Pesticide residues in food 2001

Toxicological evaluations

CARBARYL (addendum)

First draft prepared by
M.E. van Apeldoorn, G. Wolterink, E. Turkstra, M.T.M. van Raaij, P.H. van Hoeven-Arentzen and J.G.M. van Engelen
Centre For Substances and Risk Assessment
National Institute of Public Health and the Environment,
Bilthoven, The Netherlands

Explanation

Evaluation for acceptable daily intake

Biochemical aspects

Absorption, distribution and excretion

Biotransformation

Effects on enzymes and other biochemical parameters

Toxicological studies

Short-term studies of toxicity

Long-term studies of toxicity and carcinogenicity

Reproductive toxicity

Multigeneration studies

Developmental toxicity

Special studies: Neurotoxicity

Observations in humans

Comments

Toxicological evaluation

References

Explanation

Carbaryl was evaluated for toxicological effects by the Joint Meeting in 1963, 1965, 1966, 1967, 1969, 1973 and 1996 (Annex 1, references 2, 3, 6, 8, 12, 20 and 77). An ADI of 0–0.02 mg/kg bw was established in 1963 on the basis of a 1-year study in dogs, and this ADI was confirmed in 1965, 1966 and 1967. In 1969, a temporary ADI of 0–0.01 mg/kg bw was established, which incorporated an extra safety factor because of concern about effects on the male reproductive system in a 1-year study in rats treated by gavage, with a NOAEL of 2 mg/kg bw per day, and because a dose of 0.12 mg/kg bw per day may have affected renal function in a 6-week study in volunteers. In 1973, the Meeting established an ADI of 0–0.01 mg/kg bw. In 1996, carbaryl was reviewed as part of the periodic review programme of the Codex Committee on Pesticide Residues, and the Meeting established an ADI of 0–0.003 mg/kg bw on the basis of a LOAEL of 15 mg/kg bw per day in a study of carcinogenicity in mice and a safety factor of 5000, which included an extra safety factor of 50 to account for the presence of vascular tumours in male mice at all doses tested. The Meeting stated that the resulting ADI provided an adequate margin of safety, taking into account the LOAEL (3.1 mg/kg bw per day for maternal toxicity) in a study of developmental toxicity in dogs and uncertainties about effects on the male reproductive system.

The following information was available to the present Meeting: new studies on metabolism in mice and rats; a 14-day study of effects on some enzyme activities in rats; a 6-month study in p53 knockout mice; a (re-)evaluation of the incidence of bladder tumours in the 2-year study of toxicity and carcinogenicity in rats; a (re-)evaluation of all slides from the 2-year study of carcinogenicity in mice and the 2-year study of toxicity and carcinogenicity in rats; and an extended and updated epidemiological study. Furthermore, additional studies were available on the neurotoxicity, developmental toxicity, and reproductive toxicity of carbaryl.

Evaluation for acceptable daily intake

1. Biochemical aspects

(a) Absorption, distribution and excretion

Mice

A study was conducted to investigate the possible reasons for the increased incidence of tumours seen at high doses of carbaryl during the final year of a study in CD-1 mice. In this study, groups of 10 male CD-1 mice, 4 weeks of age, received a diet containing carbaryl at a concentration of 0, 11, 110, 1100 or 7980 ppm, equivalent to 0, 1.5, 16, 160 and 1100 mg/kg bw per day, for 14 days, followed by a single oral dose of 50 mg/kg bw [¹⁴C- naphthalene ring]carbaryl (radiochemical purity, 100%) by gavage on day 15. Body weights were measured on the day before the first dietary administration and on days 1, 8 and 14 thereafter. After dosing with [¹⁴C]carbaryl, urine, faeces and cage washes were collected at 24-h intervals and analysed for radiolabel content. Food consumption was assessed weekly. All animals were killed by exsanguination 168 h after administration of [¹⁴C]carbaryl, and blood and carcass samples were analysed for radiolabel. Statements of adherence to good laboratpry practice (GLP) and quality assurance (QA) were included. The radiolabel was eliminated mainly in urine (73–84%, assuming that the radiolabel found in cage washes had also been excreted in urine) and to a lesser extent in the faeces (12–19%). By 168 h after dosing, very small amounts ( 0.8%) of radiolabel were found in the carcass and blood (Table 1) (Vallès, 1999).

Table 1. Cumulative recovery of radiolabel (% dose administered) after a single oral dose of [¹⁴C]carbaryl to mice

Sample	Time after administration of carbaryl (h)	Dose (mg/kg bw)
Sample	Time after administration of carbaryl (h)	0	11	110	1100	8000
Urine	0–24	58	45	55	55	58
	0–48	66	53	63	66	67
	0–168	69	59	65	69	69
Faeces	0–24	1.2	9.8	16	15	17
	0–48	11	14	17	16	18
	0–168	12	15	18	16	19
Blood^a	168	0.01	0.01	0.01	0.01	0.01
Carcass^a	168	0.45	0.50	0.82	0.34	0.24
Cage wash	168	15	17	16	15	10
Total recovery		96	89	100	101	98

From Vallès (1999)

^{a Converted by the Meeting from micrograms of equivalents per gram
The Meeting noted that much more radiolabel was excreted in the faeces during the first 24 h after treatment by mice given the diet containing carbaryl than by controls that received only the single oral dose of [¹⁴C]carbaryl, but the significance of this finding, e.g. decreased absorption or increased excretion via the bile, was not discussed by the study author. Furthermore, the Meeting noted that it can be calculated from data in appendices to the report that the animals at the highest dietary concentration gained almost no weight over the 14 days of treatment, in contrast to the animals at the other doses.
Rats
A study was conducted to investigate the role of the metabolism of carbaryl in the causation of tumours in rats in a 2-year study, by qualitative and quantitative comparisons of the metabolic profiles in older rats after a single oral dose or short-term dietary administration of carbaryl. Five male Sprague-Dawley rats aged about 15 months and fed a normal diet received [¹⁴C-naphthalene ring]carbaryl at a single dose of 50 mg/kg bw by gavage. Their excreta were collected daily for 168 h after dosing; then, the animals were killed, and tissue samples were collected for determination of radiolabel content. Statements of adherence to GLP and QA were included.
Most of the radiolabel was excreted in the urine (86%, assuming that the radiolabel in the cage wash had also been excreted in urine), with 11% in the faeces. Most of the radiolabel was excreted during the first 48 h after treatment. By 168 h, little residue was found in tissues, and 4.3% was present in the skin and fur (see Table 2), probably as a result of contamination of the fur with urine (Totis, 1997).
Table 2. Distribution and excretion of radiolabel (% of total administered dose) after a single oral dose of [¹⁴C]carbaryl at 50 mg/kg bw to rats

Sample

Time after dosing (h)

6

24

48

72

96

120

144

168

Urine

27

50

63

67

68

69

69

69

Faeces

0.3

0.3

4.7

8.0

10

11

11

11

Cage wash

17

Tissues

0.4

Skin and fur

4.3

From Totis (1997)
Groups of male Sprague-Dawley rats aged 15 months received a diet containing unlabelled carbaryl at a nominal concentration of 0, 250, 1500 or 7500 ppm, equivalent to 0, 12, 75 and 380 mg/kg bw per day, for 83 days. At the end of this period, five animals per dose received [¹⁴C-naphthalene ring]carbaryl at 2 mg/kg bw per day by gavage for 7 days, and their excreta were collected daily up to 72 h after the last dose. The liver, kidneys, thyroid, urinary bladder and skin with fur were removed from each animal after they were killed by exsanguination and retained for determination of radiolabel content with the residual carcass. Most the radiolabel was found in the urine (85–93%, assuming that radiolabel found in the cage wash had also been excreted in urine), with 7.4–10% in the faeces; < 1% was found in the tissues (see Table 3) (Totis, 1997).
Table 3. Distribution and excretion of radiolabel (% of total administered dose) 72 h after 7 daily oral doses of [¹⁴C]carbaryl at 2 mg/kg bw to rats

Sample

Dose (mg/kg of diet)

0

250

1500

7500

Urine + cage wash

92

92

93

85

Faeces

10

7.4

9.9

10

Tissues

0.4

0.4

0.4

0.8

From Totis (1997)
(b) Biotransformation
Mice
In the study with CD-1 male mice (Vallès, 1999), urine samples collected over 0–24, 24–48 and 48–96 h were analysed for metabolites. Out of a total of 20 metabolites, four were present in relatively large quantities in the urine (see Table 4). Mice given the diet containing carbaryl at 8000 ppm had more dihydro, dihydroxynaphthyl sulfate and hydroxy-carbaryl glucuronide metabolites, which may have been formed via epoxidation and conjugation.
Table 4. Metabolites of carbaryl found in mouse urine 0–96 h after treatment (% of administered dose)

Metabolite

Dose (mg/kg of diet)

0

11

110

1100

8000

Dihydro, dihydroxynaphthyl sulfate

3.2

3.0

3.7

3.8

6.8

Hydroxy-carbaryl glucuronide

14

12

15

14

19

alpha-Naphthyl sulfate

14

11

11

12.

12

alpha-Naphthyl beta-D-glucuronide

17

11

13

20

20

From Vallès (1999). The metabolites were identified by liquid chromatography with mass spectrometry.
The Meeting noted that the study author claimed that 21 metabolites of carbaryl were formed. However, one of these metabolites was not detected in any of the samples.
Rats
In the study with 15-month-old male Sprague-Dawley rats (Totis, 1997), the urine and faecal samples collected from five rats on days 1, 2, 3, 4, 5, 6 and 7 were pooled and analysed for metabolites. In the same study, additional groups of 10 male rats received the same treatment (unlabelled carbaryl in the diet at 0, 250, 1500 or 7500 ppm for 83 days and then [¹⁴C]carbaryl at 2 mg/kg bw per day by gavage for 7 days), but were killed by exsanguination 6 h after the last dose, and liver, kidney, spleen, urinary bladder and thyroid were collected for determination of metabolites. As very low concentrations of radiolabel were found in the tissues, the metabolites could be identified only qualitatively. A glucuronide conjugate of naphthol found in kidney and urinary bladder and a sulfonate conjugate of naphthol found in plasma, kidney and urinary bladder were the two main metabolites in tissues. Investigation of the metabolism of carbaryl in urine and faecal extracts revealed the presence of up to 23 radiolabelled components in the urine and up to 20 in the faeces. Overall, the radioalabel in the faeces represented < 1.5–1.7% of the total administered dose in the controls and rats at the low and high doses and < 3.6% in those at the intermediate dose. One of the main analytes was the parent compound. In the urine, most of the recovered radiolabel was associated with three metabolites (Table 5).
Table 5. Radiolabelled components in the urine of rats (% daily administered dose) given [¹⁴C]carbaryl

Metabolite

Dose

(mg/kg of diet)

Day after treatment

1

2

3

4

5

6

7

Glucuronide of dihydro, dihydroxy-1-naphthyl-N-methylcarbamate

0

15

19

16

18

16

17

18

250

16

16

15

15

20

23

23

1500

21

20

19

21

26

27

19

7500

28

20

17

19

21

18

10

alpha-Naphthyl beta-D-glucuronide

0

16

15

15

14

13

15

15

250

16

15

15

15

22

22

17

1500

14

11

19

16

14

12

10

7500

15

12

13

15

17

15

12

Sulfonate conjugate of naphthol

0

24

23

26

22

19

24

25

250

27

23

21

9

ND

ND

15

1500

23

25

30

30

30

27

25

7500

12

18

13

ND

ND

ND

9

From Totis (1997)
ND, not detected
In the control group, the relative quantities of the three main metabolites remained stable over the 7 days of measurement. In the group at 250 ppm, the concentration of the glucuronide of dihydro, dihydroxy-1-naphthyl-N-methylcarbamate tended to increase over the 7 days and those of the sulfonate conjugate of naphthol to decrease as compared with controls, especially on days 4, 5 and 6. In the group at 1500 ppm, the concentrations of both the glucuronide and the sulfonate were slightly higher than in the control group on most days. In the group at 7500 ppm, the concentration of the sulfonate conjugate was lower than that in controls, especially on days 4, 5 and 6. Typically, in the groups at 250 and 7500 ppm, the concentrations of the sulfonate conjugate of naphthol were below the limit of detection on days 5 and 6 but were found in relatively large amounts on day 7. It is notable that the metabolite profile of rats at 250 and 7500 ppm was different from that of controls, while that of rats at 1500 ppm was similar. In all groups, the concentrations of alpha-naphthyl beta-D-glucuronide remained more or less stable.
On the basis of the data for day 1, the study author suggested that the metabolism of carbaryl shifted from formation of the sulfonate conjugate of naphthol to the glucuronide of dihydro, dihydroxy-1-naphthyl-N-methylcarbamate after 83 days of pretreatment with carbaryl (Totis, 1997). The Meeting was not convinced. Overall, the quantitative changes in metabolic profiles observed were variable, and the data for rats at 1500 ppm clearly do not support the conclusion of the author. Furthermore, the increased concentration of the glucuronide was observed only on day 1, although that of the sulfonate conjugate remained lower in rats at 250 and 7500 ppm throughout the 7-day period.
(c) Effects on enzymes and other biochemical parameters
Rats
A study was conducted to determine whether the induction of hepatic enzymes might account for the formation of tumours in the liver and thyroid of rats at the highest dose (375 mg/kg bw per day) in the long-term study of toxicity and carcinogenicity. In this 14-day study, groups of 10 male and 10 female Sprague-Dawley rats aged about 9 weeks (body weights, 320–350 g for males and 220–240 g for females) received carbaryl (purity, 98.4%) by gavage at a dose of 0, 10 or 40 mg/kg bw per day in 0.5% carboxymethylcellulose and 0.1% Tween 80. Five animals of each sex per group were killed on day 4 and the remaining animals on day 15 after an overnight fast. All groups were checked daily for deaths, morbidity and clinical signs; they were weighed on days –1, 1, 7 and 14, and their food consumption was determined on days 7 and 14. All animals killed on days 4 and 15 were examined macroscopically, and their livers were weighed. Hepatocellular proliferation was measured by proliferating cell nuclear antigen (PCNA) staining, and the livers were examined histologically on days 4 and 15. Additionally, induction of liver enzymes (cytochrome P450s) and triodothyronine and thyroxine UDP-glucuronidyl transferase (UGT) activities were assessed in the livers of animals killed on day 15. Statements of adherence to GLP and QA were included.
No deaths were observed. Most of the animals at 40 mg/kg bw per day showed reduced motor activity throughout the study, and tremors, staggering, increased salivation, piloerection, soft faeces and polypnoea were seen sporadically. Males at 40 mg/kg bw per day weighed significantly less than controls on days 7 and 15 (–8.4 and –7.5%, respectively) and consumed less food during the first week. The liver weights were unchanged by treatment, and no macroscopic or microscopic changes were observed. The total cytochrome P450 content was not changed by carbaryl treatment, and the activities of the cytochrome P450 enzymes benzoxyresorufin O-debenzylase and pentoxyresorufin O-depentylase and of lauric acid hydroxylase were not affected. However, the activity of ethoxyresorufin O-deethylase was increased significantly (5.5 times) in males at 40 mg/kg bw per day, and those of thyroxine-UGT and triiodothyronine-UGT were increased significantly in males at 40 mg/kg bw per day and in females at 10 and 40 mg/kg bw per day, with a 1.5-fold increase in thyroxine-UGT activity and a 1.8-fold increase in triiodothyronine-UGT activity in both males and females. A moderate increase in cells in G₁ phase was observed in males at 40 mg/kg bw per day on day 4 and and an increase in cells in G₁ and S phases on day 15. In females, an increase in cells in G₁ and S phases was observed at 10 and 40 mg/kg bw per day on day 15. In view of the increased UGT activities and the increase in cell cycling at 10 and 40 mg/kg bw per day, no NOAEL could be identified (Berthe, 1997).
2. Toxicological studies
(a) Short-term studies of toxicity
Rats
In the study of Totis (1997), groups of five male Sprague-Dawley rats, about 15 months old, received a diet containing carbaryl at a concentration of 0, 250, 1500 or 7500 ppm, equivalent to 0, 12, 75 and 380 mg/kg bw per day, for 90 days. During treatment, the groups were observed for deaths, morbidity (twice a day on week days, once a day at weekends), body weight and food consumption (weekly). One day after treatment, the animals were killed and the liver, kidneys and thyroid were weighed and examined histologically. Liver tissue was also examined biochemically. Statements of adherence to GLP and QA were included.
Decreased growth was seen for animals at 1500 and 7500 ppm. Carbaryl induced follicular-cell hypertrophy of the thyroid at concentrations > 250 ppm, and pericholangitis, centrilobular hypertrophy of the liver and transitional-cell hyperplasia of the renal pelvis at 7500 ppm (Table 6).
Table 6. Results of 90-day dietary study in 15-month-old male Sprague-Dawley rats

Effect

Dose (mg/kg of diet)

0

250

1500

7500

Mortality rate

No toxicologically relevant effect

Body weight (g)

78

820

690^a

650^a

Food consumption

No toxicologically relevant effect

Dermal reactions

No toxicologically relevant effect

Biochemistry in liver tissue

Protein (mg/g liver)

100

120

77^a

89

Glutathione (µmol/g liver)

4.7

5.8

3.6^a

8.3^b

Glutathione (µmol/g protein)

47

50

48

94^b

GST (µmol/min per g liver)

72

64

49^a

71

GST (µmol/min per g protein)

720

540^a

640

810

Glutathione peroxidase (IU/g liver)

220

220

240

160

Glutathione peroxidase (IU/g protein)

2100

1900

3100^a

1900

Organ weight (absolute, in g)

Liver

18

18

17

22^a

Spleen

1.2

1.1

1.4

1.6

Thyroid

0.031

0.041

0.052^a

0.059^a

Histopathology^c

Liver

Centrilobular hypertrophy

0

0

0

5

Pericholangitis

0

0

0

3

Thyroid

Follicular-cell hypertrophy

0

3

5

5

Kidney

Transitional-cell hyperplasia

0

0

0

2

From Totis (1997). GST, glutathione S-transferase

^{a Significantly increased}

^{b Significantly decreased}

^{c Number of animals, out of five, displaying effect}

Dogs
Groups of six beagles of each sex, about 6 months old, were fed diets containing technical-grade carbaryl (purity, 99.3%) at a concentration of 0, 20, 45 or 125 ppm (equal to 0, 0.6, 1.4 and 3.8 mg/kg bw per day for males and 0, 0.6, 1.5 and 4.1 mg/kg bw per day for females) for 5 weeks. The groups were observed daily throughout treatment for deaths and clinical signs. Body weights and food consumption were recorded weekly, and ophthalmological examinations were performed before and during treatment. Plasma and erythrocyte cholinesterase activity was measured in each dog before treatment and on days 14 and 32, about 2 h after feeding. Complete gross necropsies were performed, and brain samples were taken for determination of acetylcholinesterase activity. Statements of adherence to GLP and QA were included. The only finding was inhibition of plasma cholinesterase activity on day 14 in males at 20 and 125 ppm, which was considered incidental and of no toxicological relevance. The NOAEL was 125 ppm, equal to 3.8 mg/kg bw per day, the highest dose tested. (Hamada, 1991).
(b) Long-term studies of toxicity
Mice
A 2-year study with carbaryl in CD-1 mice (Hamada, 1993a) was summarized by the JMPR in 1996 (Annex 1, reference 79). The authors concluded that the lowest concentration tested (100 ppm, equal to 15 mg/kg bw per day) was the LOAEL, on the basis of an increased incidence of vascular tumours in males. The increased incidence was found only for mice killed at the end of the study or which died off schedule; the tumours were located predominantly in the liver and spleen and to a lesser degree in other tissues. An increased incidence of haemangioma and haemangiosarcoma was also seen in females at 8000 ppm (Table 7).
Table 7. Incidences of haemangiomas and haemangiosarcomas in various tissues of CD-1 mice, by concentration of carbaryl in the diet (mg/kg)

Vascular tumours

Males

Females

0

100

1000

8000

0

100

1000

8000

Total no. of tumours

2/70

9/70

13/70

18/70

5/70

6/70

5/70

10/70

No. of tumour-bearing animals

2/70

6/70

10/70

10/70

3/70

3/70

4/70

9/70

From Hamada (1993a)
Furthermore, at 8000 mgkg of diet, the incidences of renal tubule-cell adenomas and carcinomas were increased in males and the incidences of hepatocellular adenomas and carcinomas were increased in females. The NOAEL for non-neoplastic lesions was 100 ppm, equal to 15 mg/kg bw per day, on the basis of inhibition of erythrocyte and brain acetylcholinesterase activity and histopathological changes in the bladder.
As the initial histological examination of tissues of groups of 10 mice of each sex per group at the interim kill at week 53 did not reveal any changes associated with the tumours found at the terminal kill, the slides of the livers and kidneys of controls and mice at the highest dose were re-evaluated. Again, no microscopic changes that could be associated with the tumours were seen after 104 weeks (Debruyne & Irisarri, 1996).
Subsequently, an independent panel of pathologists (Hardisty, 1996a) re-evaluated all microscopic tissue sections containing proliferative vascular lesions to confirm the accuracy of the original diagnoses (according to the guidelines of the Pathology Workgroup of the USA’s Environmental Protection Agency). The slides for 10% of controls and male and female mice at the highest dietary concentration were also re-evaluated to ensure that all proliferative lesions had been identified. Furthermore, all sections of liver and kidneys from all mice in all groups were re-examined to confirm the presence or absence of proliferative changes. The re-evaluation generally confirmed the original evaluation of the study pathologist for the kidneys of male mice and the livers of female mice, as did the re-evaluation of vascular lesions. The few differences between the original diagnosis and that at re-evaluation concerned the classification of vascular neoplasms as benign (haemangioma) and malignant (haemangiosarcoma). The incidences of vascular neoplasms as observed by the panel are shown in Table 8.
Table 8. Incidences of haemangioma and haemangiosarcoma in CD-1 mice treated with carbaryl, by concentration in the diet (mg/kg), in the study of Hamada (1993a), as observed by an independent panel of pathologists

Vascular tumours

Males

Females

0

100

1000

8000

0

100

1000

8000

Haemangioma (no. of tumours)

1/70

1/70

2/70

2/70

2/70

1/70

1/70

0/70

Single site (no. of tumour-bearing animals)

1/70

1/70

2/70

2/70

2/70

1/70

1/70

0/70

Multiple sites (no. of tumour-bearing animals)

0/70

0/70

0/70

0/70

0/70

0/70

0/70

0/70

Haemangiosarcoma (no. of tumours)

1/70

9/70

11/70

16/70

4/70

6/70

4/70

10/70

Single site (no. of tumour-bearing animals)

1/70

5/70

6/70

6/70

0/70

3/70

2/70

8/70

Multiple sites (no. of tumour-bearing animals)

0/70

1/70

2/70

2/70

2/70

1/70

1/70

1/70

No. of animals bearing both haemangioma and haemangiosarcoma

0/70

1/70

0/70

0/70

0/70

1/70

0/70

0/70

Total no. of vascular neoplasms

2/70

10/70

13/70

18/70

6/70

7/70

5/70

10/70

No. of vascular tumour-bearing animals

2/70

7/70

10/70

10/70

4/70

4/70

4/70

9/70

From Hardisty (1996a)
The re-evaluation did not reveal a significant positive trend or an increased incidence of haemangioma in male mice; the incidence of haemangiosarcoma in male mice at all dietary concentrations was increased significantly, but there was no significant positive trend and the combined incidence of haemangioma and haemangiosarcoma in male mice was significantly increased only at the two higher concentrations. The incidence of haemangiosarcoma in female mice was significantly increased at the highest concentration, and the trend was statistically significant even at lower concentrations, at which the incidences were similar to those of controls owing to a ‘threshold-like’ behaviour (Hardisty, 1996a).
The Meeting noted that Hardisty conducted statistical analyses of vascular tumour incidences (number of tumour-bearing animals) in a total of 80 mice, whereas the total should have been 70 mice, i.e. those killed at the end of the study plus unscheduled deaths. The 10 mice that were killed after 53 weeks should not have been included.
The vascular tumour incidences found in the 2-year study in male CD-1 mice at 100 and 1000 ppm were compared with the incidences in controls in previous studies (Klonne, 1995; reported in Annex 1, reference 79). The incidences for males at 8000 ppm were not analysed by Klonne because that concentration exceeded the maximum tolerated dose (MTD ). The vascular tumour incidences in liver and spleen were compared with the tumour incidences in the same organs in several databases, because vascular tumours in other organs are rare and are generally not listed or routinely evaluated microscopically. The Meeting noted that, in the paper of Klonne (1995), the percentages of tumours in mice treated with carbaryl were calculated on the basis of a total of 80 animals, which also included the animals killed after 53 weeks, whereas only the unscheduled deaths and animals killed terminally should have been taken into account. In the comparisons shown below, the incidences of haemangioma, haemangiosarcoma and haemangioma and/or haemangiosarcoma were compared with those in databases of controls. Only values that exceeded the range are given.
Comparison with database of Cornington Hazleton Virginia (24 studies lasting 78 weeks and one 104-week study, 1988–93): Incidences of haemangiosarcoma in liver and spleen at 1000 ppm and combined incidences of haemangioma and haemangiosarcoma in spleen at 1000 ppm in the study with carbaryl exceeded the range in these controls.
Comparison with database of Cornington Hazleton Wisconsin (11 studies lasting 78, 91 and 104 weeks, 1986–93 [studies could not be distinguished]): Incidences of haemangiosarcoma in liver and spleen at 100 and 1000 ppm and of haemangiosarcoma in spleen in controls in the study with carbaryl exceeded the range in these controls. The combined incidence of haemangioma and haemangiosarcoma in the liver in animals at 100 and 1000 ppm and in the spleen in animals at 1000 ppm exceeded the range in these controls.
Comparison with database of Charles River Laboratories (unknown number of 24-month studies, 1981–91): Incidences of haemangiosarcoma in spleen at 100 and 1000 ppm exceeded the range in these controls. Haemangiomas in spleen were not listed in this database.
Comparison with database of Pharmaco LSR (17 studies lasting 88–104 weeks, 1986–93): Incidences of haemangiosarcoma in spleen at 1000 ppm exceeded the range in these controls. Combined incidences of haemangioma and haemangiosarcoma were not listed.
Comparison with database of Maita et al. (1988) (11 studies lasting 104 weeks, 1982–87): Incidences of haemangiosarcoma in both liver and spleen at 100 and 1000 ppm exceeded the mean percentage incidences in these controls. The combined incidences of haemangioma and haemangiosarcoma were not listed.
The Meeting noted that the incidences of haemangiosarcomas in liver and spleen of animals given diets containing carbaryl at 100 ppm also exceeded those of controls in an affiliated laboratory in which the 2-year study in mice was performed. Furthermore, the incidence of haemangiosarcoma in spleen in mice at 100 ppm exceeded the range of controls in the laboratories of the animal breeders. Two important phenomena emerge:

As the incidence of spontaneous vascular tumours increased with age, a bias was introduced bycomparing data for controls in a 78-week study with those in a 104-week study .
Comparison of the data from the Charles River laboratories for 1978–85 with those for 1981–91 showed that the upper end of the range of incidence of vascular tumours had increased by up to four times. Therefore, control data from about the same period should be used in making comparisons.

Klonne (1995) stated that the incidence of vascular tumours in males at 100 ppm fell within the range for controls in previous studies in other laboratories. The Meeting noted that, although the incidence of vascular tumours in male controls in the study with carbaryl was low, no data were available on other controls in the same laboratory in studies of the same duration of exposure. Therefore, the possibility that 100 ppm is the LOAEL for vascular tumours in males cannot be excluded.
In the 2-year study with carbaryl in CD-1 mice, vascular tumours were observed in males even at the lowest concentration of 100 ppm, equal to 15 mg/kg bw per day. To better understand the carcinogenic potential of carbaryl, especially with regard to vascular tumours, a carcinogenicity study was performed with carbaryl in p53 knockout mice, in which one allele of the p53 tumour suppressor gene has been inactivated. As it is assumed that a mutation at the intact p53 allele is needed to accelerate carcinogenicity, compounds that induce tumours by non-genotoxic mechanisms should be inactive in this model. The model was evaluated with two compounds, the genotoxin urethane, which causes vascular, pulmonary and hepatocellular tumours in standard bioassays for carcinogenicity, and d-limonene, which is not genotoxic and not carcinogenic in mice but is carcinogenic in male rats by a well-described non-genotoxic mechanism. Urethane was given orally by gavage for 180 days to groups of 20 male p53 knockout mice at a dose of 0, 1, 10 or 100 mg/kg bw per day in 0.5% methylcellulose in sterile water and 0.2% Tween 80. d-Limonene was given at one dose of 250 mg/kg bw in the same vehicle. A control group of wild-type mice was given the vehicle only. None of the controls developed tumours, and one spontaneous tumour of the prostate was seen in the group given d-limonene. On the basis of the results seen with urethane (Table 9), the authors concluded that the p53 knockout mouse was useful for investigating mechanisms of vascular tumour formation (Bigot, 1999; Carmichael et al., 1999).
Table 9. Incidences of vascular proliferative changes in p53 knockout mice treated with urethane

Tissue and change

Dose (mg/kg bw per day)

0

1

10

100^a

Liver

Endothelial hyperplasia

0/20

0/20

0/20

2/20

Haemangioma, benign

0/20

0/20

1/20

14/20

Haemangiosarcoma, malignant

0/20

0/20

0/20

8/20

Combined benign and malignant neoplasms

0/20

0/20

0/20

4/20

Combined benign and/or malignant neoplasms

0/20

0/20

1/20

18/20

Spleen

Malignant haemangiosarcoma

0/20

0/20

0/20

1/20

Heart

Endothelial (haemangioma-like) proliferation

0/20

0/0

0/20

1/20

Haemangioma, benign

0/20

0/0

0/20

1/20

Abdomonal cavity

Haemangiosarcoma, malignant

0/0

0/0

0/0

1/1

From Bigot (1999) and Carmichael et al. (1999)
^{a Five out of 20 animals were killed when moribund between days 139 and 177, and 12 out of 20 were found dead between day 104 and the end of the study.
The Meeting considered that the power of the model to discriminate between genotoxins and non-genotoxins is not as clear as suggested by the sponsor and that for weak genotoxic carcinogens the length of exposure of 6 months used in the protocol was too short to obtain conclusive results. Furthermore, the validation of the model was limited, because:

No positive control group consisting of wild-type mice exposed to urethane was included for comparison.
The compound chosen as the negative control was unfortunate, as d-limonene is carcinogenic only in the male rat kidney.
The highest dose of urethane used, 100 mg/kg bw per day, far exceeded the MTD, as indicated by the early deaths of 17/20 mice at this dose. At the intermediate dose of 10 mg/kg bw per day, only one haemangioma in the liver was observed, with no haemangiosarcomas at any site. At the lowest dose, 1 mg/kg bw per day, no vascular tumours were seen. Thus, the range of doses used may not have been well chosen.

In a study with carbaryl in the p53 knockout mouse model, groups of 20 males aged 9–11 weeks (weighing 22–29 g) received a diet containing carbaryl (purity, 99%) at a concentration of 0, 10, 30, 100, 300, 1000 or 4000 ppm, equal to 0, 1.7, 5.2, 18, 52, 160 and 720 mg/kg bw per day (mean for weeks 1–25), for at least 180 days. All groups were observed twice daily for deaths, and morbidity and clinical signs were recorded at least once per day. Detailed physical examinations were performed at least weekly. Body weights and food consumption were determined weekly for the first 14 weeks and monthly thereafter. All animals were killed on days 181–184 and examined macroscopically. The absolute weights and the organ:body and organ:brain weight ratios of brain, heart, liver, spleen, kidneys, thymus and testes were determined. About 40 tissues from all mice at 0 and 4000 ppm and of all animals that died during the study were examined microscopically. The liver, spleen, thymus, urinary bladder and all lesions seen macroscopically in animals at the intermediate dose were examined microscopically. Adherence to GLP and QA was stated.
The deaths seen during the study (one at 10 ppm, two at 30 ppm and two at 300 ppm) and the clinical signs in these animals (reduced motor activity, cold to touch and hunched posture before death) were considered by the authors not to be related to treatment. The food consumption of animals at 4000 ppm was significantly lower than that of controls during the first 8 weeks and was correlated with lower body weights. Some changes in absolute and/or relative organ weights were seen at 1000 and 4000 ppm. The only treatment-related non-proliferative change observed was the presence of intracellular globular deposits in the urinary bladder epithelium at doses > 100 ppm, with a dose-related increase in the severity of the accumulation of globular deposits. No local irritation or hypertrophy of the urinary bladder was observed. No effects were seen at 30 ppm, equal to 5.2 mg/kg bw per day.
Only a few animals at the intermediate dose had spontaneous neoplasms. No neoplastic or preneoplastic changes were observed in the vascular tissue in any organ. The authors concluded that no tumours were induced at any dose, and, further, that these results demonstrated a lack of genotoxic potential of carbaryl (Chuzel, 1999).
The Meeting noted that no tumours of the vascular system developed in this 6-month study with carbaryl, in contrast to the results of the 2-year study in CD-1 mice. This finding indicates a non-genotoxic mechanism of vascular tumour induction by carbaryl in mice. Donehower (1999) stated, however, that an 8-month study would have provided better assurance of detecting subtle carcinogens.
Rats
Hamada (1993b; reported in Annex 1, reference 79) conducted a 2-year study in Sprague-Dawley rats with carbaryl. The initial report stated that neoplasms were observed only at the highest dietary concentration (7500 ppm) in the thyroid, liver and urinary bladder, and 1996 JMPR concluded that carbaryl induced tumours in rats at a dose that exceeded the MTD. The NOAEL for non-neoplastic findings was 250 ppm, equal to 10 mg/kg bw per day, on the basis of inhibition of erythrocyte and brain acetylcholinesterase activity and a decrease in mean body weight.
As histological examination of animals at the interim kill revealed no changes associated with the tumours found at the terminal kill, the slides of the target organs (liver, kidney, thyroid gland and urinary bladder) of animals at the 53-week interim kill and at a 57-week kill after a 4-week recovery period were re-evaluated. Microscopic changes were found in the urinary bladder (transitional epithelial hyperplasia in males and females, not reversed within 4 weeks), kidney (pelvic urothelial hyperplasia in males, reversed within 4 weeks), thyroid (thyroid follicular hypertrophy in males, reversed within 4 weeks) and liver (hepatocellular hypertrophy in males and females, reversed within 4 weeks) (Debruyne & Irisarri, 1996).
Subsequently, an independent panel of pathologists (Hardisty, 1996b) re-evaluated all slides from the 53-week, 57-week and terminal kills (according to the the guidelines of the Pathology Workgroup of the USA’s Environmental Protection Agency) to confirm the accuracy of the diagnoses (see Table 10). The results of the independent group confirmed the findings of Debruyne & Irisarri (1996).
Table 10. Incidences of tumours in Sprague-Dawley rats treated with carbaryl, by concentration in the diet (mg/kg), in the study of Hamada (1993b), as observed by an independent panel of pathologists

Time of kill

Males

Females

0

250

1500

7500

0

250

1500

7500

Hyperplasia, papilloma and carcinoma in the urinary bladder

53-week interim

Transitional-cell hyperplasia

0/9

0/10

0/10

7/9

1/10

0/10

0/10

9/10

57-week recovery

Transitional-cell hyperplasia

0/10

–

–

4/10

0/10

–

–

3/10

104-week terminal kill plus unscheduled deaths

Transitional-cell hyperplasia

8/71

7/70

11/70

51/71

6/70

4/70

4/70

56/70

Transitional-cell papilloma

0/71

0/70

0/70

14/71

1/70

0/70

0/70

8/70

Squamous-cell papilloma

0/71

0/70

0/70

2/71

0/70

0/70

0/70

0/70

Transitional-cell carcinoma

0/71

0/70

0/70

10/71

0/70

0/70

0/70

5/70

Epithelial hyperplasia and carcinoma in the renal pelvis

53-week interim

Pelvic epithelial hyperplasia

1/10

0/10

1/10

7/10

0/10

1/10

2/10

1/10

57-week recovery

Pelvic epithelial hyperplasia

2/10

–

–

0/10

1/10

–

–

0/10

104-week terminal plus unscheduled deaths

Pelvic epithelial hyperplasia

13/70

10/70

13/70

29/70

22/70

39/70

29/70

21/70

Transitional-cell carcinoma

0/70

0/70

0/70

1/70

0/70

0/10

0/70

0/70

Hepatocellular hypertrophy and neoplasms in the liver

53-week interim

Hepatocellular hypertrophy

0/10

0/10

0/10

6/10

0/10

0/10

0/10

7/10

57-week recovery

Hepatocellular hypertrophy

1/10

–

–

0/10

0/10

–

–

0/10

104-week terminal plus unscheduled deaths

Hepatocellular hypertrophy

0/70

1/70

2/70

38/70

7/70

6/70

10/70

34/70

Hepatocellular adenoma

1/70

1/70

1/70

1/70

1/70

0/70

3/70

7/70

Hepatocellular carcinoma

0/70

2/70

3/70

1/70

0/70

0/70

0/70

0/70

Follicular-cell hypertrophy, adenoma and carcinoma in the thyroid gland

53-week interim

Follicular-cell hypertrophy

1/10

0/10

2/10

2/10

0/10

0/10

1/10

1/10

57-week recovery

Follicular-cell hypertrophy

0/10

–

–

0/10

0/10

–

–

0/10

104-week terminal plus unscheduled deaths

Follicular-cell hypertrophy

2/70

1/70

1/70

9/70

3/70

4/70

2/70

33/70

Follicular-cell adenoma

0/70

2/70

0/70

9/70

1/70

0/70

0/70

1/70

Follicular-cell carcinoma

0/70

0/70

1/70

0/70

0/70

0/70

0/70

0/70

From Hardisty (1996b)
The mechanism by which the bladder tumours were induced was not investigated, but Cohen (1995) reviewed all the relevant data on the urinary bladder tumours found in this study and concluded that carbaryl causes urinary bladder tumours in rats by a non-genotoxic mechanism. Hyperplastic and neoplastic lesions in the urinary bladders of males and females were induced only at the highest dietary concentration of 7500 ppm, which was far in excess of the MTD. He concluded that carbaryl induces tumours by increasing cell proliferation, probably by a direct mitogenic effect of the parent compound or one or more of its metabolites. He also concluded that the effect may be specific to rats. A similar mitogenic effect on rat bladder urothelium of another aromatic carbamate, propoxur, has been studied extensively (Cohen et al., 1994). Cohen (1995) proposed that systemic toxicity could alter the metabolism and excretion of growth factors involved in bladder proliferation, such as epidermal growth factor or interleukin-6.
Studies were therefore conducted with PCNA staining to determine whether cellular proliferation occurred in those organs in which tumours had developed in both rats and mice, i.e. in the liver of female rats, the thyroid gland of male rats and the urinary bladder of male rats, and in the liver and kidney of male and female mice. PCNA staining can be used to identify cells that are actively dividing even though clear increases in mitosis are not seen by conventional histology. The samples preserved at the 52-week interim kill of control animals and those at the high dose were examined. Statements of adherence to GLP and QA were included.
In rats, a significant increase in the number of PCNA-positive urothelial cells was seen in the urinary bladder of males (3.3 ± 5.0 compared with 0.33 ± 0.71 in controls). No or only a slight increase in the number of cycling cells was observed in the thyroid glands of males (0.56 ± 1.7 compared with 0 in controls) and in the livers of females (1.0 ± 2.2 compared with 0.60 ± 1.6).
In mice, a trend towards a minimal-to-slight increase in the number of PCNA-positive cortical tubule cells was seen in the kidneys of males and females after 52 weeks at the highest dietary concentration: treated males, 3.9 ± 2.2; control males, 1.2 ± 1.8; treated females, 2.2 ± 1.7; control females, 1.3 ± 0.80. The toxicological or biological significance of this phenomenon is uncertain. No increase in the number of PCNA-positive cells was observed in the livers of male and female mice (treated males, 6.2 ± 4.6; control males, 12 ± 6.0; treated females, 8.3 ± 3.8; control females, 4.6 ± 7.7) (Irisarri, 1996; Debruyne, 1998).
(c) Reproductive toxicity
(i) Multigeneration studies
Rats
In a two-generation (one litter per generation) study of reproductive toxicity, groups of 30 Sprague-Dawley CD rats of each sex were fed diets containing carbaryl (purity, 99.1%) at a concentration of 0, 75, 300 or 1500 ppm, equal to 0, 4.7, 24 and 93 mg/kg bw per day for males and 0, 4.8, 21 and 96 mg/kg bw per day for females, for both generations. At least 70 days after the beginning of treatment, the F₀ parents were mated in a 1:1 ratio for 2 weeks, to produce the F₁ litters. The dams were allowed to rear their young to day 21 post partum. The F₁ and F₂ litters were culled, if necessary, to a total of five male and five female pups on day 4. After weaning, the F₀ parents were killed, and groups of 30 male and 30 female weanlings per dose were used as the F₁ parents and were given carbaryl by the regimen described above, to produce the F₂ litters. The F₁ adults and the F₂ weanlings were killed at the end of the study. All animals, including pups, were observed daily. The food consumption of the F₀ and F₁ parents was determined weekly before mating and during gestation and lactation. Body weights were determined weekly, except that the body weights of the females were determined on days 0, 7, 15 and 20 of gestation, on the day of parturition, and on days 4, 7, 15 and 21 after delivery. All parental animals were examined after death, both grossly and histologically. Antibodies against respiratory syncytial and sialodacryoadenitis virus were found at necropsy in five F₁ parental animals. The infection had probably occurred near the start of the pre-breeding period and had resolved a number of weeks before mating, although no clinical signs of sialodacryoadenitis were observed. The authors considered that this viral infection had no significant effect on the results. Cholinesterase activity was not measured. Statements of adherence to GLP and QA were included.
No treatment-related deaths were observed. Signs of systemic toxicity were seen in F₀ and F₁ parents at 300 and 1500 ppm. The effects at 300 ppm included reduced food consumption, reduced body-weight gain in F₁ males before breeding (neither statistically significant), and reduced body weights in F₁ females during lactation. At 1500 ppm, all parental animals had decreased body weights and food consumption. At necropsy, the F₀ females at 300 and 1500 ppm had increased absolute and relative liver weights, and F₁ females had increased relative liver weights. Treatment had no effect on mating, fertility, pregnancy or gestational indices, numbers of implants, total, live or dead pups per litter, or on the per cent postimplantation loss. No treatment-related gross or histological lesions were found in the reproductive organs of either sex in any generation. The body weights per litter of F₁ and F₂ pups were reduced at 1500 ppm during lactation. The survival indices and mortality rates of F₁ and F₂ pups are shown in Table 11.
Table 11. Survival indices and mortality rates during lactation of F₁ and F₂ pups of rats given carbaryl

Observation

Dietary concentration (ppm)

0

75

300

1500

F1 pups

4-day survival index (%)^a

98

99

95

98

Lactation index (%)^b

99

100

98

95

Mortality, days 0–4 (no. of pups/no. of litters)

11/6

7/6

20/9

15/13

Mortality, days 5–21 (no. of pups/no. of litters)

2/2

0/0

5/3

16/6

F2 pups

4-day survival index (%)^a

98

99

92

89

Lactation index (%)^b

99

99

94

90

Mortality, days 0–4 (no. of pups/no. of litters)

12/8

13/9

43/13

55/14

Mortality, days 5–21 (no. of pups/no. of litters)

4/3

3/3

14/3

15/6

From Tyl et al. (2000)
^{a Number of surviving pups at 4 days divided by number of live pups at day 0
^{b Number of surviving pups at day 21 divided by number of live pups at day 4
In F₁ pups, effects on the lactational index and mortality rate were observed at the highest dietary concentration on days 5–21. These effects were not statistically significant. Vaginal opening and preputial separation were delayed by 2.1 and 1.4 days, respectively, in F₁ offspring at 1500 ppm, and these effects were related to lowered body weights. F₂ pups showed dose-related decreases in 4-day survival and lactational index. The trends were statistically significant, but no statistical significance was reached at 300 or 1500 ppm. The mortality rate was increased in a dose-related fashion on days 0–4. The absolute weights of the thymus and spleen were decreased in most weanlings of rats at 1500 ppm, to the same degree as body weights. No treatment-related malformations were observed.
The NOAEL for parental toxicity based on effects on body and liver weights in parental animals was 75 ppm, equal to 4.7 mg/kg per day. The NOAEL for toxicity in the offspring, based on increased F₂ pup mortality, was 75 ppm, equal to 4.7 mg/kg per day (Tyl et al., 2001).
(ii) Developmental toxicity
Rats
In a study of developmental toxicity, 25 pregnant Sprague-Dawley Crl:CD (SD) BR rats received carbaryl (purity, 99%) in a 0.5% solution of methylcellulose 400 by gavage at a dose of 0, 1, 4 or 30 mg/kg bw per day on days 6–20 of gestation. Statements of adherence to GLP and QA were included.
No deaths were observed. Increased salivation was observed at least once in 18/25 dams at 30 mg/kg bw per day within 20 min of treatment, the effect disappearing about 1 h after treatment. This effect was generally observed between days 14 and 20, although one animal showed increased salivation from the first day of treatment. Body-weight gain and food consumption were significantly reduced among dams at 30 mg/kg bw per day. There were no treatment-related effects on pre- or post-implantation loss, number of fetuses per litter or fetal sex ratio. Fetal body weights were significantly reduced at the highest dose. The fetal and litter incidences of incomplete or absent ossification of the seventh cervical centrum, incomplete ossification of the fifth sternebra and non-ossification of the first metacarp were increased at 30 mg/kg bw per day, a maternally toxic dose. There were no treatment-related changes in the incidences of malformations. The NOAEL for maternal toxicity was 4 mg/kg bw per day, on the basis of salivation and reduced body-weight gain and food consumption. The NOAEL for embryo- and fetotoxicity was 4 mg/kg bw per day, on the basis of decreased body weight and delayed ossification (Repetto-Larsay, 1998). The Meeting noted that inhibition of acetylcholinesterase activity in erythrocytes and/or brain was not measured.
Rabbits
Groups of 22 pregnant New Zealand white rabbits received carbaryl (purity, 99%) in a 0.5% an aqueous solution of methylcellulose by gavage at a dose of 0, 5, 50 or 150 mg/kg bw per day on days 6–29 of gestation. On day 25, about 1 h after dosing, blood was collected, and cholinesterase activity was measured in plasma and erythrocytes. Statements of adherence to GLP and QA were included.
One control doe, one doe at 5 mg/kg bw per day and two does at 50 mg/kg bw per day died, but these deaths were considered not to be related to treatment. The body-weight gain of does at the highest dose was reduced. Plasma cholinesterase activity was reduced by 13, 46 and 68% and erythrocyte cholinesterase activity by 6, 19 and 27% at 5, 50 and 150 mg/kg bw per day, respectively, the reductions being statistically significant at the two higher doses. As inhibition of plasma cholinesterase activity reflected more closely the expected inhibition in brain than did erythrocyte acetylcholinesterase activity (see studies of neurotoxicity, below), the 19% inhibition of erythrocyte acetylcholinesterase activity seen at the intermediate dose was considered to be toxicologically relevant. There were no treatment-related effects on pre- or post-implantation loss, the number of fetuses per litter or the fetal sex ratio. Fetal body weights were significantly reduced at 150 mg/kg bw per day. There was no treatment-related change in the incidence of malformations. The NOAEL for maternal toxicity was 5 mg/kg bw per day, on the basis of decreased erythrocyte cholinesterase activity at 50 mg/kg bw per day. The NOAEL for embryo- and fetotoxicity was 50 mg/kg bw per day, on the basis of reduced fetal body weight per litter (Tyl et al., 1999).
(d) Special studies: Neurotoxicity
(i) Single exposure
Rats
Groups of 24 Sprague-Dawley rats of each sex received technical-grade carbaryl (purity, 99.1%) in an aqueous solution containing 0.5% carboxymethylcellulose and 0.1% Tween 80 by gavage at a single dose of 0, 10, 30 or 90 mg/kg bw. Six animals of each sex per dose were killed 1, 8, 24 and 48 h after dosing, and blood samples were retained for analysis. Body weights were measured before dosing and before termination of the study. Gross clinical examinations were performed twice daily, and detailed clinical examinations were performed before dosing and just before the terminal kill. At necropsy, the whole brain, the left hemisphere and the frontal cortex, hippocampus, cerebellum and caudate/putamen of the right hemisphere were weighed. Cholinesterase activity was determined in the left hemisphere, as a measure of whole brain activity, in the four brain structures of the right hemisphere and in blood samples. Statements of adherence to GLP and QA were included.
The body weight of males at 90 mg/kg bw was significantly reduced 24 h after treatment. No deaths occurred. No clinical effects were observed in animals at 10 mg/kg bw. In animals at 30 mg/kg bw, tremors, salivation and fur staining or wetness around the muzzle were observed 1 h after treatment. In animals at 90 mg/kg bw, tremors, salivation, fur staining and wetness around the muzzle, urogenital and periorbital areas, decreased activity and abnormal breathing sounds were observed 1–48 h after treatment. The severity and frequency of the clinical signs were related to dose and decreased with time.
In general, cholinesterase activity was reduced to a similar extent in all the brain regions examined and was reduced in a dose-related fashion 1 h after dosing by about 35, 70 and 80% in the groups at 10, 30 and 90 mg/kg bw, respectively. Complete recovery was observed 8 h after treatment with 10 mg/kg bw, whereas the activity was reduced by about 22 and 30% at 30 and 90 mg/kg bw, respectively. By 24 h after dosing, only the group at 90 mg/kg bw showed a reduction in brain cholinesterase activity (by 30%), and by 48 h after dosing the activity had returned to control values in all treated groups. The cholinesterase activity in whole blood, plasma and erythrocytes followed similar patterns. Erythrocyte acetylcholinesterase activity was reduced by 32% 1 h after dosing at 10 mg/kg bw, by 56% and 33% 1 and 8 h after dosing at 30 mg/kg bw and by 65%, 20% and 33% 1, 8 and 24 h after dosing at 90 mg/kg bw. The reduction in plasma cholinesterase activity after 1 h more closely reflected the findings in brain than did erythrocyte acetylcholinesterase activity, the inhibition in plasma cholinesterase activity at 1 h being 38%, 68% and 86% at 10, 30 and 90 mg/kg bw, respectively. On the basis of the effects on brain and erythrocyte acetylcholinesterase activity, the LOAEL was 10 mg/kg bw. No clinical signs were observed at this dose (Brooks & Broxup, 1995a).
Groups of two male and two female Sprague-Dawley rats received technical-grade carbaryl (purity, 99.1%) in an aqueous solution containing 0.5% carboxymethylcellulose and 0.1% Tween 80 by gavage at a single oral dose of 10, 50, 100, 250, 500 or 1000 mg/kg bw, followed by a 3-day observation period. No control groups were used. Body weights were measured twice before dosing and on days 0, 1 and 3. Gross clinical examinations were performed twice daily before dosing. Detailed physical examinations were performed on day 0 before dosing and 0.5, 1, 2, 4 and 8 h and 1, 2 and 3 days after dosing. Cholinesterase activity was not assessed. Statements of adherence to GLP and QA were included.
One male and both females at 500 mg/kg bw and all animals at 1000 mg/kg bw died within 24 h of treatment. On the day of treatment, all animals at doses > 50 mg/kg bw showed weight loss, moderate to severe salivation and tremors, lachrymation and/or periorbital staining, staining and/or wetness of the muzzle and urogenital area, decreased activity and decreased respiration with abnormal breathing sounds (except animals at 50 mg/kg bw) and a weakened condition (except at 50 and 100 mg/kg bw). No effects were seen at 10 mg/kg bw (Brooks & Broxup, 1995b).
Groups of eight male and eight female Sprague-Dawley rats received technical-grade carbaryl (purity, 99.1%) in an aqueous solution containing 0.5% carboxymethylcellulose and 0.1% Tween 80 by gavage at a single dose of 0, 10, 50 or 125 mg/kg bw. Three animals of each sex per dose were tested in a functional observational battery (FOB) before dosing on day 0 and 0.5, 1, 2, 4, 6, 8 and 24 h after dosing, at which time they were killed. Three animals of each sex per dose were killed 0.5, 1, 2, 4 or 8 h after dosing. Blood samples and brains were taken from all animals for determination of cholinesterase activity. Body weights were measured once before dosing and just before termination of the study. Clinical examinations were performed on all animals before dosing and before termination. Statements of adherence to GLP and QA were included.
No deaths occurred. Animals at 50 and 125 mg/kg bw had decreased body weights 4, 8 and 24 h after treatment. Animals at 50 mg/kg bw showed slight to moderate tremors (0.5–2 h after dosing), slight to severe salivation (0.5–1 h after dosing) and muzzle staining (at all times). In animals at 125 mg/kg bw, slight to severe tremors, slight to severe salivation and muzzle staining (0.5–8 h after dosing) were observed. Piloerection, forepaw staining, urogenital staining, pupil constriction, periorbital staining and discharge were observed occasionally.
The animals at 50 and 125 mg/kg bw groups that were subjected to the FOB showed not only the clinical effects described above but also a dose-dependent deterioration of gait, ranging from slight ataxic gait to severe impairment or incapacity 0.5–4 h after dosing. The respiration of these animals was dose-dependently decreased 0.5–2 h after dosing. Activity and arousal were dose-dependently decreased at 50 and 125 mg/kg bw, the greatest reductions being seen at 0.5–2 h.
Brain cholinesterase activity was decreased in all groups. At 10 mg/kg bw, decreases of 50% and 34% were observed 0.5 and 1 h after dosing, respectively, with almost complete recovery by 2 h after dosing. At 50 mg/kg bw, brain cholinesterase activity was decreased 0.5–8 h after dosing (by 75% 0.5–1 h after dosing and by 36% after 8 h). At 125 mg/kg bw, brain cholinesterase activity was reduced 0.5–24 h after dosing (by 80% 0.5–1 h after dosing and by 34% reduction at 24 h). The timing of the reductions in cholinesterase activity in whole blood, plasma and erythrocytes followed a similar pattern, with maximal reductions 0.5–2 h after dosing. Cholinesterase activity in plasma was reduced to approximately the same extent as that in brain during the first 0.5–4 h after dosing. In erythrocytes, the reduction was slightly smaller, the maximal reductions at 0.5 h being approximately 24%, 40% and 51% at 10, 50 and 125 mg/kg bw, respectively. No consistent differences were found between males and females with regard to the effect of carbaryl on brain or blood cholinesterase activity. On the basis of the effects on brain and erythrocyte cholinesterase activity, the LOAEL was 10 mg/kg bw. No clinical signs were observed at this dose (Brooks & Broxup, 1995c).
Groups of 12 Sprague-Dawley rats of each sex received technical-grade carbaryl (purity, 99.1%) in an aqueous solution containing 0.5% carboxymethylcellulose and 0.1% Tween 80 by gavage at a single dose of 0, 10, 50 or 125 mg/kg bw. Body weights were measured before dosing and during behavioural testing. Food consumption was measured weekly. Gross clinical examinations were performed twice daily on all animals, and detailed clinical examinations were performed on days 1, 8 and 15. All animals were subjected to a FOB test and a motor activity test before dosing, and on days 0 (0.5–1.5 h after dosing), 7 and 14. On day 15, six rats of each sex per group were perfused for neuropathological examination, the remaining six rats being subjected to complete necropsy. Cholinesterase activity was not assessed. Statements of adherence to GLP and QA were included.
No deaths occurred. Males and females at 125 mg/kg bw group showed decreased body-weight gain and food consumption during the first week after treatment. Animals at 125 mg/kg bw showed staining of the muzzle, lower jaw, urogenital region and ventral surface, females having a higher incidence. A few females also had ocular discharge, opaque eyes, periorbital staining, red liquid in the urine and cervical swelling. Staining of the urogenital region and ventral surface was also found in a few females at 50 mg/kg bw. In the FOB, the animals at 50 and 125 mg/kg bw showed dose-dependent increases in the frequency of tremors, gait disturbance, salivation, passivity, acoustic startle response and hind-limb splay, and dose-dependent decreases in motor and rearing activity, arousal, defaecation, urination, extensor thrust, toe and tail pinch response, grip strength of the fore- and hindlimbs and body temperature, 0.5–1.5 h after dosing. Dose-dependent reductions in body temperature were observed in females on days 7 and 14 after treatment, reaching statistical significance in the group given 125 mg/kg bw. The group at 10 mg/kg bw showed no effects in the FOB at any time. No effects on gross appearance or on gross or microscopic neurological appearance were found. The NOAEL was 10 mg/kg bw on the basis of the clinical effects (Brooks et al., 1995).
(ii) Repeated exposure
Rats
Groups of 27 Sprague-Dawley rats of each sex received technical-grade carbaryl (purity, 99.1%) in an aqueous solution containing 0.5% carboxymethylcellulose and 0.1% Tween 80 by gavage at a dose of 0, 1, 10 or 30 mg/kg bw per day for 13 weeks. Body weights and food consumption were measured weekly. Gross clinical examinations were performed twice daily on all animals, and detailed clinical examinations were performed on day 2 or 3, and weekly thereafter, within 0.5–1 h of treatment. An FOB and a motor activity test were administered to 12 animals of each sex per dose before treatment and in weeks 4, 8 and 13 (0.5–1.5 h after dosing). Five animals at each dose were used to determine blood cholinesterase activity before and at the end of the study (week 13). At the end of the study, six rats of each sex per group were perfused for neuropathological examination, the remaining six rats of each sex per group being subjected to complete necropsy. Three subgroups of five animals of each sex per dose were used to determine cholinesterase activity in plasma, erythrocytes and whole blood, in the left hemisphere and in the frontal cortex, hippocampus, cerebellum and caudate/putamen of the right hemisphere of the brain. One subgroup was bled at week 0 and at week 4, a second was bled at week 0 and at week 8 and the third was bled at weeks 4, 8 and 13. Blood samples were collected 1 h after treatment. Before terminal blood collection, the brains were removed. Statements of adherence to GLP and QA were included.
No deaths occurred. Rats at 30 mg/kg bw per day showed decreased body-weight gain during the first week of treatment and a slight reduction in food consumption throughout the study. These animals occasionally showed increased salivation and slight to moderate tremors. In the FOB, animals at 10 and 30 mg/kg bw per day had decreased pupil size and increased frequencies of tremors and (in females) reduced rearing activity. Animals at 30 mg/kg bw per day also showed increased salivation and reduced defaecation. The females in this group had reduced tail and toe pinch response and extensor thrust, and the males showed reduced pinna response. The body temperature was dose-dependently decreased, reaching statistical significance in the group at 30 mg/kg bw per day at all times of assessment and occasionally in females at 10 mg/kg bw per day. At 4 and 8 weeks, locomotor activity was decreased in females (not significant in males) at 30 mg/kg bw per day.
Cholinesterase activity was significantly decreased by more than 20% in erythrocytes, whole blood, plasma and whole brain and individual brain structures in rats at 10 and 30 mg/kg bw per day at 4, 8 and 13 weeks. In rats at 10 mg/kg bw per day, the reductions in activity did not always reach statistical significance. A nonsignificant reduction of 31% in cholinesterase activity in the hippocampus in females at week 13 was the only finding at 1 mg/kg bw per day, and was due mainly to an extremely low value in one female. As the inhibition seen at the lowest single dose of 10 mg/kg bw was reversed rapidly (within 4 h), as would be expected of a carbamate, the inhibition should not increase markedly with duration of dosing. Thus, at the highest dose, very little change was seen after week 4. Therefore, a statistically nonsignificant reduction of more than 20% was considered not to be a biologically relevant effect. No treatment-related effects on gross appearance or gross or microscopic neuropathological appearance were found. The NOAEL was 1 mg/kg bw per day on the basis of the effects in the FOB and the reductions in cholinesterase activity (Robinson & Broxup, 1996).
Groups of 32 mated F₀female Sprague-Dawley rats received technical-grade carbaryl (purity, 99.1%) in an aqueous solution containing 0.5% carboxymethylcellulose and 0.1% Tween 80 by gavage at a dose of 0, 0.1, 1 or 10 mg/kg bw per day from day 6 of gestation to day 10 post partum. Gross clinical examinations of the dams were performed twice daily. Body weights were measured on days 0, 6, 9, 12, 15, 18 and 20 of gestation and on days 0, 4, 7, 11, 13 and 21 post partum. On these days (except day 0 post partum), the animals were subjected to a FOB test 0.5–2 h after dosing. Six animals per dose were bled on day 6 of gestation (before dosing), 1 h after dosing on days 6, 15 and 20, on day 4 post partum and terminally on day 10 post partum for measurement of cholinesterase activity. After the terminal blood sample collection, the brains were weighed and analysed. The time of onset and completion of littering and the females’ behaviour post partum were recorded. At termination, all females underwent complete necropsy.
The numbers of live, malformed and dead F₁ pups were recorded. Live pups were weighed on days 0, 4, 7, 11, 13 and 21 post partum. The litters were culled to eight pups (four males and four females when possible) on day 4 post partum. From days 4–21, the times of tooth eruption and eye opening were recorded, and the pups were subjected to a FOB and motor activity assessment. One pup of each sex per litter was removed on day 11 post partum, and six of each sex per group were selected for neuropathological and neuromorphological evaluation. The total weights of the brain and brain regions were recorded for the unselected pups. The remaining pups (three males and three females per litter) were weaned on day 21 to form the F₁ adult generation. Gross clinical examination of the F₁ adult generation was performed twice daily. Body weights were measured once a week. The days of vaginal opening and preputial separation were recorded. The F₁ adults were tested for motor activity (day 60), startle habituation (days 22 and 60), passive avoidance (days 23–24) and activity in a water maze (days 60–65). At termination, six animals of each sex per group were perfused, and their organs were sampled for histological examination. The brains of an additional six animals of each sex at 0 and 10 mg/kg bw per day were retained for neuromorphometric examination. Statements of adherence to GLP and QA were included.
No treatment-related clinical signs or deaths were observed in the dams. Those at 10 mg/kg bw per day showed a reduction in body weight on days 6–9 of gestation and also increased incidences of pupil size reduction, slight tremors and disturbed gait in the FOB throughout testing (day 6 of gestation to day 10 post partum). Dams at 10 mg/kg bw showed occasionally decreased cholinesterase activity in erythrocytes (by 28%), whole blood (by 35%) and plasma (by 39%, not significant) from day 20 of gestation to day 10 post partum, and in the brain (42% reduction) at termination. No other treatment-related effects were observed in the dams.
A small but statistically significant increase in the number of stillborn pups was observed in the group at 10 mg/kg bw per day, but the cause of death was not established. As the mean number per litter (0.3) was within the range of control groups in other studies (0–0.9 between October 1989 and January 1995), this effect was considered by the study authors to be of no toxicological significance. The Meeting endorsed that view. No treatment-related effects were observed in the F₁ adult generation. A few changes were observed during neuromorphometric analysis, and some reached statistically significance (Table 12 and see below). Generally, the changes were not seen consistently in males and females, and their direction was inconsistent; the Meeting considered that they were not related to treatment. The NOAEL for maternal toxicity was 1 mg/kg bw per day, based on effects on body weight, observations in the FOB and the reduction in cholinesterase activity. The NOAEL for toxicity in offspring was 10 mg/kg bw per day, the highest dose tested (Robinson & Broxup, 1997).
Table 12. Statistically significant morphometric changes (µm) in right and left sub-locations of the brain in rats in a study of developmental neurotoxicity with carbaryl

Morphometric measurement

Males

Females

0

10 mg/kg bw

0

10 mg/kg bw

per day

per day

Pups, day 11

Parietal cortex, right forebrain

1500

1610*

1550

1550

Parietal cortex, left forebrain

1550

1560

1600

1640

Length of right cerebellum

4380

4730

4440

3750*

Length of left cerebellum

3930

4710*

4600

3580**

Adults, day 70

Frontal cortex, right forebrain

1740

1610

1820

1720

Frontal cortex, left forebrain

1820

1640*

1760

1640

Length of right cerebellum

6200

6720*

62201

6280

Length of left cerebellum

6140

6190

6220

6290

Width of right cerebellum

4570

4870

4720

4440

Width of left cerebellum

4600

4890

4560

4470

Additional evaluation^a

Length of right cerebellum

6480

6780

5210

5260

Length of left cerebellum

6640

6750

6000

5670

Width of right cerebellum

5280

4960

4420

5260**

Width of left cerebellum

4970

4770

4220

5480***

From Robinson & Broxup (1997). Data rounded to three significant figures because of the degree of inter-animal variation and experimental error
* p < 0.05; ** p < 0.01; *** p < 0.001 (Dunnett’s test)
^{a As the slides prepared from the left cerebellum of four of six animals at the highest dose were not suitable for measuring the length of the cerebellum, additional histomorphometric evaluation was performed on the left and right cerebelli of the ani
In their review of the final report of the study, the USA’s Environmental Protection Agency indicated that "... the bilateral decrease in the length of the cerebellum accompanied by a non-statistically significant 5% decrease in cerebellar weights in the day 11 females and the bilateral increase in the width of the cerebellum in the day 70 female animals at the highest dose (10 mg/kg bw per day) may possibly be treatment related Further some forebrain measurements may have also been affected." (The Meeting noted that the cerebellar weights of females were decreased [not statistically significantly] on day 11 by 6%, 11% and 5% at the lowest, intermediate and highest doses, respectively.) Therefore, supplemental histomorphometric evaluations were performed.
A preliminary study was undertaken in order to examine the morphological changes that occur normally in the developing rat brain, without any treatment. The forebrain (frontal and parietal cortex, left and right side and thickness of corpus callosum) and cerebellum (lobule base thickness and thickness of internal and external granular layers) of 10- and 13-day old pups were compared by histomorphometry. This study indicated that the changes occurring in the right and left forebrain between day 10 and day 13 were similar in male and female pups, and the cerebellar changes observed were consistent with the expected migration of cells from the external towards the internal layer at that time. Additionally, the study showed that morphometric changes in the developing brain of rat pups occur bilaterally in the frontal and parietal cortex and in the cerebellum (Hamelin & Yipchuck, 2001).
In the main study, supplemental histomorphometric evaluations were performed on the existing slides of the cerebelli of six pups and six adults of each sex per dose in the control and highest-dose groups. Further, the bilateral measurements of the parietal cortex in the forebrains of male pups and the frontal cortex in the forebrains of male adults were re-evaluated. The data were presented as the means for the two sides. No statistically significant difference was found between treated and control animals in any of the measures in the cerebellum or in the mean measures in the neocortex. The Meeting concluded that the new morphometric results, which revealed no changes at the highest dose, are in agreement with a lack of treatment-related effects on terminal body weight, brain and cerebellar weights, the results of behavioural and motor activity tests and gross and histopathological appearance of male and female pups and adults (Robinson & Broxup, 2001a,b).
3. Observations in humans
Pastides (1993; evaluated in Annex 1, reference 79) reported the results of an epidemiological study of employees who had worked in a carbaryl unit and had been hired between the start-up of the unit in 1960 and 1978. Workers in the manufacturing unit, those in maintenance and those in packaging and distribution could have been exposed to carbaryl. Information on vital status and cause of death obtained in 1988 was used. Data on a total of 448 employees representing 7532 person–years were included. Data on employees specifically exposed to carbaryl were not analysed separately. The 25 deaths identified resulted in elevated standardized mortality ratios (SMRs) for cancer of the pancreas, unspecified cancer and cancer of the brain and other parts of the nervous system. The SMRs for cancer of the pancreas and for unspecified cancer showed only slight excesses and were based on only one case each. The SMR for cancer of the brain and other parts of the nervous system was based on only two observed cases with tumours of different histological origins, reducing the probability that the two malignancies were caused by the same exposure. For all categories, the very wide confidence intervals indicated the low precision of the SMR estimates.
In a subsequent phase of this study, the analysis was based on the vital status of employees through 1994. The cohort consisted of 765 men who had been employed between 1960 and 1994 (including employees from the previous study who were hired before 1978 and additional employees who were hired after 1978), representing 12 580 person–years. A subcohort from which all men who had been employed for < 1 year were eliminated was also evaluated, resulting in a group of 599 men with 9634 person–years. The SMRs were calculated in relation to the rates for all white males in the USA and to all white males in West Virginia, the production site. Fewer deaths from all causes and from all malignancies than expected were found for both the large cohort and the subcohort. Some of the SMRs slightly exceeded 100 but were well within the 95% confidence interval. This was also the case for SMRs for malignant neoplasms of the brain and other nervous system cancers (Pastides & Zorn, 1997).
Comments
To elucidate the role of metabolism in the formation of tumours in the long-term studies in mice and rats, metabolite profiles were determined in mice and 15-month-old rats. The kinetics and metabolism of carbaryl in mice and rats were found to be comparable in studies evaluated by the 1996 JMPR. Some evidence was obtained that mice formed more metabolites via epoxidation and conjugation at a high, probably toxic, dietary concentration of carbaryl (about 8000 ppm). In the study in 15-month-old rats, no convincing evidence was found for a shift in the urinary metabolite pattern at the high (toxic) dietary concentration of 7500 ppm.
The 1996 Meeting could not identify a NOAEL for vascular tumours (haemangiomas and haemangiosarcomas) in male mice. The highest dose in this 2-year study also increased the incidence of renal tubule-cell adenomas and carcinomas in males and the incidences of vascular tumours and of hepatocellular adenomas and carcinomas in females. Re-evaluation of the histological slides from the 2-year study in mice confirmed the original findings of the study pathologists, namely, increased incidences of vascular tumours in males at all doses. Although the incidence of vascular tumours found at the lowest dietary concentration of 100 ppm, equal to 15 mg/kg bw per day, in this study was within the range of incidences in some control groups in 104-week studies performed in other laboratories, it was not possible to exclude the possibility that the incidences were outside the range of values for control groups in similar studies performed in the same laboratory, in the absence of data on such studies.
In 1996, the Meeting concluded that carbaryl is not genotoxic. Some support for a non-genotoxic mechanism of vascular tumour formation by carbaryl in mice was found in a newly available 6-month study in the p53 knockout mouse model, in which tumours are induced readily by genotoxic compounds. In this model, carbaryl did not induce vascular tumours, whereas the genotoxic carcinogen urethane, used as a positive control in the validation study, did.
The Meeting concluded that the increased incidence of vascular tumours was likely to be species- and sex-specific, but, in view of the rarity and malignancy of these tumours, they could not be discounted in human risk assessment.
In 1996, the Meeting concluded that carbaryl is carcinogenic in rats only at doses that exceed the maximal tolerated dose. Increased incidences of tumours were observed in the thyroid in males, the liver in females and the urinary bladder in males and females at the highest dietary concentration of 7500 ppm. Re-evaluation of the histological slides from the 2-year study in rats confirmed the original findings of the study pathologist that increased incidences of tumours in the thyroid (follicular-cell adenomas and carcinomas in males), liver (hepatocellular adenoma in females) and urinary bladder (transitional-cell papillomas and carcinomas in males and females) occurred at the highest dietary concentration of 7500 ppm. The finding of extensive hyperplasia in the urinary bladder without associated necrosis, inflammation or regeneration suggested that the tumorigenic response was due to cell proliferation associated with a mitogenic effect of carbaryl or one of its metabolites, which appears to be an effect specific to rats. On the basis of this new information, the Meeting reaffirmed the conclusion of the 1996 JMPR.
In 1996, the Meeting recommended that a new two-generation study of reproductive toxicity be carried out in rats, with special attention to the male reproductive tract, as effects on this system were observed in some long-term studies of toxicity in rats at doses given by gavage that were significantly lower than those given in the diet in the studies of reproductive toxicity (which suffered from various shortcomings in study design). In the newly available two-generation study of reproductive toxicity, treatment with carbaryl had effects on the offspring at maternally toxic doses. No effects on reproductive parameters were found in animals of either sex at any dietary concentration up to the highest, equal to 92 mg/kg bw per day. At a concentration of 300 ppm, equal to 21 mg/kg bw per day, effects were seen on the weights of the body and liver of parental animals and on the mortality rate of pups of the F₂ generation. The NOAEL for parents and offspring was 75 ppm, equal to 4.7 mg/kg bw per day. On the basis of this new study, and taking into account that all the previously evaluated studies were of limited significance and validity for evaluating reproductive effects, the Meeting concluded that carbaryl does not impair fertility or reproduction and has no adverse effects on the male or female reproductive system.
In 1996, the Meeting concluded that carbaryl has developmental toxicity, manifested as deaths in utero, reduced fetal weight and malformations, but only at doses that are overtly maternally toxic. The shortcomings of these studies were such that NOAELs for developmental toxicity that could be used for risk assessment could not be identified. In the newly available studies of developmental toxicity in rats and rabbits, treatment with carbaryl caused fetal effects only at maternally toxic doses. In a study in rats, the highest dose of 30 mg/kg bw per day resulted in maternal toxicity, reduced fetal body weights and delayed ossification. The NOAEL for maternal, embryo- and fetotoxicity was 4 mg/kg bw per day. Effects on cholinesterase activity were not determined. In a study in rabbits, doses > 50 mg/kg bw per day were maternally toxic (inhibition of erythrocyte acetylcholinesterase activity). Fetotoxic effects (reduced fetal body weight per litter) were seen at 150 mg/kg bw per day, the highest dose tested. The NOAEL for maternal toxicity was 5 mg/kg bw per day, and that for embryo- and fetotoxicity was 50 mg/kg bw per day. The compound did not induce irreversible structural effects in either rats or rabbits at doses up to 30 and 150 mg/kg bw per day, respectively. On the basis of the new studies and taking into account the limited significance and validity of the previously evaluated studies for evaluating developmental effects, the Meeting concluded that carbaryl is not teratogenic.
In studies of neurotoxicity in rats, a single dose > 30 mg/kg bw or repeated administration of doses > 10 mg/kg bw per day for 13 weeks by gavage resulted in dose-dependent clinical effects, including decreased pupil size, tremors, salivation, reduced body temperature and fur staining. A single oral dose of 500 or 1000 mg/kg bw was lethal. Single oral doses > 10 mg/kg bw (lowest dose tested) induced marked, dose-dependent reductions in cholinesterase activity in plasma, erythrocytes, whole blood and brain, which returned to pretreatment values within 4, 24 and 48 h after doses of 10, 30 and 90 mg/kg bw, respectively. A NOAEL could not be identified in these studies with single doses. The LOAEL was 10 mg/kg bw. In the 13-week study of neurotoxicity, cholinesterase activity was significantly decreased by more than 20% in erythrocytes, whole blood, plasma, whole brain and individual brain structures at doses of 10 and 30 mg/kg bw at weeks 4, 8 and 13 in a dose-related fashion. The NOAEL was 1 mg/kg bw per day.
In a study of developmental neurotoxicity, treatment of pregnant rats from day 6 of gestation to day 10 post partum at a dose of 10 mg/kg bw per day induced clinical effects and reductions in erythrocyte and brain cholinesterase activity. Treatment of the dams had no effect on clinical, developmental or behavioural parameters in offspring up to 4 months of age. The NOAEL for offspring was 10 mg/kg bw per day, the highest dose tested. The NOAEL for neurotoxic effects in the dams was 1 mg/kg bw per day.
Two epidemiological studies of carbaryl production workers employed between 1978 and 1994 showed no increase in the mortality rate from cancer when compared with that of unexposed workers. After re-evaluating a 6-week study in volunteers (which was reported in the evaluations of the 1973 and 1996 JMPR), the Meeting concluded that an increased ratio of amino acid nitrogen to creatinine in urine observed at a dose of 0.13 mg/kg bw per day was an equivocal, inconsistent effect and was not relevant for risk assessment.
Toxicological evaluation
The Meeting concluded that the existing database on carbaryl was adequate to characterize potential hazards to fetuses, infants and children.
The critical effect of carbaryl is inhibition of brain acetylcholinesterase activity. This is a rapidly reversible effect (recovery within 4 h in rats at the lowest single dose of 10 mg/kg bw), which is driven by the peak concentration in plasma rather than by the area under the plasma concentration–time curve (AUC). Therefore, in terms of brain acetylcholinesterase activity, the ADI could be the same value as the acute reference dose (RfD). However, carbaryl was considered to be a non-genotoxic carcinogen in mice, causing vascular tumours in one sex (males) at all doses tested. The Meeting established an ADI of 0-0.008 mg/kg bw on the basis of the LOAEL of 100 ppm, equal to 15 mg/kg bw per day, and a safety factor of 2000, which incorporated an extra safety factor of 20 in view of the occurrence of this rare and malignant type of tumour, for which a no-effect level could not be identified.
Carbaryl is moderately toxic to rats after a single oral dose (LD₅₀ = 220–720 mg/kg bw). In studies with single and repeated oral doses, the critical effect of carbaryl was of an acute nature, i.e. inhibition of acetylcholinesterase activity. A NOAEL could not be identified for this effect in studies with single doses in rats; the lowest effective dose was 10 mg/kg bw, at which no clinical signs were observed. As dogs and rats have been shown to be equally sensitive to cholinesterase inhibition by carbaryl in short-term and long-term studies, the Meeting established an acute RfD of 0.2 mg/kg bw, on the basis of the NOAEL of 125 ppm, equal to 3.8 mg/kg bw per day, in a 5-week study in dogs. A safety factor of 25 was used because the effects were rapidly reversible and were driven by the peak concentration in plasma (see Annex 1, reference 91, Annex 5).
Levels relevant to risk assessment

Species

Study

Effect

NOAEL

LOAEL

Mouse

2-year study of carcinogenicitya

Carcinogenicity

–

100 ppm, equal to 15 mg/kg bw per day

Rat

2-year study of toxicity and carcinogenicitya

Toxicity

250 ppm, equal to 10 mg/kg bw per day

1500 ppm, equal to 60 mg/kg bw per day

Carcinogenicity

1500 ppm, equal to 60 mg/kg bw per day

7500 ppm, equal to 350 mg/kg bw per dayb

13-week study of neurotoxicityc

Neurotoxicity

1 mg/kg bw per day

10 mg/kg bw per day

Multigeneration study of reproductive toxicitya

Parental toxicity

75 ppm, equal to 4.7 mg/kg bw per day

300 ppm, equal to 21 mg/kg bw per day

Offspring toxicity

75 ppm, equal to 4.7 mg/kg bw per day

300 ppm, equal to 21 mg/kg bw per day

Developmental toxicityc

Maternal toxicity

4 mg/kg bw per day

30 mg/kg bw per day

Embryo- and fetotoxicity

4 mg/kg bw per day

30 mg/kg bw per day

Acute neurotoxicityc

Neurotoxicity

–

10 mg/kg bw

Rabbit

Developmental toxicityc

Maternal toxicity

5 mg/kg bw per day

50 mg/kg bw per day

Embryo- and fetotoxicity

50 mg/kg bw per day

150 mg/kg bw per day

Dog

5-week study of toxicitya

Toxicity

125 ppm, equal to 3.8 mg/kg bw per day

400 ppm, equal to 10 mg/kg bw per day

1-year study of toxicitya

Toxicity

125 ppm, equivalent to 3.1 mg/kg bw per day

400 ppm, equal to 10 mg/kg bw per day

a Diet
b Above the MTD
c Gavage
Estimate of acceptable daily intake
0–0.008 mg/kg bw
Estimate of acute reference dose
0.2 mg/kg bw
Studies that would provide information useful for continued evaluation of the compound

Study of carcinogenicity in mice
Studies of the mechanism of formation of vascular tumours in mice¹
Further observations in humans
List of relevant end-points for setting guidance values for dietary and non-dietary exposure

Absorption, distribution, excretion, and metabolism in mammals

Rate and extent of oral absorption

Up to 95% absorption within 24-48 h

Dermal absorption

Slow in rats, 16-34% at low doses, 1.2-4% at high doses within 24 h. In humans, 45% within 8 h after application in acetone

Distribution

Uniformly distributed; highest concentrations of residues in carcass, kidney, and blood

Potential for accumulation

None

Rate and extent of excretion

Rapid and nearly complete within 24 h at low dose, within 48 h at high dose. Mainly via urine (90-95%)

Metabolism in animals

Extensive, with only 2.9% unchanged in urine. Three main metabolic pathways:

(i) arene oxide formation with subsequent hydrolysis to dihydrodihydroxycarbaryl and conjugation via the mercapturic acid

(ii) carbamate hydrolysis to form 1-naphthol

(iii) oxidation of N-methyl moiety (alkyl oxidation)

The metabolites formed via these pathways were conjugated with sulfate or glucuronic acid.

Toxicologically significant compounds

Parent compound

Acute toxicity

Rat, LD₅₀,oral

220-720 mg/kg bw (pretreatment with carbaryl increased LD₅₀); cats most sensitive species

Rat, LD₅₀,dermal

> 2000 mg/kg bw

Rat, LC₅₀, inhalation

No data

Skin irritation

Classification unknown

Eye irritation

Classification unknown

Skin sensitization

Classification unknown

Short-term toxicity

Target / critical effect

Cholinesterase inhibition; effects on liver

Lowest relevant oral NOAEL

3.8 mg/kg bw per day (5 weeks, dogs)

Lowest relevant dermal NOAEL

No data

Lowest relevant inhalation NOAEL

10 mg/m³ (90 days, rats)

Genotoxicity

Weight of evidence suggests no genotoxic concern

Long-term toxicity and carcinogenicity

Target/critical effect

Cholinesterase inhibition; liver, kidney, urinary bladder

Mice: vascular tumours

Rats: thyroid, body weight

Lowest relevant NOAEL

< 15 mg/kg bw per day (2 vyears, mice, LOAEL)

10 mg/kg bw per day (2 years; rats)

Carcinogenicity

Mice: vascular tumours in males at lowest dietary concentration (15 mg/kg bw per day). At maximum tolerated dose: renal tubular cell-adenoma in males and hepatocellular carcinoma and vascular tumours in females

Rats: Thyroid follicular-cell adenoma (males) hepatocellular carcinoma (females), urinary bladder transitional-cell carcinoma (both sexes) at maximum tolerated dose

Reproductive toxicity

Reproduction target / critical effect

Deaths of pups in F₂ generation at parentally toxic doses (cholinesterase activity not examined)

Lowest relevant (reproductive) NOAEL

4.7 mg/kg bw per day

Developmental target / critical effect

Rats: decreased fetal body weight, delayed ossification at maternally toxic dose (cholinesterase activity not examined)

Rabbits: decreased fetal body weight at maternally toxic dose

Lowest relevant developmental NOAEL

4 mg/kg bw per day (rats)

Neurotoxicity / Delayed neurotoxicity

Acute; NOAEL

< 10 mg/kg bw; inhibition of cholinesterase activity (rats, single dose)

3.8 mg/kg bw; inhibition of cholinesterase activity (5 weeks, dogs)

90-day; NOAEL

1 mg/kg bw per day; inhibition of cholinesterase activity (rats)

Delayed neuropathy

Negative

Other toxicological studies; observations in humans

No reliable data

Medical data

Several cases of poisoning dominated by symptoms of inhibition of cholinesterase activity

Summary

Value

Study

Safety factor

ADI

0-0.008 mg/kg bw

Mice, carcinogenicity (LOAEL)

2000

Acute reference dose

0.2 mg/kg bw

Dogs, 5 weeks

25

References
Berthe, P. (1997) 14-day toxicity study in the rat by gavage. Unpublished report No. SA 95515, dated 9 January 1997, from Rhône-Poulenc Agrochimie, Centre de Recherche, Sofia Antipolis, France. Submitted to WHO by Rhône-Poulenc Agrochimie, Lyon, France.
Bigot, D. (1999) Validation on p53 knockout mice to predict rodent carcinogenicity. Unpublished draft report No. SA 97040, dated 21 July 1999, from Rhône-Poulenc Agrochimie, Centre de Recherche, Sofia Antipolis Cedex, France. Submitted to WHO by Rhône-Poulenc Agrochimie, Lyon Cedex, France.
Brooks, W. & Broxup. B. (1995a) An acute study of the time course of cholinesterase inhibition by orally administered carbaryl, technical grade, in the rat. Unpublished report No. 97392 from ClinTrials BioResearch, Senneville, Quebec, Canada. Submitted to WHO by Aventis CropsScience, Lyon Cedex, France.
Brooks, W. & Broxup. B. (1995b) An acute benchmark-dose toxicity study of orally administered carbaryl, technical grade, in rats. Unpublished report No. 97387 from ClinTrials BioResearch, Senneville, Quebec, Canada. Submitted to WHO by Aventis CropsScience, Lyon Cedex, France.
Brooks, W. & Broxup. B. (1995c) A time of peak effects study of a single orally administered dose of carbaryl, technical grade, in the rat. Unpublished report No. 97388 from ClinTrials BioResearch, Senneville, Quebec, Canada. Submitted to WHO by Aventis CropsScience, Lyon Cedex, France.
Brooks, W., Robinson, K. & Broxup. B. (1995) An acute study of the potential effects of a single orally administered dose of carbaryl, technical grade, on behavior and neuromorphology in rats. Unpublished report No. 97389 from ClinTrials BioResearch, Senneville, Quebec, Canada. Submitted to WHO by Aventis CropsScience, Lyon Cedex, France.
Carmichael, N.G., Debruyne, E.L.M. & Bigot-Lasserre, D. (1999) The p53 knockout mouse as a model for chemical carcinogenesis in vascular tissue. Unpublished report. Submitted to WHO by Rhône-Poulenc Agrochimie, Lyon Cedex, France.
Chuzel, F. (1999) Carbaryl. 6-month carcinogenicity study in p53 knockout mice by dietary administration. Unpublished report from Rhône-Poulenc Agro, Centre de Recherche, Sofia Antipolis Cedex, France. Submitted to WHO by Rhône-Poulenc Agro, Lyon Cedex, France.
Cohen, S.M. (1995) Evaluation of the urinary bladder carcinogenicity of carbaryl in rats. Unpublished report from the Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska, USA. Dated 2 November 1995. Submitted to WHO by Rhône-Poulenc Agro, Lyon Cedex, France.
Cohen, S.M., Cano, M., Johnson, L.S., St John, M.K., Asamaoto, M., Garland, E.M., Thyssen, J.H., Sangha, G.K. & Van Goethem, D.L. (1994) Mitogenic effects of propoxur on male rat bladder urothelium. Carcinogenesis, 15, 2593–2597.
Debruyne, E. (1998) Carbaryl. 52-week toxicity study in the CD1 mouse: target organs cell cycling assessment.. Unpublished report No. SA 97529, amended report dated 2 December 1998, from Rhône-Poulenc Agro, Centre de Recherche, Sofia Antipolis Cedex, France. Submitted to WHO by Rhône-Poulenc Agro, Lyon Cedex, France.
Debruyne, E. & Irisarri, E. (1996) Carbaryl technical—Chronic toxicity studies in the rat (HWA study No.656-139) and the mouse (HWA study No. 656-138) evaluation of histological slides Unpublished report, document No. 601389, study No. R&D/CRSA/TOX-HPA-4, dated 19 March 1996, from Rhône-Poulenc Secteur Agro. Submitted to WHO by Rhône-Poulenc Agro, Lyon Cedex, France.
Donehower, L.A. (1999) ILSI/HESI grant progress report, December 1999.
Hamada, N.N. (1991) Subchronic toxicity study in dogs with carbaryl technical. Unpublished report No. HLA 656-152, dated 28 March 1991, from Hazleton Laboratories America Inc., Virginia, USA. Submitted to WHO by Rhône-Poulenc Ag Co., North Carolina, USA.
Hamada, N.N. (1993a) Oncogenicity study with carbaryl technical in CD-1 mice. Unpublished report No. HWA 656-138, dated 20 May 1993, from Hazleton, Washington, Inc. Submitted to WHO by Rhône-Poulenc Ag Co., North Carolina, USA.
Hamada, N.N. (1993b) Combined chronic toxicity and oncogenicity study with carbaryl technical in Sprague Dawley rats. Unpublished report No. HWA 656-139, dated 6 August 1993 from Hazleton, Washington, Inc. Submitted to WHO by Rhône-Poulenc Ag Co., North Carolina, USA.
Hamelin, N. & Yipchuck, G. (2001) Morphometric evaluation of rat brain areas for developmental neuropathology. Unpublished report No. 99579 from ClinTrials BioResearch, Senneville, Quebec, Canada. Submitted to WHO by Aventis CropsScience, Lyon Cedex, France.
Hardisty, J.F. (1996a) Pathology working group review report for the oncogenicity study with carbaryl technical in CD-1® mice. Unpublished report EPL project No. 259-011from Experimental Pathology Laboratories Inc. to Rhône-Poulenc Ag Co.. Submitted to WHO by Rhône-Poulenc Ag Co., North Carolina 27709, USA.
Hardisty, J.F. (1996b) Pathology working group review report for the combined chronic toxicity and oncogenicity study with carbaryl technical in Sprague-Dawley rats. Unpublished report EPL project No. 259-012, dated 16 December 1996, from Experimental Pathology Laboratories Inc. to Rhône-Poulenc Ag Co.,. Submitted to WHO by Rhône-Poulenc Agro, Lyon Cedex, France.
Irisarri, E. (1996) Carbaryl. 52-week toxicity study in the rat and mouse target organs cell cycling assessment. Pathology report (post-mortem). Unpublished report No. SA 95493, dated 18 March 1996, from Rhône-Poulenc Agro, Centre de Recherche, Sofia Antipolis Cedex, France. Submitted to WHO by Rhône-Poulenc Agro, Lyon Cedex, France.
Klonne, D.R. (1995) Carbaryl—Mouse historical control data position paper. Unpublished report from Rhône-Poulenc. Dated November 1995. Submitted to WHO by Rhône-Poulenc Agro, Lyon Cedex, France.
Maita, K., Hirano, M., Haroda, T., Mitsumori, K., Yoshida, A., Takahashi, K., Nakashima, N., Kitazawa, T., Enomoto, A., Inui, K. et al. (1988) Mortality, major causes of moribundity, and spontaneous tumors in CD-1 mice. Toxicol. Pathol., 16, 340–349.
Pastides, H. (1993) Standardized mortality ratio analysis of employees exposed to carbaryl at the Rhône-Poulenc Institute, West Virginia Plant. Unpublished report dated 21 January 1997. Submitted to WHO by Rhône-Poulenc Agro, Lyon Cedex, France.
Pastides, H. & Zorn, M. (1997) An evaluation of the mortality experience of carbaryl unit employees at the Rhône-Poulenc Institute, West Virginia plant. Unpublished report. Project No. 00RP497, dated 18 June 1997, from University of Massachusetts, Department of Biostatistics and Epidemiology, Amherst, to Rhône-Poulenc Ag Co., North Carolina, USA. Submitted to WHO by Rhône-Poulenc Agro, Lyon Cedex, France.
Repetto-Larsay M. (1998) Carbaryl developmental toxicology study in the rat by gavage. Unpublished report No. SA 98070, dated 21 October 1998, from Rhône-Poulenc Agro, Centre de Recherche, Sofia Antipolis Cedex, France. Submitted to WHO by Rhône-Poulenc Agro, Lyon Cedex, France.
Robinson, K. & Broxup. B. (1996) A 13 week study of the potential effects of orally administered, carbaryl technical grade, on behavior, neurochemistry and neuromorphology in rats. Unpublished report No. 97390 from ClinTrials BioResearch, Senneville, Quebec, Canada. Submitted to WHO by Aventis CropsScience, Lyon Cedex, France.
Robinson, K. & Broxup. B. (1997) A developmental neurotoxicity study of orally administered carbaryl, technical grade, in the rat. Unpublished report No. 97391 from ClinTrials BioResearch, Senneville, Quebec, Canada. Submitted to WHO by Aventis CropsScience, Lyon Cedex, France.
Robinson, K. & Broxup. B. (2001a) A developmental neurotoxicity study of orally administered carbaryl, technical grade, in the rat. Unpublished report No. 97391 (Final report amendment No, 1, dated 6 July 2001) from ClinTrials BioResearch, Senneville, Quebec, Canada. Submitted to WHO by Aventis CropsScience, Lyon Cedex, France.
Robinson, K. & Broxup. B. (2001b) A developmental neurotoxicity study of orally administered carbaryl, technical grade, in the rat. Unpublished report No. 97391 (Final report amendment No. 2, dated 10 July 2001) from ClinTrials BioResearch, Senneville, Quebec, Canada. Submitted to WHO by Aventis CropsScience, Lyon Cedex, France.
Totis, M. (1997) Investigation of the metabolism of [¹⁴C]-carbaryl in the 15 month old rat following dietary administration. Unpublished report No. SA 95288, amended 3 October 1997, from Rhône-Poulenc Agrochimie. Submitted to WHO by Rhône-Poulenc Ag Co., North Carolina, USA.
Tyl R.W., Marr M.C. & Myers C.B. (1999) Developmental toxicity evaluation (with cholinesterase assessment) of carbaryl administered by gavage to New Zealand white rabbits. Unpublished report No. 65C-7297-200/100, dated 3 June 1999, from Research Triangle Institute, Research Triangle Park, North Carolina, USA. Submitted to WHO by Aventis CropsScience, Lyon Cedex, France.
Tyl R.W., Myers C.B. & Marr M.C. (2001) Two-generation reproductive toxicity evaluation of carbaryl (RPA007744) administered in the feed to CD® (Sprague-Dawley) rats. Unpublished report No. 65C-07407-400, dated 24 May 2001, from Research Triangle Institute, Research Triangle Park, North Carolina, USA. Submitted to WHO by Aventis CropsScience, Lyon Cedex, France.
Vallès, B. (1999) Carbaryl: Investigation of the metabolism of [¹⁴C]-carbaryl following 14 days administration to the male CD1 mouse. Unpublished report study No. SA 97481, dated 16 June 1999, from Rhône-Poulenc Agro. Submitted to WHO by Rhône-Poulenc Ag Co., North Carolina, USA.

ENDNOTES
¹ Before such a study is begun, it is recommended that the mechanisms of tumour formation be evaluated within the ‘conceptual framework for cancer risk assessment’ (developed by an IPCS working group).

See Also:
Toxicological Abbreviations
Carbaryl (EHC 153, 1994)
Carbaryl (HSG 78, 1993)
Carbaryl (ICSC)
Carbaryl (PIM 147)
Carbaryl (FAO Meeting Report PL/1965/10/1)
Carbaryl (FAO/PL:CP/15)
Carbaryl (FAO/PL:1967/M/11/1)
Carbaryl (FAO/PL:1968/M/9/1)
Carbaryl (FAO/PL:1969/M/17/1)
Carbaryl (AGP:1970/M/12/1)
Carbaryl (WHO Pesticide Residues Series 3)
Carbaryl (WHO Pesticide Residues Series 5)
Carbaryl (Pesticide residues in food: 1976 evaluations)
Carbaryl (Pesticide residues in food: 1977 evaluations)
Carbaryl (Pesticide residues in food: 1979 evaluations)
Carbaryl (Pesticide residues in food: 1984 evaluations)
Carbaryl (Pesticide residues in food: 1996 evaluations Part II Toxicological)
Carbaryl (IARC Summary & Evaluation, Volume 12, 1976)}}}}}

Sample	Time after dosing (h)
Sample	6	24	48	72	96	120	144	168
Urine	27	50	63	67	68	69	69	69
Faeces	0.3	0.3	4.7	8.0	10	11	11	11
Cage wash								17
Tissues								0.4
Skin and fur								4.3

Sample	Dose (mg/kg of diet)
Sample	0	250	1500	7500
Urine + cage wash	92	92	93	85
Faeces	10	7.4	9.9	10
Tissues	0.4	0.4	0.4	0.8

Metabolite	Dose (mg/kg of diet)
Metabolite	0	11	110	1100	8000
Dihydro, dihydroxynaphthyl sulfate	3.2	3.0	3.7	3.8	6.8
Hydroxy-carbaryl glucuronide	14	12	15	14	19
alpha-Naphthyl sulfate	14	11	11	12.	12
alpha-Naphthyl beta-D-glucuronide	17	11	13	20	20

Metabolite	Dose (mg/kg of diet)	Day after treatment
Metabolite	Dose (mg/kg of diet)	1	2	3	4	5	6	7
Glucuronide of dihydro, dihydroxy-1-naphthyl-N-methylcarbamate	0	15	19	16	18	16	17	18
250	16	16	15	15	20	23	23
1500	21	20	19	21	26	27	19
7500	28	20	17	19	21	18	10
alpha-Naphthyl beta-D-glucuronide	0	16	15	15	14	13	15	15
250	16	15	15	15	22	22	17
1500	14	11	19	16	14	12	10
7500	15	12	13	15	17	15	12
Sulfonate conjugate of naphthol	0	24	23	26	22	19	24	25
250	27	23	21	9	ND	ND	15
1500	23	25	30	30	30	27	25
7500	12	18	13	ND	ND	ND	9

Effect	Dose (mg/kg of diet)
Effect	0	250	1500	7500
Mortality rate	No toxicologically relevant effect
Body weight (g)	78	820	690^a	650^a
Food consumption	No toxicologically relevant effect
Dermal reactions	No toxicologically relevant effect
Biochemistry in liver tissue
Protein (mg/g liver)	100	120	77^a	89
Glutathione (µmol/g liver)	4.7	5.8	3.6^a	8.3^b
Glutathione (µmol/g protein)	47	50	48	94^b
GST (µmol/min per g liver)	72	64	49^a	71
GST (µmol/min per g protein)	720	540^a	640	810
Glutathione peroxidase (IU/g liver)	220	220	240	160
Glutathione peroxidase (IU/g protein)	2100	1900	3100^a	1900
Organ weight (absolute, in g)
Liver	18	18	17	22^a
Spleen	1.2	1.1	1.4	1.6
Thyroid	0.031	0.041	0.052^a	0.059^a
Histopathology^c
Liver
Centrilobular hypertrophy	0	0	0	5
Pericholangitis	0	0	0	3
Thyroid
Follicular-cell hypertrophy	0	3	5	5
Kidney
Transitional-cell hyperplasia	0	0	0	2

Vascular tumours	Males				Females
Vascular tumours	0	100	1000	8000	0	100	1000	8000
Total no. of tumours	2/70	9/70	13/70	18/70	5/70	6/70	5/70	10/70
No. of tumour-bearing animals	2/70	6/70	10/70	10/70	3/70	3/70	4/70	9/70

Tissue and change	Dose (mg/kg bw per day)
Tissue and change	0	1	10	100^a
Liver
Endothelial hyperplasia	0/20	0/20	0/20	2/20
Haemangioma, benign	0/20	0/20	1/20	14/20
Haemangiosarcoma, malignant	0/20	0/20	0/20	8/20
Combined benign and malignant neoplasms	0/20	0/20	0/20	4/20
Combined benign and/or malignant neoplasms	0/20	0/20	1/20	18/20
Spleen
Malignant haemangiosarcoma	0/20	0/20	0/20	1/20
Heart
Endothelial (haemangioma-like) proliferation	0/20	0/0	0/20	1/20
Haemangioma, benign	0/20	0/0	0/20	1/20
Abdomonal cavity
Haemangiosarcoma, malignant	0/0	0/0	0/0	1/1

Time of kill	Males				Females
Time of kill	0	250	1500	7500	0	250	1500	7500
Hyperplasia, papilloma and carcinoma in the urinary bladder
53-week interim
Transitional-cell hyperplasia	0/9	0/10	0/10	7/9	1/10	0/10	0/10	9/10
57-week recovery
Transitional-cell hyperplasia	0/10	–	–	4/10	0/10	–	–	3/10
104-week terminal kill plus unscheduled deaths
Transitional-cell hyperplasia	8/71	7/70	11/70	51/71	6/70	4/70	4/70	56/70
Transitional-cell papilloma	0/71	0/70	0/70	14/71	1/70	0/70	0/70	8/70
Squamous-cell papilloma	0/71	0/70	0/70	2/71	0/70	0/70	0/70	0/70
Transitional-cell carcinoma	0/71	0/70	0/70	10/71	0/70	0/70	0/70	5/70
Epithelial hyperplasia and carcinoma in the renal pelvis
53-week interim
Pelvic epithelial hyperplasia	1/10	0/10	1/10	7/10	0/10	1/10	2/10	1/10
57-week recovery
Pelvic epithelial hyperplasia	2/10	–	–	0/10	1/10	–	–	0/10
104-week terminal plus unscheduled deaths
Pelvic epithelial hyperplasia	13/70	10/70	13/70	29/70	22/70	39/70	29/70	21/70
Transitional-cell carcinoma	0/70	0/70	0/70	1/70	0/70	0/10	0/70	0/70
Hepatocellular hypertrophy and neoplasms in the liver
53-week interim
Hepatocellular hypertrophy	0/10	0/10	0/10	6/10	0/10	0/10	0/10	7/10
57-week recovery
Hepatocellular hypertrophy	1/10	–	–	0/10	0/10	–	–	0/10
104-week terminal plus unscheduled deaths
Hepatocellular hypertrophy	0/70	1/70	2/70	38/70	7/70	6/70	10/70	34/70
Hepatocellular adenoma	1/70	1/70	1/70	1/70	1/70	0/70	3/70	7/70
Hepatocellular carcinoma	0/70	2/70	3/70	1/70	0/70	0/70	0/70	0/70
Follicular-cell hypertrophy, adenoma and carcinoma in the thyroid gland
53-week interim
Follicular-cell hypertrophy	1/10	0/10	2/10	2/10	0/10	0/10	1/10	1/10
57-week recovery
Follicular-cell hypertrophy	0/10	–	–	0/10	0/10	–	–	0/10
104-week terminal plus unscheduled deaths
Follicular-cell hypertrophy	2/70	1/70	1/70	9/70	3/70	4/70	2/70	33/70
Follicular-cell adenoma	0/70	2/70	0/70	9/70	1/70	0/70	0/70	1/70
Follicular-cell carcinoma	0/70	0/70	1/70	0/70	0/70	0/70	0/70	0/70

Observation	Dietary concentration (ppm)
Observation	0	75	300	1500
F1 pups
4-day survival index (%)^a	98	99	95	98
Lactation index (%)^b	99	100	98	95
Mortality, days 0–4 (no. of pups/no. of litters)	11/6	7/6	20/9	15/13
Mortality, days 5–21 (no. of pups/no. of litters)	2/2	0/0	5/3	16/6
F2 pups
4-day survival index (%)^a	98	99	92	89
Lactation index (%)^b	99	99	94	90
Mortality, days 0–4 (no. of pups/no. of litters)	12/8	13/9	43/13	55/14
Mortality, days 5–21 (no. of pups/no. of litters)	4/3	3/3	14/3	15/6

Morphometric measurement	Males		Females
Morphometric measurement	0	10 mg/kg bw	0	10 mg/kg bw
	per day		per day
Pups, day 11
Parietal cortex, right forebrain	1500	1610*	1550	1550
Parietal cortex, left forebrain	1550	1560	1600	1640
Length of right cerebellum	4380	4730	4440	3750*
Length of left cerebellum	3930	4710*	4600	3580**
Adults, day 70
Frontal cortex, right forebrain	1740	1610	1820	1720
Frontal cortex, left forebrain	1820	1640*	1760	1640
Length of right cerebellum	6200	6720*	62201	6280
Length of left cerebellum	6140	6190	6220	6290
Width of right cerebellum	4570	4870	4720	4440
Width of left cerebellum	4600	4890	4560	4470
Additional evaluation^a
Length of right cerebellum	6480	6780	5210	5260
Length of left cerebellum	6640	6750	6000	5670
Width of right cerebellum	5280	4960	4420	5260**
Width of left cerebellum	4970	4770	4220	5480***

Species	Study	Effect	NOAEL	LOAEL
Mouse	2-year study of carcinogenicitya	Carcinogenicity	–	100 ppm, equal to 15 mg/kg bw per day
Rat	2-year study of toxicity and carcinogenicitya	Toxicity	250 ppm, equal to 10 mg/kg bw per day	1500 ppm, equal to 60 mg/kg bw per day
		Carcinogenicity	1500 ppm, equal to 60 mg/kg bw per day	7500 ppm, equal to 350 mg/kg bw per dayb
	13-week study of neurotoxicityc	Neurotoxicity	1 mg/kg bw per day	10 mg/kg bw per day
	Multigeneration study of reproductive toxicitya	Parental toxicity	75 ppm, equal to 4.7 mg/kg bw per day	300 ppm, equal to 21 mg/kg bw per day
		Offspring toxicity	75 ppm, equal to 4.7 mg/kg bw per day	300 ppm, equal to 21 mg/kg bw per day
	Developmental toxicityc	Maternal toxicity	4 mg/kg bw per day	30 mg/kg bw per day
		Embryo- and fetotoxicity	4 mg/kg bw per day	30 mg/kg bw per day
	Acute neurotoxicityc	Neurotoxicity	–	10 mg/kg bw
Rabbit	Developmental toxicityc	Maternal toxicity	5 mg/kg bw per day	50 mg/kg bw per day
		Embryo- and fetotoxicity	50 mg/kg bw per day	150 mg/kg bw per day
Dog	5-week study of toxicitya	Toxicity	125 ppm, equal to 3.8 mg/kg bw per day	400 ppm, equal to 10 mg/kg bw per day
	1-year study of toxicitya	Toxicity	125 ppm, equivalent to 3.1 mg/kg bw per day	400 ppm, equal to 10 mg/kg bw per day

Absorption, distribution, excretion, and metabolism in mammals
Rate and extent of oral absorption	Up to 95% absorption within 24-48 h
Dermal absorption	Slow in rats, 16-34% at low doses, 1.2-4% at high doses within 24 h. In humans, 45% within 8 h after application in acetone
Distribution	Uniformly distributed; highest concentrations of residues in carcass, kidney, and blood
Potential for accumulation	None
Rate and extent of excretion	Rapid and nearly complete within 24 h at low dose, within 48 h at high dose. Mainly via urine (90-95%)
Metabolism in animals	Extensive, with only 2.9% unchanged in urine. Three main metabolic pathways:
	(i) arene oxide formation with subsequent hydrolysis to dihydrodihydroxycarbaryl and conjugation via the mercapturic acid
	(ii) carbamate hydrolysis to form 1-naphthol
	(iii) oxidation of N-methyl moiety (alkyl oxidation)
	The metabolites formed via these pathways were conjugated with sulfate or glucuronic acid.
Toxicologically significant compounds	Parent compound
Acute toxicity
Rat, LD₅₀,oral	220-720 mg/kg bw (pretreatment with carbaryl increased LD₅₀); cats most sensitive species
Rat, LD₅₀,dermal	> 2000 mg/kg bw
Rat, LC₅₀, inhalation	No data
Skin irritation	Classification unknown
Eye irritation	Classification unknown
Skin sensitization	Classification unknown
Short-term toxicity
Target / critical effect	Cholinesterase inhibition; effects on liver
Lowest relevant oral NOAEL	3.8 mg/kg bw per day (5 weeks, dogs)
Lowest relevant dermal NOAEL	No data
Lowest relevant inhalation NOAEL	10 mg/m³ (90 days, rats)
Genotoxicity	Weight of evidence suggests no genotoxic concern
Long-term toxicity and carcinogenicity
Target/critical effect	Cholinesterase inhibition; liver, kidney, urinary bladder
	Mice: vascular tumours
	Rats: thyroid, body weight
Lowest relevant NOAEL	< 15 mg/kg bw per day (2 vyears, mice, LOAEL)
	10 mg/kg bw per day (2 years; rats)
Carcinogenicity	Mice: vascular tumours in males at lowest dietary concentration (15 mg/kg bw per day). At maximum tolerated dose: renal tubular cell-adenoma in males and hepatocellular carcinoma and vascular tumours in females
	Rats: Thyroid follicular-cell adenoma (males) hepatocellular carcinoma (females), urinary bladder transitional-cell carcinoma (both sexes) at maximum tolerated dose
Reproductive toxicity
Reproduction target / critical effect	Deaths of pups in F₂ generation at parentally toxic doses (cholinesterase activity not examined)
Lowest relevant (reproductive) NOAEL	4.7 mg/kg bw per day
Developmental target / critical effect	Rats: decreased fetal body weight, delayed ossification at maternally toxic dose (cholinesterase activity not examined)
	Rabbits: decreased fetal body weight at maternally toxic dose
Lowest relevant developmental NOAEL	4 mg/kg bw per day (rats)
Neurotoxicity / Delayed neurotoxicity
Acute; NOAEL	< 10 mg/kg bw; inhibition of cholinesterase activity (rats, single dose)
	3.8 mg/kg bw; inhibition of cholinesterase activity (5 weeks, dogs)
90-day; NOAEL	1 mg/kg bw per day; inhibition of cholinesterase activity (rats)
Delayed neuropathy	Negative
Other toxicological studies; observations in humans	No reliable data
Medical data	Several cases of poisoning dominated by symptoms of inhibition of cholinesterase activity

Summary	Value	Study	Safety factor
ADI	0-0.008 mg/kg bw	Mice, carcinogenicity (LOAEL)	2000
Acute reference dose	0.2 mg/kg bw	Dogs, 5 weeks	25