4.5 Chlorpropham (201)

TOXICOLOGY

Chlorpropham is the ISO approved name for 1-methylethyl (3-chlorophenyl) carbamate, which is a plant growth regulator used for pre-emergence and early post-emergence control of grass weeds. It is also used to inhibit potato sprouting. The toxicity of chlorpropham was evaluated by the JMPR in 1963, 1965 and 2000. In 2000, the Meeting established an ADI of 0-0.03 mg/kg bw based on a NOAEL of 10 mg/kg bw per day in a 90-day study of toxicity in Wistar rats, this NOAEL being identified on the basis of a significant decrease in erythrocyte counts and an increase in methaemoglobin formation at the next higher dose of 47 mg/kg bw per day. A safety factor of 300 was applied, which included an additional safety factor of 3 to account for inadequacies in the assessment of methaemoglobinaemia (lack of measurements of methaemoglobin formation at early time-points, a concern since adaptation to this effect can occur), the critical toxicological effect. This ADI also provided an adequate margin of safety for the effects on the thyroid observed in dogs (NOAEL, 5 mg/kg bw per day). An ARfD equal to the maximum ADI was also established.

The sponsor conducted a study of acute toxicity in female beagle dogs in order to refine the ARfD, in order to address concerns with respect to the extent of investigation of methaemoglobinaemia. The 2005 JMPR was asked by the CCPR to review the ARfD for chlorpropham, and as a consequence of this review, the Meeting also reconsidered the ADI.

The new study of acute toxicity complied with GLP.

Toxicological data

In 2000, the JMPR determined that chlorpropham has low acute toxicity: the oral LD₅₀ in rats was > 2000-4200 mg/kg bw, and the dermal LD₅₀ in both rats and rabbits was > 2000 mg/kg bw. Chlorpropham is also only weakly toxic after inhalation since there were no deaths at 0.47 mg/L, the highest attainable concentration.

Chlorpropham was not irritating to the eyes or skin of rabbits. It did not sensitize the skin of guinea-pigs in a Bühler test, in an open epicutaneous test, or in a Magnusson & Kligman test. Although chlorpropham sensitized the skin of 30% of the guinea-pigs tested in a split adjuvant test, the 2000 JMPR concluded that chlorpropham is unlikely to cause sensitization in humans.

After an evaluation of short- and long-term studies of the effects of chlorpropham in mice, rats, and dogs, the 2000 JMPR determined that the haematopoietic system was the main toxicological target; changes were observed in the morphology and parameters of erythrocytes, including increased formation of methaemoglobin, and changes in the spleen and liver consistent with a haemolytic effect. In a study of dermal toxicity in rabbits, chlorpropham also produced haematopoietic effects. In dogs fed diets containing chlorpropham for 28 days or fed capsules containing chlorpropham for 90 days, effects were also seen on the thyroid gland at doses similar to or lower than those that affected erythrocytes. In dogs given capsules containing chlorpropham for 60 weeks, a NOAEL of 5 mg/kg bw per day was identified on the basis of effects on the thyroid gland, including increased weight, decreased concentrations of thyroxine (in a test for stimulation by thyroid-stimulating hormone), and, occasionally, decreased concentrations of tri-iodothyronine. In 90-day and 2-year dietary studies in rats, reduced thyroid weights were seen at doses higher than those that caused haematotoxic effects.

Chlorpropham was not a reproductive toxicant in rats and was not teratogenic in rats and rabbits. In 2000 the JMPR had concluded that while chlorpropham may be weakly genotoxic in vitro, it was unlikely to present a risk to humans, although it was noted that this conclusion should be validated in adequate studies of genotoxicity in vivo.

The present Meeting evaluated a study of acute oral toxicity in female dogs given capsules containing chlorpropham as single doses at up to 625 mg/kg bw. Chlorpropham produced clinical signs of toxicity manifested as vomiting and reduced activity at 125 mg/kg bw and above, apparent within 2 h after dosing, but these signs were no longer evident by 4-6 h after dosing. The NOAEL was 50 mg/kg bw. Chlorpropham also produced increased formation of methaemoglobin in all treated groups. However, the increases in methaemoglobin levels were very small, reaching a maximum of 0.8% in one of four animals at the highest dose. The effects were possibly treatment-related at 125 and 625 mg/kg bw, but the small increases at 50 mg/kg bw resulted in levels that were no higher and no more prolonged than those seen in control animals. None of the increases in methaemoglobin levels were toxicologically significant at any dose. With respect to the maximum increase in methaemoglobin seen in this study (0.8%), it should be noted that in 2004 the JMPR recommended that for acute exposure to xenobiotics that induce methaemoglobin formation, only an increase in methaemoglobin formation of 4% (or higher) above background in dogs should be considered to be relevant for setting an ARfD.

The primary effects of repeated doses of chlorpropham appear to be on the haematopoietic system and on the thyroid. In rats, haematological effects appeared at lower doses than did thyroid effects, while in dogs thyroid effects appeared at lower doses than did haematological effects. In a 90-day study of toxicity in rats, the NOAEL for increased methaemoglobin formation was 10 mg/kg bw per day, while in a 90-day study of toxicity in dogs, the NOAEL was 25 mg/kg bw per day. These apparent differences in NOAEL are likely to be caused by artefacts of dose selection rather than to any increased sensitivity of rats over dogs. Thus the study of acute toxicity in dogs was considered to be adequate to assess the effects of acute dosing with chlorpropham on the formation of methaemoglobin.

Toxicological evaluation

The Meeting reconsidered the previously established ADI on the basis of the new study providing information on methaemoglobin measurements at early time-points. Because the new study in dogs addressed previous concerns about the induction of methaemoglobin at early time-points, the Meeting established an ADI of 0-0.05 mg/kg bw based on the NOAEL of 5 mg/kg bw per day in a 60-week study in dogs fed with chlorpropham, on the basis of changes in the thyroid at 50 mg/kg bw per day, and using a safety factor of 100. This ADI provided an adequate margin of safety for the haematotoxic effects seen in the studies of repeated doses in rats.

The Meeting established an ARfD of 0.5 mg/kg bw, on the basis of a NOAEL of 50 mg/kg bw in the study of acute toxicity in dogs given capsules containing chlorpropham identified on the basis of clinical signs of toxicity at the higher doses of 125 and 625 mg/kg bw, and using a safety factor of 100. Slight increases in methaemoglobin levels in this study were not considered to be toxicologically significant at any dose.

An addendum to the toxicological monograph was prepared.

Levels relevant to risk assessment

^a Dietary administration
^b Highest dose tested
^c Gavage administration
^d Capsule

Estimate of acceptable daily intake for humans

0-0.05 mg/kg bw

Estimate of acute reference dose

0.5 mg/kg bw

Information that would be useful for the continued evaluation of the compound

Results from epidemiological, occupational health and other observational studies of human exposures

Critical end-points for setting guidance values for exposure to chlorpropham

DIETARY RISK ASSESSMENT

Long-term intake

The 2001 JMPR had calculated the International Estimated Daily Intake (IEDI) for chlorpropham for animal products and potatoes (and for their processed fractions) for which MRLs were estimated and for which consumption data was available using the previous ADI of 0-0.03 mg/kg bw.

The Meeting established an ADI of 0-0.05 mg/kg bw for chlorpropham. The IEDIs of chlorpropham, on the basis of the estimated STMRs, were 2-30% of the maximum ADI for the five GEMS food regional diets. The results are shown in Annex 3. The Meeting concluded that long-term intake of residues of chlorpropham from use on potatoes is unlikely to present a public health concern.

Short-term intake

The 2001 JMPR had calculated the International Estimated Short-term Intake (IESTI) for chlorpropham for animal products and potatoes (and their processed fractions) for which MRLs were estimated and for which consumption data was available using the previous ARfD of 0.03 mg/kg bw.

The Meeting established an ARfD of 0.5 mg/kg bw for chlorpropham. The IESTI represented 0-20% of the ARfD for the general population and 0-60% of the ARfD for children. The values of 20 and 60% represent the estimated short-term intake of cooked potatoes with skin.

The Meeting concluded that the short-term intake of residues of chlorpropham from uses that have been considered by the JMPR is unlikely to present a public health concern.

4.6 Clofentezine (156)

TOXICOLOGY

Clofentezine is an acaricide that is used in plant protection products for the control of spider mites on a wide range of crops. It acts primarily as an ovicide, but it has some activity against early motile stages of mites. The International Union of Pure and Applied Chemistry (IUPAC) chemical name for clofentezine is 3,6-bis(2-chlorophenyl)-1,2,4,5-tetrazine. It was last evaluated by the JMPR in 1986, when an ADI of 0-0.02 mg/kg bw was established based on a NOAEL of 40 ppm (equivalent to 2 mg/kg bw per day) for hepatotoxicity in rats and a NOAEL of 50 ppm (equal to 1.72 mg/kg bw per day) for hepatotoxicity in dogs.

Clofentezine was considered by the present Meeting as part of the periodic review programme of the CCPR. Some GLP-compliant studies of absorption, distribution, metabolism and excretion, toxicity in dogs and effects on the rat thyroid were considered for the first time.

Biochemical aspects

Pharmacokinetic studies in laboratory animals showed that oral doses of clofentezine were quickly absorbed from the gut lumen, with peak concentrations occurring in the plasma after a maximum of 4-6 h in rats. At least half the administered oral dose was absorbed. The liver was the major site for distribution in all species investigated, with high concentrations of radiolabel also being found in the kidneys. Residues were persistent in several tissues, with low concentrations of radiolabel still being present in the liver and adipose tissue of rats at 25 days after the last dose of radiolabelled clofentezine. Radiolabel from orally administered [¹⁴C]clofentezine crossed the placental barrier of rats to reach the fetuses of pregnant rats, but concentrations of radiolabel in the fetuses were about five times lower than in the mothers.

Primary metabolism occurred by two major pathways:

• hydroxylation of the phenyl ring at the 3, 4 and/or 5 position;

• hydroxylation at the 3-phenyl position and replacement of the chlorine atom on the same phenyl ring with a methylthio group.

The relative importance of the two pathways differed from species to species, with hydroxylation being the main route in calves and baboon, but methylthiolation being more important in rodents and rabbits. The primary metabolites could be conjugated with glutathione, mercapturic acid or cysteine before excretion in the bile or urine.

Clofentezine and/or its metabolites were found in the urine and faeces of treated animals with up to about three-quarters of an oral dose being voided in the faeces. About 50% of the radiolabel was associated with unchanged clofentezine. The chemical identity of the rest of the radioactivity in the faeces was not investigated. The possible occurrence of enterohepatic circulation was not investigated.

Studies of the effects of oral doses on liver enzymes showed that clofentezine is a potent inducer of several enzymes, including uridine diphosphoglucuronyl transferase (UDPGT) in rats and cytochrome P450 in mice and rats. The NOEL for effects on these enzymes in rats was 1 mg/kg bw per day.

Toxicological data

Clofentezine has low acute oral toxicity in all species tested (mouse, rat, Syrian hamster and dog), causing no serious adverse effects at any dose tested (up to 5200 mg/kg bw in mice and rats). It also has low acute toxicity in rats exposed dermally (LD₅₀ > 2100 mg/kg bw) or by inhalation (LD₅₀ > 0.89 mg/L).

Clofentezine was not an irritant to the skin of guinea-pigs or the eyes of rabbits. It gave a negative result in a Magnusson & Kligman maximization test for skin sensitization in guinea-pigs.

The main toxicological effects seen in short-term studies in mice, rats or dogs given repeated doses of clofentezine in the diet were hepatotoxicity (changes in histopathology and clinical chemistry) and changes to the thyroid, including follicular hyperplasia. The lowest NOAEL identified from short-term feeding studies was 40 ppm (equal to 2.65 mg/kg bw per day) for effects on the liver in a 90-day study of toxicity in rats. In mice, the NOAEL was 200 ppm (equal to 30.3 mg/kg bw per day) for increased weights of the thyroid and the liver. In dogs, the lowest NOAEL identified was 50 ppm (equal to 1.72 mg/kg bw per day) for hepatotoxicity in a 12-month feeding study.

In a study of carcinogenicity in mice, non-neoplastic changes to the liver included vacuolation and eosinophilia of the hepatocytes. There were no consistent or dose-dependent effects on any tumour type.

The Meeting concluded that there was no evidence of a tumourigenic response in mice.

In the long-term study of toxicity/carcinogenicity in rats, there was limited evidence to suggest that prolonged high doses of clofentezine could cause thyroid follicular cell adenomas and carcinomas in this species. A marginal increase in the incidence of these tumours was seen only in the males at the highest dietary concentration (400 ppm), and was only slightly greater than the incidence in control male rats in a different long-term study of toxicity/carcinogenicity performed in the same laboratory. No changes in the thyroid were seen in the long-term study of toxicity/carcinogenicity in rats at 40 ppm (equal to 1.72 mg/kg bw per day). The results of studies of effects on hormones, enzymes and morphological changes associated with thyroid homeostasis did not clearly establish a mode of action for the development of thyroid tumours.

The Meeting concluded that there was no risk of thyroid tumours developing in rats given oral doses of 1.72 mg/kg bw per day or less.

Clofentezine gave negative results in an adequate range of tests for genotoxicity in vitro and in vivo.

The Meeting concluded that clofentezine is unlikely to be genotoxic.

Noting the absence of genotoxicity, the Meeting concluded that the marginal increase in incidence of thyroid follicular cell tumours in males at the highest dose did not indicate a carcinogenic risk to humans at the levels of exposure likely to be experienced by consumers or workers.

The results of a two-generation study of reproduction in rats showed that exposure to clofentezine at a dietary concentration of 400 ppm caused decreased body-weight gains in pups during lactation, resulting in low body-weights of pups in the weeks following lactation. A transient marginal decrease in pup weight of the F₂ generation males at 40 ppm at 1 week after weaning was not considered to be toxicologically significant. The NOAEL for the study was 40 ppm (equivalent to 2.7 mg/kg bw per day) on the basis of decreased pup weight.

Studies of developmental toxicity in rats and rabbits treated by gavage showed that clofentezine was neither teratogenic nor embryotoxic. The only indication of fetotoxicity was low fetal body weight in rats at maternally toxic doses. The NOAEL for maternal toxicity in these studies was 320 mg/kg bw per day in rats and 250 mg/kg bw per day in rabbits.

No evidence of neurotoxicity was apparent from the available studies of toxicity.

Routine monitoring of workers in a factory producing clofentezine has shown no adverse effects attributable to exposure to clofentezine.

The Meeting concluded that the existing database on clofentezine was adequate to characterize the potential hazards to fetuses, infants and children.

Toxicological evaluation

The Meeting established an ADI of 0-0.02 mg/kg bw based on the NOAEL of 1.72 mg/kg bw per day for thyroid changes in a long-term study of toxicity/carcinogenicity in rats and also for hepatotoxicity in a 12-month study in dogs, and using a safety factor of 100.

The Meeting concluded that it was not necessary to set an ARfD for clofentezine, since clofentezine has low acute toxicity and does not cause developmental toxicity or any other toxicological effect that would be elicited by a single exposure.

A toxicological monograph was prepared.

Levels relevant to risk assessment

^a Highest dose tested
^b Oral gavage administration

Estimate of acceptable daily intake for humans

0-0.02 mg/kg bw

Estimate of acute reference dose

Unnecessary

Information that would be useful to the continued evaluation of the compound

Results from epidemiological, occupational health and other observational studies of human exposures

Critical end-points for setting guidance values for exposure to clofentezine