Toxicological evaluations

Guinea-pigs

One guinea-pig was given [5-¹⁴C]racemic methoprene (purity, 96.9%) at a single dose of 49 mg/kg bw by gavage, and urine, faeces and expired CO₂ were collected for 24 h. At that time, the animal was killed, and samples of blood, fat and muscle were taken. The animal excreted 50% of the radiolabel within 24 h, with 24% in urine, 9% in faeces and 17% in expired air. The peak concentration of radiolabel in urine was reached 5.5 h after dosing. By 24 h after treatment, the blood contained 19 mg equivalent per ml, and muscle and fat contained 3.3 and 11 mg equivalent per gram wet tissue, respectively (Chamberlain et al., 1975).

Cows

A Hereford steer weighing 277 kg, housed in a metabolism room, was given a single oral dose of 2 g of [5-¹⁴C]racemic methoprene, equal to 7.2 mg/kg bw, and urine and faeces were collected for 2 weeks. Room air samples were collected every 3 h for the first 2 days, every 6 h for the next 2 days and once a day for the next 10 days. Blood samples were taken regularly. After 14 days, the animal was killed, and samples of tissues were collected for measurement of radiolabel. The steer excreted about 22% of the administered radiolabel in urine and 39% in faeces over 14 days. Although part of the radiolabel was reported to have been expired as ¹⁴CO₂, the amount was not quantified. The concentration of radiolabel in blood peaked at 72 h and then very slowly declined. After 14 days, the concentration of radiolabel in blood was about half the maximum value. The highest concentrations of radiolabel were found in bile, gall-bladder, liver, kidney, lung, adrenal, spleen and fat. (Chamberlain et al., 1975).

A Jersey cow weighing 338 kg was given a single oral dose of 210 mg of [5-¹⁴C]racemic methoprene, equal to 0.61 mg/kg bw, and urine, faeces and milk were collected for 7 days. Expired air was sampled continuously for the first 4 h and for 1 h every fourth hour for the next 3 days. Blood samples were taken 6 and 48 h and 7 days after treatment. After 7 days, 73% of the radiolabel had been eliminated, with 20% in urine, 30% in faeces, 15% in expired air and 8% in the milk, indicating that 27% may have remained in the body. The concentrations of radiolabel in expired air, urine, faeces and milk peaked about 24–48 h after treatment. By day 7 after treatment, the highest concentrations of radiolabel were found in bile, gall-bladder, liver, kidney, ovary, lung, spleen and fat (Chamberlain et al., 1975).

Laying hens were given [5-¹⁴C]racemic methoprene (purity, 95.9%) as single oral doses of 0.6–77 mg/kg bw. Excreta, separated into ‘urine’ and faeces, expired air and eggs were collected from three hens for 14 days, and urine, faeces and expired air were collected from eight hens for 48 h, after which time tissue samples were taken for measurement of radiolabel. Chickens given low doses of methoprene (0.6–3.4 mg/kg bw) excreted most of the radiolabel in expired air (35–44% within 48 h), while those given higher doses (59–64 mg/kg bw) eliminated most of the radiolabel in urine (34–38%) and faeces (17–19%) within 48 h. Over 14 days after administration, up to 19% of the radiolabel was eliminated in eggs, mainly in the yolk. After 48 h, most of the radiolabel was found in liver, kidney, intestines and lungs (Davison, 1976).

(b) Biotransformation

Three rats (sex not specified) with cannulated bile ducts received a single oral dose of [5-¹⁴C]racemic methoprene at 25 mg/kg bw. Bile, urine and faeces were collected for 48 h after treatment. At least 12 unidentified metabolites were detected in urine, and two major unidentified metabolites were detected in bile. Intact methoprene was found only in faeces as a small fraction of the total excreted radiolabel; this probably represented unabsorbed compound, as the bile contained no intact methoprene. Since a large proportion of the radiolabel was excreted as ¹⁴CO₂, methoprene appears to be extensively metabolized (Chasseaud et al., 1974; Hawkins et al., 1977). A statement of quality assurance was included in the study of Chasseaud et al. (1974).

Guinea-pigs

In the study of Chamberlain et al. (1975) in which one guinea-pig was given [5-¹⁴C]racemic methoprene as a single dose of 49 mg/kg bw by gavage, urine and faeces were examined for metabolites. In urine collected 3–6 h after dosing, 95–99% of the recovered radiolabel consisted of glucuronic acid conjugates and other polar compounds. After treatment with glucuronidase, the two main metabolites identified were 11-methoxy-3,7,11-trimethyldodeca-2,4-dienoic acid and 11-hydroxy-3,7,11-trimethyldodeca-2,4-dienoic acid, accounting for 75% of the radiolabel in urine. Isopropyl-11-hydroxy-3,7,11-trimethyldodeca-2,4-dienoate and 7-methoxy citronellic acid were also identified. No intact methoprene was found in urine. In faeces, 77% of the recovered radiolabel represented intact methoprene; small quantities (3–8%) of 11-hydroxy-3,7,11-trimethyldodeca-2,4-dienoic acid, 11-methoxy-3,7,11-trimethyldodeca-2,4-dienoic acid and isopropyl-11-hydroxy-3,7,11-trimethyldodeca-2,4-dienoate were found.

Cows

In the study of Chamberlain et al. (1975) in which a Hereford steer was given a single oral dose of [5-¹⁴C]racemic methoprene at 7.2 mg/kg bw, a large proportion of the radiolabel in faeces represented intact methoprene. In urine, the main metabolites were isopropyl 11-hydroxy-3,7,11-trimethyldodeca-2,4-dienoate and 11-methoxy-3,7,11-trimethyldodeca-2,4-dienoic acid; no intact methoprene was found. Part of the radiolabel was expired as ¹⁴CO₂, and part was incorporated in cholesterol and bile acids, indicating extensive metabolism and biodegradation of methoprene (Quistad et al., 1974, 1975a).

In the study of Chamberlain et al. (1975) in which a Jersey cow was given a single oral dose of [5-¹⁴C]racemic methoprene at 0.61 mg/kg bw, 15% of the radiolabel was eliminated in expired air over 7 days. Radiolabelled acetic acid and cholesterol were present in blood. In milk, radiolabelled fatty acids, lactose, lactalbumin and casein were detected; only 1% consisted of intact methoprene. The primary metabolites of methoprene were not detected in milk (Quistad et al., 1975b).

In the study of Davison (1976) in which laying hens were given [5-¹⁴C]racemic methoprene at single oral doses of 0.6–77 mg/kg bw, faeces collected on the first day contained 39% intact methoprene, while no methoprene was detected in urine or blood. In urine, 5% of the radiolabel represented conjugated 11-methoxy-3,7,11-trimethyl-dodeca-2,4-dienoic acid, 4% was associated with 11-hydroxy-3,7,11-trimethyl-dodeca-2,4-dienoic acid, 5% was incorporated in uric acid, and 85% of the compounds containing radiolabel were not identified. In blood at 48 h, < 0.1% of the radiolabel represented methoprene or its primary metabolites. About 1% of the radiolabel in blood was incorporated in cholesterol, and 96% of the compounds containing radiolabel were not identified. Muscle and fat contained only trace amounts of methoprene. Most of the radiolabel in egg yolk was incorporated in natural compounds such as cholesterol and fatty acids, and only 2% represented methoprene residues (Quistad et al., 1976).

(a) Acute toxicity

The acute toxicity of the racemate and S-methoprene is summarized in Table 2.

Methoprene was injected intraperitoneally to Sprague-Dawley rats at doses of 3.0–52 g/kg. The clinical signs included depression, tremors, lachrymation, diarrhoea and distended abdomen. The histopathological findings included fibrinous peritonitis. Rats treated intraperitoneally with 3 g/kg bw on two subsequent days or at an interval of 48 h showed no increase in mortality rate as compared with rats treated once at a dose of 3 g/kg bw (Jorgenson & Sasmore, 1972a).

Two male and two female beagle dogs received methoprene by gavage at a dose of 10 g/kg bw. The two females died within 30 and 40 min of treatment, one male died after 109 min. and the other male was killed for humane reasons after 3 h. The clinical signs observed were aggressive behaviour, piloerection, pupil dilatation, salivation, increased respiratory frequency followed by shallow respiration, loss of gait, convulsions, vomiting, opisthotonos and nystagmus. Gross examination revealed congestion of the kidneys, liver, lungs and scleral vessels; telangiectasia was observed in the liver. Two animals had signs of cardiac effects (ecchymosis, ventricular dilatation). Signs of minor congestion were found in the central nervous system, haematopoietic tissues, the reproductive tract and the digestive tract (Hill, 1972a).

Groups of one male and one female dog received methoprene at an oral dose of 1, 2 or 5 g/kg bw. No clinical signs were observed. After sacrifice on day 21 after dosing, no treatment-related pathological changes were found on gross examination (Hill, 1972b).

No deaths occurred when dogs were exposed for 6 h to a mist (particle size, 2–5 µm) of technical-grade methoprene as a 2% aqueous solution, providing an estimated total dose of 30 mg/kg bw for males, or 29 mg/kg bw when corrected for purity. However, the actual concentration of methoprene in air was not reported. The clinical signs observed during exposure were increased heart rate and respiration frequency, vomiting, salivation and exhaustion. It was not clear whether these signs were associated with the compound or the procedure. Food intake and body weight were reduced during the first 1–2 days after exposure (Saito, 1975a).

No deaths, no clinical signs and no irritation were seen in New Zealand white rabbits treated dermally with technical-grade S-methoprene. Statements of compliance with GLP and QA were included (Brown, 1984a).

In one study of oral administration (Hill, 1972c) and two by inhalation (Hiddemen, 1972; Olson, 1972a), the purity of the technical-grade (racemic) methoprene was not reported. These studies were not evaluated.

A number of studies were performed with formulations of S-methoprene. The acute toxicity of formulations containing 2% (Blasczak, 1994a,b), 4% (Blasczak, 1994c,d), 5% (Hiles & Collins, 1984a,b) or 20% (Schindler & Baldwin, 1991a,b) S-methoprene was low after oral or dermal administration. No deaths occurred after inhalation of a 20% S-methoprene formulation at the highest concentration tested (5.2 mg/l) (Collins & Procter, 1984).

Undiluted technical-grade racemic methoprene (purity unspecified) was administered into the right conjunctival sac of five male New Zealand white rabbits at a volume of 0.1 ml. The eyes were examined and scored for irritation 24, 48 and 72 h after administration. No signs of irritation were observed (Hill, 1971).

Eight female New Zealand white rabbits received 0.1 ml of technical-grade racemic methoprene (purity unspecified) into one eye. The eyes of five animals were washed with lukewarm saline 5 min after dosing, and the eyes of the remaining three animals were washed similarly 24 h after dosing. Slight erythema of the conjunctivae was seen in all treated eyes 1 h after dosing. No signs of irritation were seen 1, 2, 3 or 7 days after dosing (Hill, 1973a).

The conjunctival sacs of the eyes of three male and three female New Zealand white rabbits were instilled with technical-grade S-methoprene (purity, 90%) and examined and scored for irritation according to the method of Draize after 1, 24, 48 and 72 h and 4 and 7 days. Slight irritation (score 1 for erythema and chemosis) was observed in two females and three males after 1 h, but all signs of irritation had subsided within 48–72 h. Statements of compliance with GLP and QA were included (Brown, 1984b).

Further studies of ocular irritation were performed with liquid or solid formulations of S-methoprene. In studies with a 2% ground briquet formulation (Blaszcak, 1992e) or a 4% ground pellet formulation (Blaszcak, 1994f), transient, moderate-to-severe ocular irritation was observed, which disappeared within 3–7 days. In the two studies with liquid formulations containing 5% (Hiles & Collins, 1984c) or 20% (Schindler & Baldwin, 1991c) S-methoprene, conjunctival erythema was observed, which disappeared within 1 and 3 days, respectively.

Racemic methoprene (purity, 68.9%) was applied in a volume of 0.5 ml onto the intact or abraded clipped skin of six female New Zealand white rabbits, and the site was covered with gauze sponges and rubberized cloth for 24 h. No signs of skin irritation were observed immediately or 48 h after removal of the occlusive dressing (Hill, 1972d)

The clipped intact or abraded skin of six New Zealand white rabbits was exposed to 0.5 ml of technical-grade racemic methoprene (purity unspecified), and the site was occluded for 24 h. Slight erythema was observed immediately after removal of the dressing at four of six abraded sites and two of six intact sites. Barely perceptible erythema was observed next to the abrasions at five of six sites 48 h after removal of the dressing (Hill, 1973b).

The clipped intact skin of three male and three female New Zealand white rabbits was exposed for 4 h to 0.5 ml of technical-grade S-methoprene (purity, 90%), covered with a gauze. Skin irritation was assessed according to the scores of Draize. No signs of skin irritation and no toxic effects were observed in any animal 1, 24, 48 or 72 h after the end of treatment. Statements of compliance with GLP and QA were included (Schindler & Brown, 1984b).

A number of studies of dermal irritation were performed with formulations of S-methoprene. Formulations containing 2% (Blasczak, 1994g), 4% (Blasczak, 1994h), 5% (Hiles & Collins, 1984c) or 20% (Schindler & Baldwin, 1991d) S-methoprene did not irritate the skin.

The skin sensitizing properties of racemic methoprene (purity, 95.7%) were tested in groups of five male guinea-pigs according to the method of Landsteiner. In a preliminary range-finding test with an intradermal injection of 0.05 ml of an oily solution, the highest concentration of methoprene that did not cause skin irritation was 0.3%. Therefore, for the induction phase, the animals were given a total of 10 intradermal injections every 48 h of a cottonseed oil solution containing 0.03, 0.1 or 0.3% methoprene. The first injection comprised 0.05 ml of solution, and the nine subsequent injections contained 0.1 ml of solution. After 2 weeks, an intradermal challenge injection was given. During the induction phase, mild, temporary erythema was observed in some animals shortly after injection of methoprene or the vehicle, but the intensity of the dermal reactions did not increase with repeated administration. The type and intensity of dermal reactions after the challenge injection of methoprene did not differ from those observed during the induction phase (Nakayoshi, 1975). The Meeting noted that the appearance of reactions both in animals challenged with the vehicle and in those challenged with methoprene complicates evaluation of the results. It should be noted that the test was not performed according to current guidelines, and the number of animals per group may not have been sufficient for a sound statistical evaluation.

Three studies were performed on the skin sensitizing properties of formulations of S-methoprene. In a standard Buehler skin sensitization assay, 0.3 ml of a formulation containing 2% or 4% methoprene diluted with 0.3 ml of physiological saline did not induce skin sensitisation (Blaszcak, 1994i,j). However, in a modified Buehler test with a 20% liquid formulation of S-methoprene in groups of five male and five female Hartley albino guinea-pigs, nine of 10 animals showed significant erythema after challenge with undiluted test substance, while none of the uninduced animals showed this response. Under the conditions of this test, the formulation of S-methoprene was classified as an extreme skin sensitizer. Statements of compliance with GLP and QA were included (Schindler & Baldwin, 1991e).

(b) Short-term studies of toxicity

Rats

In a range-finding study, groups of five male and five female young Sprague-Dawley rats weighing 59–73 g received a diet containing technical-grade racemic methoprene (purity, 68.9%) at a nominal concentration of 1000, 5000, 10 000, 20 000 or 40 000 ppm, equivalent to 100, 500, 1000, 2000 and 4000 mg/kg bw per day (or 70, 340, 690, 1400 and 2800 mg/kg bw per day when corrected for purity) for 2 weeks. After treatment, all animals were returned to normal food for an additional week. All rats receiving methoprene refused their diets on the first day of the study. Those at 1000, 5000 and 10 000 ppm resumed their normal feeding habits, whereas those at 20 000 and 40 000 ppm continued to have greatly reduced food consumption and body growth. The effect was ascribed to poor palatability. In the third week, when the rats received basal diet, all animals resumed normal feeding. Gross examination at the end of the third week showed no abnormalities (Jorgenson & Sasmore, 1972a).

The Meeting noted, however, that the treatment period was followed by a 1-week recovery period, during which any treatment-induced gross pathological changes might have disappeared.

In a limited study, groups of 15 male and 15 female Sprague-Dawley rats (approximately 28 days old) were fed diets containing technical-grade racemic methoprene (purity, 68.9%) at a nominal concentration of 0, 250, 500, 1000 or 5000 ppm, equivalent to 12, 25, 50 and 250 mg/kg bw per day (or 9, 17, 34 and 170 mg/kg bw per day when corrected for purity) for 90 days. No treatment-related deaths occurred, and grossly observed behaviour was normal. Body weight, food consumption and haematological end-points at weeks 4, 8 and 13 were comparable to those of controls, and blood chemistry values were not affected in a dose-related pattern at the end of the study. Urine analysis at 13 weeks showed normal values. At termination, animals at the highest dietary concentration showed increased organ:body weight ratios for liver (both sexes) and kidney (males only). Selected tissues from 10 males and 10 females from the control group and that at the highest dietary concentration, and kidney and liver from the remaining five animals of each sex in these groups and from animals at 1000 ppm were examined microscopically. A slightly higher incidence than in controls of a kidney lesion characterized by vacuoles within swollen convoluted tubules was seen in males at 5000 ppm. In addition, renal tubule regeneration, not seen in concurrent controls or in females, was present in three males at 1000 ppm and seven males at 5000 ppm. The kidneys of animals at doses < 1000 ppm were not examined histologically. The minor histopathological changes in the kidneys were considered to be of no toxicological significance (Jorgenson & Sasmore, 1972b).

The Meeting noted that the design of the study deviated in several respects from current guidelines. For instance no ophthalmic examinations were performed, and detailed reports of clinical and behavioural examinations were not given. Haematological, clinical biochemical and urinary end-points were investigated in only five animals of each sex per group.

In a range-finding study, groups of three male beagle dogs were fed diets containing technical-grade racemic methoprene (purity, 68.9%) at a nominal concentration of 0, 1000, 5000, 10 000 or 20 000 ppm, equivalent to 25, 120, 250 and 500 mg/kg bw per day, for 2 weeks. One male at 20 000 ppm was killed at the end of the 2-week period and necropsied, while the remaining animals were returned to basal diet for an additional week. The animals at 10 000 and 20 000 ppm maintained their weight or showed slight weight loss, while those at 1000 and 5000 ppm showed a slight increase in weight, but less than that of the control animals. The food intake of dogs at 10 000 and 20 000 ppm was markedly decreased. On return to a basal diet in the third week, the food consumption in all treated groups increased. Gross examination at 3 weeks showed an increased relative liver weight with increasing concentration of methoprene in the diet. Histological evaluation showed vacuolization and swelling of hepatocytes in livers of animals at 10 000 or 20 000 ppm, whereas sections from dogs at 1000 and 5000 ppm showed no difference from controls (Jorgenson & Sasmore, 1972a).

The Meeting noted that treatment was followed by a 1-week recovery period, during which any treatment-induced gross pathological changes might have disappeared.

Groups of four male and four female beagles, about 19 weeks old, were fed diets containing technical-grade racemic methoprene (purity, 68.9%) at a nominal concentration of 0, 250, 500 or 5000 ppm, equivalent to 6.2, 12 and 120 mg/kg bw per day, for 90 days. No deaths occurred. Behaviour, body weight, food consumption and haematological end-points were not adversely affected. Urine analysis at weeks 4, 8 and 13 and ocular examinations at termination were reported to show no abnormal findings. Serum alkaline phosphatase activity was elevated in animals of each sex at 5000 ppm at weeks 4, 8 and 13. At terminal sacrifice, the organ:body weight ratio of the liver was increased in both sexes at 5000 ppm. Gross pathological examination of all animals and microscopic evaluation of selected tissues, including the liver, from all animals in the control group and at the highest dietary concentration showed no treatment-related changes. The NOAEL was 500 ppm, equivalent to 12 mg/kg bw per day (8.6 mg/kg bw per day when corrected for purity), on the basis of the increased liver weight and the increase in alkaline phosphatase activity (Jorgenson & Sasmore, 1972b).

The Meeting noted that, in contrast to the current OECD guideline, detailed clinical and behavioural observations, ophthalmic observations and the results of urine analysis were not reported.

In a 30-day study, technical-grade racemic methoprene (purity, 95.7%) was applied to the clipped, unabraded skin of five male and five female Japanese rabbits, without occlusion. Undiluted methoprene was applied at a nominal dose of 100, 300, 900 or 2700 mg/kg bw per day over a circular area 10 cm in diameter. It is not clear what treatment the control animals received. The animals were weighed and observed for clinical signs daily. Haematological and blood chemical end-points were measured before treatment and at necropsy. Food and water intake was assessed every 5 days. Urine was analysed weekly. At necropsy, gross and histopathological examinations were performed.

Redness of the skin was observed in animals at the highest dose on days 4–29, and erythema was observed occasionally during treatment in some animals at 300 and 900 mg/kg bw per day. No erythema was seen in controls or animals at 100 mg/kg bw per day. Males at doses > 300 mg/kg bw per day and females in all treated groups showed reduced weight gain or weight loss during the study and an increased neutrophil count at termination. The leukocyte counts were increased in all treated groups at termination. The treated patch of skin was injured by scratching in all treated groups. The absolute and relative weights of the kidney tended to be dose-dependently increased. The absolute and relative liver weights were increased in animals at the highest dose. Gross and histopathological examinations showed that the only compound-related findings were in the treated skin sites. The study authors suggested that the effects on body weight and leukocyte counts in the treated animals were the result of the skin injury. The LOAEL was 100 mg/kg bw per day (97 mg/kg bw per day when corrected for purity) on the basis of effects on body-weight gain, kidney weight and leukocyte count (Nakasawa, 1975).

The Meeting noted that the study design did not meet current OECD guidelines. As the treated dermal patch was not occluded, the animals were able to scratch it. Furthermore, the skin was not washed between treatments, so that residual test compound could have built up, and oral uptake of the test substance could have occurred. The study was considered to be of limited value.

Groups of 10 male and 10 female rats (strain unspecified) were exposed by inhalation to an aerosol of racemic methoprene (purity, 68.9%) at a nominal chamber concentration of 0, 2 or 20 mg/l air, 4 h/day, 5 days per week for 3 consecutive weeks. None of the animals died. Animals at 20 mg/l air had a nasal discharge during each exposure. The weekly body weights and terminal haematological values were comparable to those of controls. Gross necropsy and histopathological evaluation of the liver, lung, kidney and trachea showed no treatment-related changes. Although the Meeting in 1984 (Annex 1, reference 43) concluded that the biochemical parameters in blood did not indicate a consistent pattern of toxicity, re-evaluation of the data by the current Meeting indicated that the total blood bilirubin concentrations were dose-dependently and significantly increased in males at both doses (p < 0.001, Wilcoxon). Serum alkaline phosphatase activity was significantly enhanced at 20 mg/l in both males and females (p < 0.05, Wilcoxon). No other indication of liver damage was found (aspartate and alanine aminotransferase activity, gross or histopathological appearance). In view of the overall pattern of toxicity in other studies in which the liver was the primary target tissue, the NOAEC was 2 mg/l (1.4 mg/l when corrected for purity) (Olson, 1972b).

The Meeting noted that, in contrast to the current OECD guideline, only two concentrations were used, the duration of exposure was only 4 h/day instead of 6 h/day, and the actual room concentrations and particle size of the aerosol were not determined. This study was considered of limited value.

Groups of three male and three female beagles were exposed by inhalation via the nose only to technical-grade racemic methoprene (purity, 95.7%) in 2% ethanol solution as an aerosol at a nominal dose of 0.012, 0.0250 or 0.062 mg/kg bw per day, given for 3 min, 6 days/week, for 4 weeks. The particle size of the aerosol was 0.5–2.5 µm. Groups of two males and two females recived the vehicle only or were untreated. There were no deaths. Except for salivation in two animals during exposure on the first day, no compound-related effects on body weight, food or water consumption, haematological, blood chemical or urinary end-points or gross or histopathology were reported. The NOAEC was 0.062 mg/kg bw per day (0.06 mg/kg bw per day when corrected for purity), the highest dose tested (Saito, 1975b).

Owing to the very short daily exposure, this study is of limited value for evaluating the toxic potential of repeated exposure by inhalation.

(c) Long-term studies of toxicity and carcinogenicity

Groups of 50 male and 50 female Charles River CD-1 mice received diets containing racemic methoprene (purity, 86.9%) at a nominal concentration of 250, 1000 or 2500 ppm, equivalent to 38, 150 and 380 mg/kg bw per day, for 78 weeks. The survival rates of males were 64% of controls, 56% at the lower dietary concentration, 54% at the intermediate concentration and 60% at the highest concentration, while those of females were 52%, 44% and 48%, respectively. As the survival rate of females at the intermediate concentration at week 72 was only 44%, the remaining animals in this group were killed. The survival rate was considered not to have been affected by treatment. Treatment also had no effects on behaviour, appearance, body weight or food consumption. Histopathological examination revealed the presence of an unidentified brown pigment in the livers of animals at 1000 or 2500 ppm, and many mice at 2500 ppm also had numerous focal accumulations of macrophages with brownish foamy cytoplasm in the liver, often associated with small necrotic foci and mononuclear inflammatory cells. The NOAEL was 1000 ppm, equivalent to 150 mg/kg bw per day (130 mg/kg bw per day when corrected for purity), on the basis of the latter effect. No treatment-related effects on tumour incidence were observed (Wazeter & Goldenthal, 1975a).

The Meeting noted that, in contrast to current guidelines, no differential blood count was performed. Furthermore, negative results for carcinogenicity should be based on a survival rate > 50% at 18 months. Although this requirement was not fully met, the Meeting considered the survival rate of controls and mice at the highest dietary concentration to be acceptable.

Groups of 50 male and 50 female Charles River CD rats were fed diets containing technical-grade racemic methoprene (purity, 86.9%) at a nominal concentration of 0, 250, 1000 or 5000 ppm, equivalent to 0, 12, 50 and 250 mg/kg bw per day, for 2 years. The survival rates of males were 38% of controls and those at the highest dietary concentration, 54% of those at the lowest concentration and 46% of those at the intermediate concentration. About 50% of control males survived up to week 97, and 50% of males at the highest dietary concentration survived up to week 102. In females, the survival rates at 104 weeks were 52%, 60%, 58% and 48% of controls and those at the low, intermediate and high concentrations, respectively. General appearance, behaviour, body weight and food consumption were not adversely affected. No compound-related effects were seen on haematological, biochemical or urinary parameters measured in five males and five females per group at five intervals during the study. Ophthalmic examination revealed no changes related to treatment. The absolute and relative weights of the liver were elevated (about 120% of control liver weight) in females at 5000 ppm. Gross examination indicated no pathological findings attributable to treatment. Histopathological evaluation of a wide range of tissues showed an increased incidence of hepatic lesions, such as bile-duct proliferation and portal lymphocyte infiltration, in males at 5000 ppm. No significant difference in the incidence of any particular tumour was found between control and treated groups. The NOAEL was 1000 ppm, equivalent to 50 mg/kg bw per day (44 mg/kg bw per day when corrected for purity) (Wazeter & Goldenthal, 1975b).

The Meeting noted that, in contrast to the current OECD guidelines, clinical, haematological and urinary parameters were assessed in only five instead of 20 animals of each sex per group. Furthermore, no satellite group was included. Negative results for carcinogenicity should be based on a survival rate > 50% at 24 months. Although this requirement was not fully met, the Meeting considered the survival rate of controls and mice at the highest dietary concentration to be acceptable.

(d) Genotoxicity

The results of studies on the genotoxicity of the racemate and S-methoprene are summarized in Table 3. One assay for dominant lethal mutations performed with the racemate in rats in vivo was available (Johnston, 1973), but it was poorly conducted and was therefore not used in the current evaluation.

(e) Reproductive toxicity

(i) Multigeneration studies

Rats

Groups of 20 male and 20 female weanling Long Evans rats were fed diets containing technical-grade racemic methoprene (purity, 86.9–87.5%) at a nominal concentration of 0, 500 or 2500 ppm, equivalent to 0, 25 and 75 mg/kg bw per day, until they were at least 100 days of age, before mating to initiate a three-generation (one litter per generation) study of reproductive toxicity. F₁ and F₂ pups were selected to become parents at weaning and, after a 70-day growth and feeding period, were mated to produce successive generations. In the parental generations, no compound-related effects were seen on mortality rate, food consumption during the growth period, maternal growth rate during gestation and lactation, mating performance, pregnancy rate or duration of gestation. Total weight gain during the growth period was slightly decreased in F₀ and F₁ animals of each sex at 2500 ppm. At this concentration, the mean pup weight was reduced in F₂ litters on day 21 and in F₃ litters on days 14 and 21. Additionally, the mean number of pups born dead per litter was increased in F₃ litters of this group. There were no treatment-related effects on other parameters, including litter size (live pups) at birth, survival of pups during lactation, sex ratio of pups and findings in F₃ weanlings at necropsy. The NOAEL was 500 ppm, equivalent to 33 mg/kg bw per day (or 29 mg/kg bw per day when corrected for purity), on the basis of reductions in weight gain and mean pup weight and the increased mean number of pups born dead per litter (Killeen & Rapp, 1974).

The Meeting noted that, in contrast to current OECD guidelines, only two dietary concentrations were used. Furthermore, no histopathological examinations were performed.

(ii) Developmental toxicity

Mice

Groups of 30 mated mice of the ICR lineage were intubated with technical-grade racemic methoprene (purity, 95.7%) in olive oil at a nominal dose of 0, 50, 200 or 600 mg/kg bw per day on days 7–14 of gestation. The pregnant dams (20–23 mice in each group) were killed on day 18 of gestation, and the fetuses were removed for external, internal and skeletal examination. There were no compound-related deaths. Food and water consumption during the gestation period was comparable in all groups. An increase in body weight (compared with controls) was noted in pregnant dams at both 200 and 600 mg/kg bw per day on day 18. The mean number of implantations and the mean number of live fetuses were both increased at the highest dose. The body weights of female fetuses in all treated groups were increased. No treatment-related effects were seen on the mean number of dead embryos or on the sex ratio of fetuses. No internal or external abnormalities were seen in fetuses of control or treated groups, although information on the number of fetuses per control or treated group examined for such abnormalities was not available. Fetuses in all treated groups showed a statistically significant increase in the number of caudal vertebrae, as compared with controls. The effects observed in the fetuses and the increased maternal body weight were considered indicative of modest advancement of development and thus not of toxicological relevance. The NOAEL for maternal toxicity, embryotoxicity and fetotoxicity was 600 mg/kg bw per day (570 mg/kg bw per day when corrected for purity), the highest dose tested (Nakasawa et al., 1975a).

In the same experiment, 10–14 pregnant mice were treated as described above, and the dams were allowed to litter and rear their young until weaning. Pups from five litters per dose group were killed at weaning, and those from the remaining litters were observed for 7 additional weeks. No deaths or abnormal signs were seen in the dams. Maternal body-weight change during gestation and after parturition was unaffected. No compound-related effects were seen on the mean number of implantations, duration of gestation, mean litter size or the survival rate of pups at birth or at weaning. No adverse effects were seen on the rate of physical development of pups before weaning, as judged by auricle development, hair growth and opening of the eyelids. In weaned pups, no dose-related effects were seen on the time of descent of testes or opening of the vagina. The behaviour of pups during 10 weeks post partum was normal. Pups necropsied at 3 or 10 weeks of age showed no gross or skeletal abnormalities. At weaning, increases in the absolute weights of the liver, kidney and lung were observed in male pups at 600 mg/kg bw per day. In female pups, the weights of these organs were also increased but the increase reached statistical significance only for lung weight. A non-dose-related decrease in the organ:body weight ratio of the testes was seen in all treated groups killed after 21 days and in those at the two higher doses killed at 70 days. At 70 days, differences from controls in the weights of the spleen, kidney, heart and lung were observed, but these were not dose-related or were seen only at the highest dose. Histological examination of the ovaries and testes of pups reportedly revealed a single case of atrophy of seminiferous tubules at 50 mg/kg bw per day (data not shown). The NOAEL for toxicity to offspring was 200 mg/kg bw per day (190 mg/kg bw per day when corrected for purity), on the basis of effects on organ weights. Methoprene was not teratogenic under the conditions of these experiments (Nakasawa et al., 1975a).

The Meeting noted that, in contrast to the current OECD guideline, dams were not treated on days 6 and 15 of gestation; therefore, the complete period of organogenesis was not covered.

Rabbits

Groups of 10 pregnant Japanese rabbits were treated by gavage with technical-grade racemic methoprene (purity, 95.7%) in olive oil at a nominal dose of 0, 50, 200 or 2000 mg/kg bw per day on days 7–18 of gestation. The does were killed on day 28 of gestation, and the fetuses were removed surgically for examination for external, internal and skeletal abnormalities. No deaths or abnormal symptoms were seen. Two does at the highest dose aborted, and maternal weight gain was depressed. Increases in the incidence of fetal deaths (6% in controls; 20.5% at 2000 mg/kg bw group) and in the proportion of female fetuses were also seen at the highest dose. Fetuses at both 200 and 2000 mg/kg showed a non-dose-related decrease in tail length. No compound-related effects were observed with respect to the mean number of implantations, litter size (live fetuses), body weight or body length of fetuses or the frequency of fetal abnormalities. The NOAEL for maternal toxicity was 200 mg/kg bw per day on the basis of reductions in weight gain and abortions. The NOAEL for fetal toxicity was also 200 mg/kg bw per day, on the basis of the increased percentage of fetal deaths. The NOAELs after correction for purity were 190 mg/kg bw per day. Methoprene was not teratogenic under the conditions of the experiment (Nakasawa et al., 1975b).

The Meeting noted that, in contrast to the current OECD guideline, the does were not treated on day 6 of gestation; therefore, the complete period of organogenesis was not covered. Furthermore, only 10 instead of 12 females per dose were used.

(f) Special studies

(i) Endocrine activity in mammals

Immature female mice (19–21 days of age) received racemic methoprene (purity unspecified) subcutaneously at a dose of 0.015 or 0.15 mg/kg bw per day for 3 days. No increase in the uterus:body weight ratio was seen. When methoprene was given subcutaneously to 21-day-old castrated male rats at a dose of 0.37 or 3.7 mg/kg bw per day for 7 days, no increase was seen in the organ:body weight ratio of seminal vesicles, ventral prostate or levator ani. In bilaterally adrenalectomized male rats, 21–23 days old, subcutaneous injection of methoprene at 0.9 or 9 mg/kg bw per day for 6 days did not affect the thymus:body weight ratio. The results of these studies suggest that methoprene has no oestrogenic, androgenic, anabolic or glucocorticoid activity (Rooks, undated).

The results of studies of the acute toxicity of metabolites of methoprene are summarized in Table 4. The methoprene metabolites 11-hydroxy-3,7,11-trimethyldodeca-2,4-dienoic acid, 11-methoxy-3,7,11-trimethyldodeca-2,4-dienoic acid, isopropyl-11-hydroxy-3,7,11-trimethyldodeca-2,4-dienoate and 7-methoxy citronellic acid have been found in both plants and animals. 7-Hydroxycitronellic acid and 7-methoxycitronellal are found exclusively in plants.

The irritation potential of the methoprene metabolite 7-methoxycitronellal (purity, 97.7%) to the eye was assessed in three male and three female New Zealand white rabbits which received 0.1 ml of the substance into the conjunctival sac of the right eye. Signs of irritation were scored according to the method of Draize. The treatment caused transient, mild conjunctival irritation (redness, chemosis and discharge) in all animals and a slight dulling of the corneal surface in one animal. The signs of ocular irritation disappeared within 2–3 days. (Wazeter & Goldenthal, 1973).

The clipped intact or abraded skin of six New Zealand white rabbits was exposed to 0.5 ml of the methoprene metabolite 7-methoxycitronellal (purity, 97.7%), and the site was occluded for 24 h. No skin irritation was observed in the animals with intact skin, and very slight erythema and oedema were observed in the animals with abraded skin. No control groups were included. This metabolite is considered not to be a primary skin irritant (Wazeter & Goldenthal, 1973).

The absorption, distribution, excretion and metabolism of racemic methoprene have been studied in mice, rats, guinea-pigs, cows and chickens given single doses. No study of metabolism after repeated doses was available. After administration of single oral doses of methoprene, the radiolabel was relatively rapidly absorbed and excreted in urine, faeces and expired air. Further, about 8% of the radiolabel administered to a cow was excreted in milk 7 days after dosing, and up to 19% of radiolabel administered to chickens was excreted in eggs 14 days after dosing. In most species investigated, the bulk of the radiolabel was excreted within 5 days or less, and the remainder was incorporated into tissues.

Substantial enterohepatic circulation occurs in rats, and a small percentage of intact, unabsorbed methoprene was found in faeces, with none in urine or bile, after its administration. Methoprene is probably extensively metabolized in rats, as a large portion of the radiolabel was excreted with CO₂. After a single oral dose to rats, the peak plasma concentration, 1.6% of the administered radiolabel, was reached by 6 h; the level declined slowly, with a half-time of about 48 h. Whole-body autoradiography and tissue analysis showed that most of a single labelled dose was located in organs concerned with absorption, biotransformation and excretion. A relatively high concentration of radiolabel was found in adrenal cortex, lachrymal glands and adipose tissue after 48 h.

Studies in guinea-pigs, cattle and chickens showed that racemic methoprene was extensively metabolized to polar conjugates (glucuronides), which were excreted in the urine and faeces, and that the C5-labelled molecule underwent rapid a and b oxidation to produce CO₂ and acetate, which was incorporated into natural products such as triglycerides, bile acids and cholesterol found in tissues, milk and eggs.

The pharmacokinetics of S-methoprene was investigated for 7–8 h in blood and fat of rats given a single oral or intravenous dose. The clearance of S-methoprene was relatively rapid after intravenous administration of 10 mg/kg bw. After oral administration of 10 or 100 mg/kg bw, S-methoprene was rapidly absorbed, and the maximum concentration of parent compound in blood was reached 2 h after dosing. In fat, the concentration of unchanged methoprene reached a plateau 3–4 h after intravenous and 4–6 h after oral administration, and then very slowly declined. Because of this slow decline, methoprene may build up in fat after repeated dosing. Most of the radiolabel in fat was unchanged methoprene, whereas in blood methoprene was degraded rapidly to other radiolabelled compounds.

The racemate and S-methoprene showed little acute toxicity. The LD₅₀ values for S-methoprene were > 5000 mg/kg bw (oral, rat) and > 2000 mg/kg bw (dermal, rabbit), and those of the racemate were > 24 000 mg/kg bw (oral, rat) and > 3400 mg/kg bw (oral, dog). The LD₅₀ for the racemate after intraperitoneal administration in rats was 3300 mg/kg bw. WHO (1999) has classified methoprene as ‘unlikely to present acute hazard in normal use’. The racemate and S-methoprene were not irritating to the eye or skin of rabbits. In a limited test in guinea-pigs, the racemate appeared to have no sensitizing properties. In a study with a formulation containing 20% S-methoprene, skin sensitization was seen; however, it was unclear whether the effect was due to S-methoprene or to another compound in the formulation.

Several studies of the toxicity of repeated doses of racemic methoprene given by oral or dermal application or inhalation were available. The design and reporting of these studies did not meet current guidelines, and the studies of dermal application or inhalation were considered inadequate for evaluation. The studies by oral administration could be used to deduce the toxicological profile of methoprene, and, despite their shortcomings, most were considered suitable for use in risk assessment.

Studies in which mice (78 weeks), rats (14 or 90 days, 104 weeks), and dogs (14 or 90 days) were given racemic methoprene in the diet showed that the compound has little toxic potential. Some effects were found on food intake and body weight, but the main effect was to increase the weight of the liver relative to body weight (in rats at doses > 5000 ppm; in dogs at doses > 1000 ppm). This effect was not always associated with histopathological changes. In the 90-day study in dogs treated in the diet, the NOAEL was 500 ppm, equivalent to 8.6 mg/kg bw per day. In the 2-year study in rats treated in the diet at 5000 ppm, the highest dose tested, increased absolute and relative liver weights and an increased incidence of hepatic lesions such as bile-duct proliferation and portal lymphocyte infiltration were observed in male rats. The NOAEL was 1000 ppm, equivalent to 44 mg/kg bw per day. Minor histopathological changes in the kidneys observed in this study were considered of no significance for human risk assessment. In the 78-week study of carcinogenicity in mice, hepatic lesions characterized by pigment deposition in the cytoplasm of parenchymal cells were seen at 1000 and 2500 ppm, with increased incidence and severity at the highest dose. Focal accumulations of macrophages with brownish foamy cytoplasm were found in the livers of survivors of each sex at 2500 ppm, and an increased frequency of amyloidosis of the intestine was seen in females at this dose. No adverse effects (the brownish pigment was considered not to be of toxicological relevance) were observed at 1000 ppm, equivalent to 130 mg/kg bw per day.

No increase in the incidence of tumours at any site was seen in either the 78-week study of carcinogenicity in mice or the 2-year study of toxicity and carcinogenicity in rats treated in the diet.

Racemic methoprene did not induce chromosomal aberrations in Chinese hamster ovary cells in vitro. No increase in the frequency of reverse mutations in Salmonella typhimurium was observed. The Meeting noted that only a limited range of concentrations were tested. No definitive conclusion can be drawn about the genotoxic potential of the racemate.

S-Methoprene did not induce reverse mutations in S. typhimurium or mitotic crossing-over, gene conversion or reverse mutations in Saccharomyces cerevisiae. On the basis of the negative results in a limited range of studies for genotoxicity and the results of the studies of carcinogenicity with methoprene, the Meeting concluded that methoprene was unlikely to pose a carcinogenic risk to humans.

In a three-generation study of reproductive toxicity with racemic methoprene in rats, the total weight gain of animals of each sex in the F₀ and F₁ generations during the growth period was slightly decreased, the mean weight of pups in the F₂ and F₃ litters was reduced, and the mean number of pups in the F₃ litters born dead per litter was increased at a dose of 2500 ppm. The NOAEL was 500 ppm, equivalent to 29 mg/kg bw per day.

In a study of developmental toxicity in which mice were treated on days 7–14 of gestation with racemic methoprene, no toxicologically relevant effects were observed in dams or fetuses at any dose; the NOAEL was 570 mg/kg bw per day, the highest dose tested. In the same experiment, several dams were allowed to litter and rear their pups until weaning; the pups of five litters at each dose were killed at weaning, and the remaining litters were observed for 7 additional weeks. Effects on organ weights were observed in pups at the highest dose. The NOAEL for toxicity to offspring was 190 mg/kg bw per day. In a study of developmental toxicity in which rabbits were treated on days 7–18 of gestation with racemic methoprene, the NOAEL for maternal, embryo- and fetotoxicity was 190 mg/kg bw per day, on the basis of reduced weight gain and an increased frequency of abortions among the does and an increased percentage of fetal deaths at 1900 mg/kg bw per day, the highest dose tested. The shortcoming of the studies—mice and rabbits were treated for 2 and 1 day less than that required in the relevant test guideline—was not considered critical for evaluating end-points of developmental toxicity. All the developmental effects were seen at very high doses or only postnatally. Therefore, the Meeting considered that additional studies were not necessary and concluded that methoprene is not teratogenic.

Several plant metabolites of methoprene showed little acute toxicity when administered orally.

The Meeting reaffirmed the basis of the ADI for racemic methoprene established in 1987, but lowered the value to 0–0.09 mg/kg bw to correct for the purity of the racemate tested. The basis for the ADI was the NOAEL of 500 ppm, equivalent to 8.6 mg/kg bw per day (corrected for purity), in the 90-day study in dogs and a safety factor of 100.

The LD₅₀ for racemic methoprene given orally was > 2000 mg/kg bw, and no toxic signs were seen at this dose. In studies with repeated oral doses (including studies of teratogenicity), racemic methoprene did not induce signs indicative of acute toxicity. The Meeting concluded that allocation of an acute reference dose was unnecessary.

S-Methoprene

No bridging studies with repeated doses were available for S-methoprene. The Meeting therefore made the conservative assumption that, in the absence of any information to the contrary, all the toxicity of the racemate was due to the S-enantiomer. On this basis, the Meeting established an ADI for S-methoprene of 0-0.05 mg/kg bw, equal to one-half the ADI for the racemate (which is a 1:1 mixture of the R and S enantiomers).

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       Toxicological Abbreviations