α-n-butyl-α-(4-chlorophenyl)-1H-1,2,4-triazole-1-propanenitrile

Endpoint summary

Description of key information

Two dietary studies with exposure of male and female rats for 13 weeks are available. Both studies are well-documented and include parameters comparable to guideline 408. NOAEL = 18.8 mg/kg bw/day

A dietary study with exposure of male and female dogs for 1 year is available. The study is well-documented and include parameters comparable to guideline 452 and 409. NOAEL = 3.09 mg/kg bw/day

Liver was found to be the main target organ in both species.

 

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05/31/84 to 06/06/85

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.4100 (Chronic Toxicity)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: approximately 4-month old.
- Housing: The dogs were housed individually in stainless steel cages.
- Diet: 300 grams of standard canine diet daily. Feed was offered to the animals for a two hour period each day ending no later than noon. Fresh diets were prepared weekly. Uneaten or unused diets were discarded as hazardous waste.
- Water: Filtered tap water via an automatic watering system, ad libitum.
- Acclimation period: six weeks.

ENVIRONMENTAL CONDITIONS
- Temperature: 66 °F (19 °C).
- Photoperiod: The room had an automatically controlled light/dark cycle of 12 hours.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

Thirty male and 30 female dogs were randomly assigned (computerized randomization based upon body weight) to five groups such that there were no significant weight differences among groups of the same sex.
Animals in Group 1 were fed control diet. Animals in Groups 2 through 5 were fed diets containing Myclobutanil at concentrations of 10, 100, 400, and 1600 ppm of Myclobutanil, respectively.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The first time diets were prepared, samples from the top, middle, and bottom of each dietary concentration were collected and submitted for analysis of active ingredient to determine uniformity of mixing and determine nominal concentration. At the end of each subsequent week, a sample of each dietary concentration was submitted for analysis. Select samples were analyzed for active ingredient content to verify the stability of Myclobutanil in the diet.
The quantity of feed consumed by each dog was determined daily, beginning two weeks prior to compound administration. The mean feed consumption for each group was calculated weekly and expressed as g/animal/day.

Duration of treatment / exposure:

One year

Frequency of treatment:

daily

Dose / conc.:

0 ppm

Remarks:

Control

Dose / conc.:

10 ppm

Remarks:

corresponding to 0.34 mg/kg bw/day and 0.40 mg/kg bw/day in males and females, respectively

Dose / conc.:

100 ppm

Remarks:

corresponding to 3.09 mg/kg bw/day and 3.83 mg/kg bw/day in males and females, respectively

Dose / conc.:

400 ppm

Remarks:

corresponding to 14.28 mg/kg bw/day and 15.68 mg/kg bw/day in males and females, respectively

Dose / conc.:

1 600 ppm

Remarks:

corresponding to 54.22 mg/kg bw/day and 58.20 mg/kg bw/day in males and females, respectively

No. of animals per sex per dose:

6 dogs/sex/group

Control animals:

yes, concurrent no treatment

Observations and examinations performed and frequency:

Each dog was observed daily for signs of ill health or reaction to treatment. Physical examinations were performed on all dogs weekly for the first month of the study and once every other week for the remainder of the study.
The weight of each dog was recorded weekly beginning two weeks prior to the initiation of dosing. Group mean body weights were calculated for each of these time periods.
Laboratory Studies was done twice during the pretest period (weeks -2 and -1) and at weeks 13, 25, 39 and 53 of compound administration. Samples of blood were collected from the jugular vein of each dog. The dogs were fasted overnight prior to blood collection.
Freshly voided urine samples were collected on each of two consecutive days from all animals during the pretest period (week -4) and from control and high dose (1600 ppm) male and female dogs after 25 and 51 weeks of compound administration.
Ophthalmoscopic examinations were performed on all dogs during the pre-test period and during weeks 26 and 52 of treatment with Myclobutanil. A Keeler indirect ophthalmoscope was used for the examination.

Sacrifice and pathology:

Following 12 months of dosing with Myclobutanil, all surviving dogs were anesthetized with intravenous sodium pentabarbital, killed by exsanguination, and necropsied. All organs and tissues were examined and gross abnormalities recorded.
Organ weights were obtained on the adrenals, brain, gonads, heart, kidneys, liver, pituitary, thyroid/parathyroid, and spleen of all animals. Relative organ weights were calculated as percentages of body weight x 100 (i.e. organ weight (g) X 10,000 / body weight (g)).
Histopathologic evaluation was performed in tissues and organs from all dogs of the control and high dose groups. Liver, gallbladder and testes were examined from all dogs.

Statistics:

Distributions of all continuous data were inspected for normality and homogeneity of variance across treatment groups.
Transformations were performed where appropriate before using a one-way analysis of variance (ANOVA) to assess the overall treatment effect. If a significant (p

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights of male dogs fed diets containing 1600 ppm myclobutanil were significantly decreased after one week of treatment but comparable to control dogs throughout the remainder of the study. Mean body weights of female dogs at 1600 ppm Myclobutanil were significantly below that of control female dogs during the first five weeks of the study. During the first week, females at 1600 ppm lost weight and they had a reduced weight gain during the second week of the study. All other statistically significant differences between the control and treated groups are spurious occurrences and not related to treatment with Myclobutanil. No treatment related changes were seen in body weights of male and female dogs fed diets containing 10, 100, and 400 ppm Myclobutanil throughout the 12 months of dosing.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Feed consumption of female dogs fed diets containing 1600 ppm Myclobutanil was consistently below that of control female dogs throughout the study. Feed consumption of males at 1600 ppm was decreased in the first week of the study but comparable to control dogs at other time periods. Feed consumption of males and females at 10, 100, and 400 ppm Myclobutanil were comparable to those of the male and female control dogs throughout the 12 months of treatment.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In male dogs fed diets containing 1600 ppm Myclobutanil a slight decrease in the number of RBC and an increase in the number of platelets were observed throughout the one year treatment period.
In female dogs no treatment related changes in hematologic parameters were observed at any of the doses tested.
All other statistically significant differences between the control and treated groups are spurious occurrences and not related to treatment with Myclobutanil.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In male and female dogs fed diets containing 1600 ppm Myclobutanil an increase in inorganic phosphorus and a decrease in serum albumin were observed. Alkaline phosphatase was increased in both sexes at 1600 ppm and in females at 400 ppm. SGPT was increased in males and GGT was increased in females at 1600 ppm.
All changes were consistent throughout the 12 month treatment period. No treatment related changes in clinical chemistry parameters were observed in dogs fed 10 or 100 ppm Myclobutanil for 12 months.

Endocrine findings:

not examined

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Increased absolute and relative liver weights were observed in both sexes at 1600 ppm and in females at 400 ppm Myclobutanil. The statistically significant increase in absolute liver weight of Group 3 (100 ppm) female dogs is attributed to the larger size of the dogs in this group when compared to the control dogs and not to treatment with Myclobutanil. Since liver weight is judged not to be increased in Group 3, the statistically significant increase in relative liver weight in Group 2 (10 ppm) female dogs is judged to be a spurious difference from controls and not related to treatment with Myclobutanil.
No other organ weight changes attributed to treatment with Myclobutanil were seen at doses up to and including 1600 ppm.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Compound related gross changes were observed in the livers of Group 5 (1600 ppm) male and female dogs. These liver changes, consisting of enlargement and/or accentuated lobular architecture, were noted in one male and three female dogs at the high dose.
Other gross changes were scattered among the groups, including the control group, with variable incidences, and were not considered to be compound related. Frequent incidental changes included reddened portions of the intestinal tract, thickened and reddened mammary glands (females only), and distended uteri (females only).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Compound related histopathologic changes occurred only in the livers of Groups 4 (400 ppm) and 5 (1600 ppm) male and female dogs. Minimal to mild hepatocellular hypertrophy was noted in one of six Group 4 (400 ppm) and five of six Group 5 (1600 ppm) males, while mild to moderate hepatocellular hypertrophy was noted in two of six Group 4 (400 ppm) and six of six Group 5 (1600 ppm) females. It was not noted in any animals in Groups 1 (control), 2 (10 ppm), or 3 (100 ppm).
Hepatocellular hypertrophy occurred with a slightly higher incidence and/or greater severity in female dogs when compared to male dogs at the same dose. "Ballooned" hepatocytes were noted sporadically in more severely affected Group 5 (1600 ppm) females, generally in centrilobular areas.
Centrilobular hepatocellular hypertrophy correlated well with gross liver changes of enlarged and accentuated lobular architecture noted in Group 5 (1600 ppm), and with increased absolute and relative liver weights noted in Groups 4 (400 ppm) and 5 (1600 ppm).

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

histopathology: neoplastic

organ weights and organ / body weight ratios

Remarks on result:

other: corresponding to 3.09 mg/kg bw/day and 3.83 mg/kg bw/day in males and females, respectively

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

400 ppm

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

presumably yes

Conclusions:

No observed effect level (NOEL) for dietary administration of Myclobutanil to Beagle dogs was 100 ppm (3.09 mg/kg/day for males and 3.83 mg/kg bw/day for females). Effects seen at 400 ppm (14.28 mg/kg bw/day for males and 15.68 mg/kg bw/day for females) included increased absolute and relative liver weights (females), hepatocellular hypertrophy (both sexes) and increased serum alkaline phosphatase levels (females).

Executive summary:

Myclobutanil was administered in the diet to 5 groups (6 dogs/sex/group) of purebred Beagle dogs for 1 year at dietary concentrations of 0, 10, 100, 400, and 1600 ppm of active ingredient (ai). All dogs were observed daily for signs of ill health or reaction to treatment. Body weights were monitored weekly beginning 2 weeks prior to treatment. Feed consumption was monitored daily beginning 2 weeks prior to treatment. Physical examinations were performed weekly for the first month of treatment and once every other week for the remainder of the study. Twice during the pre-test period and at weeks 13, 25, 39, and 53 all dogs were bled for haematology and clinical chemistry analyses. Urine samples were collected on each of two consecutive days from all animals during the pre-test period (week 4) and from control and high dose (1600 ppm Myclobutanil) male and female dogs after 25 and 51 weeks. Ophthalmology examinations were performed on all dogs during the pre-test period and during weeks 26 and 52. After 12 months of treatment all dogs were killed, necropsied, selected organ weights recorded, and tissues collected for histopathologic evaluation.

No deaths occurred and no treatment related clinical signs were evident. Body weights of male dogs were significantly decreased at 1600 ppm after one week and in female dogs during the first five weeks of treatment. Feed consumption was decreased in females at 1600 ppm throughout the study and in males during the first week only. There was a decrease in RBC and an increase in platelets in male dogs at 1600 ppm. Alkaline phosphatase was increased in both sexes at 1600 ppm and in females at 400 ppm. Both sexes had increased inorganic phosphorus and decreased serum albumin at 1600 ppm. SGPT and GGT were increased in males and females respectively at 1600 ppm. No treatment related changes were seen in the urinary parameters or in the ophthalmology examinations. Increases in absolute and relative liver weights were seen in both sexes at 1600 ppm and in females at 400 ppm. Hepatocellular hypertrophy was seen in both sexes at 400 and 1600 ppm. This change occurred in a centrilobular to panlobular distribution and was of slightly higher incidence and/or greater severity in females. More severely affected female dogs at 1600 ppm displayed "ballooned" hepatocytes, probably representing severely hypertrophied, possibly degenerating, hepatocytes. The hepatocellular hypertrophy correlated well with gross liver changes of enlargement and accentuated lobular architecture (at 1600 ppm) and with increased absolute and relative liver weights (at 400 and 1600 ppm).

The No Observed Effect Level (NOEL) for dietary administration of Myclobutanil to Beagle dogs was 100 ppm (3.09 mg/kg bw/day for males and 3.83 mg/kg bw/day for females). Effects seen at 400 ppm (14.28 mg/kg bw/day for males and 15.68 mg/kg bw/day for females) included increased absolute and relative liver weights (females), hepatocellular hypertrophy (both sexes) and increased serum alkaline phosphatase levels (females).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

3.09 mg/kg bw/day

Study duration:

chronic

Species:

dog

Quality of whole database:

The available information comprises adequate, reliable and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VII, 8.6, of Regulation (EC) No 1907/2006

System:

hepatobiliary

Organ:

liver

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A key subchronic oral repeated dose toxicity study in rats similar to OECD 408 and in compliance with GLP is available. This 13-week feeding study was carried out in Sprague Dawley (SD) rats. The test article was administered at dosages of 100, 300 and 3000 ppm in the diet. The control group received untreated diet (CRF-1) only. The following results were observed:

- No treatment-related change was observed in general signs, ophthalmological and hematological examinations at any doses.

- A depression of the body weight gain was noted in both sexes receiving 3000 ppm during the administration period.

- A decrease in food consumption was observed in males receiving 3000 ppm at week 1.

- A slight increase in round cells in the urinalysis was observed in males receiving 3000 ppm.

- The blood biochemical examination indicated statistically signtficant decreases of glucose and triglyceride in males receiving 3000 ppm and of total bilirubin in both sexes receiving 3000 ppm.

- Increased absolute (male only) and relative liver weight (both sexes) and increased absolute and relative kidney weights (male only) were seen at 3000 ppm. Decreased absolute and relative adrenal weights were seen in males at 3000 ppm.

- Histopathological examination revealed hypertrophy of hepatic cell in both sexes receiving 3000 ppm, vacuolar degeneration of tubular epithelium of the kidney, and vacuolization of cortical cells, atrophy of zona fasciculata and fine vacuolization of zona glomerulosa of the adrenal in males receiving 3000 ppm.

In conclusion, a no-observed-adverse- effect-dose level (NOAEL) of the test item is considered to be 300 ppm (approximately 18.8 and 19.5 mg/kg bw/day in male and female rats, respectively).

A second 90-day dietary study with rats similar to OECD 408 and in compliance with GLP is available. When fed to male and female rats for 3 months, myclobutanil has a no-observed effect level of 100 ppm in the diet (5.22 mg/kg bw/day) in males and 300 ppm in the diet (19.7 mg/kg bw/day) in females. The only effect seen at 300 ppm in males was an increase in hepatic mixed function oxidase activity. At 1000 ppm gross changes in the liver were seen at necropsy and liver weight changes occurred. At 3000 ppm histopathologic changes were seen in the liver, kidneys, adrenals, and thyroids; male body weights were decreased; and serum chemistry changes were evident. At 10,000 ppm decreases in feed consumption, hematologic changes, and histopathologic changes in the spleen and lung were also evident. At 30,000 ppm all the test animals died within the first 9 weeks of treatment.

Effects which are considered toxicologically significant or adverse (based on the magnitude of the changes seen) were seen at doses of 3000 ppm and greater (decreased body weight, gross changes, histopathologic changes, increased liver and kidney weights). The no adverse effect level was 1000 ppm in the diet for both sexes (51.5 mg/kg/day in males and 65.8 mg/kg/day in females).

A chronic toxictiy study in dogs is available which is similar to OECD 452 and 409 and in compliance with GLP. Myclobutanil was administered in the diet to 5 groups (6 dogs/sex/group) of purebred Beagle dogs for 1 year at dietary concentrations of 0, 10, 100, 400, and 1600 ppm of myclobutanil. All dogs were observed daily for signs of ill health or reaction to treatment. Body weights were monitored weekly beginning 2 weeks prior to treatment. Feed consumption was monitored daily beginning 2 weeks prior to treatment. Physical examinations were performed weekly for the first month of treatment and once every other week for the remainder of the study. Twice during the pre-test period and at weeks 13, 25, 39, and 53 all dogs were bled for haematology and clinical chemistry analyses. Urine samples were collected on each of two consecutive days from all animals during the pre-test period (week 4) and from control and high dose (1600 ppm Myclobutanil) male and female dogs after 25 and 51 weeks. Ophthalmology examinations were performed on all dogs during the pre-test period and during weeks 26 and 52. After 12 months of treatment all dogs were killed, necropsied, selected organ weights recorded, and tissues collected for histopathologic evaluation.

No deaths occurred and no treatment related clinical signs were evident. Body weights of male dogs were significantly decreased at 1600 ppm after one week and in female dogs during the first five weeks of treatment. Feed consumption was decreased in females at 1600 ppm throughout the study and in males during the first week only. There was a decrease in RBC and an increase in platelets in male dogs at 1600 ppm. Alkaline phosphatase was increased in both sexes at 1600 ppm and in females at 400 ppm. Both sexes had increased inorganic phosphorus and decreased serum albumin at 1600 ppm. SGPT and GGT were increased in males and females respectively at 1600 ppm. No treatment related changes were seen in the urinary parameters or in the ophthalmology examinations. Increases in absolute and relative liver weights were seen in both sexes at 1600 ppm and in females at 400 ppm. Hepatocellular hypertrophy was seen in both sexes at 400 and 1600 ppm. This change occurred in a centrilobular to panlobular distribution and was of slightly higher incidence and/or greater severity in females. More severely affected female dogs at 1600 ppm displayed "ballooned" hepatocytes, probably representing severely hypertrophied, possibly degenerating, hepatocytes. The hepatocellular hypertrophy correlated well with gross liver changes of enlargement and accentuated lobular architecture (at 1600 ppm) and with increased absolute and relative liver weights (at 400 and 1600 ppm).

The No Observed Effect Level (NOEL) for dietary administration of Myclobutanil to Beagle dogs was 100 ppm (3.09 mg/kg bw/day for males and 3.83 mg/kg bw/day for females). Effects seen at 400 ppm (14.28 mg/kg bw/day for males and 15.68 mg/kg bw/day for females) included increased absolute and relative liver weights (females), hepatocellular hypertrophy (both sexes) and increased serum alkaline phosphatase levels (females).

Based on available data it is concluded that myclobutanil induces liver weight increase associated with hepatocellular hypertrophy in rats and dogs and the liver is a target organ. Based on the observations in dogs it was concluded that classification STOT RE 2 is warranted.  

Justification for classification or non-classification

Based on the available data it is concluded that Myclobutanil meets the classification criteria for STOT-RE in Category 2 according to the Classification, Labelling and Packaging (CLP) Regulation ((EC) No 1272/2008).