs for maternal toxicity and developmental toxicity were was 1000mgkg�1 bw day�1. Mated rabbits were given methoxyfenozide at doses of up to1000mg kg�1 bw day�1. No test-material-related adverse clinical signs wereobserved or effects on reproductive or litter parameters were seen. The NOAELsfor maternal toxicity and developmental toxicity were 1000mg kg�1 bw day�1.There was no evidence of teratogenicity.
7.4.2.6 Neurotoxicity
An acute neurotoxicity study was undertaken in the rat at single doses of up to2000mg kg�1 bw by gavage . The study included an FOB. No deaths wereobserved, nor were there test-material-related clinical signs of systemic toxicityor effects on bodyweight. Hindlimb grip strength for males at the highest dosewas reduced, but it was considered that overall, there was no convincing evi-dence for a substance-related effect on grip strength or motor activity. Grossnecropsy and histopathology did not reveal any treatment-related findings inthe central or peripheral nervous systems. The NOAEL was 2000mg kg�1 (thehighest dose). A 13-week dietary study in rats at feed concentrations givingintakes of to up to 1577mg kg�1 bw day�1 was undertaken. This study includedan FOB. Deaths were not seen and there were no test-material-related adverseclinical signs. Moreover, effects on bodyweight were not seen. No test-material-related effects were seen either in the FOB or on motor activity. There was noevidence of neurotoxic or neuropathic effects in rats receiving diets at any test
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material concentration, therefore the NOAEL was the highest dietaryconcentration.
7.4.2.7 Reference Doses
The JMPR established an ADI of 0.1mg kg�1 bw based on the NOAELs fromthe long-term study in rats, and the 1-year study in dogs, and a 100-fold safetyfactor; an ARfD of 0.9mg kg�1 bw was established based on a 1-day dog studywith tebufenozide. It was considered that this was a conservative approachbecause tebufenozide was more potent than methoxyfenozide in producingadverse effects on red blood cells.39
7.5 Conclusions
The insecticides discussed in this chapter are generally of low toxicity, but arenot knock-down insecticides and do not usually treat established infestations ofadult insects.
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