Methyl (R)-2-(4-(3-chloro-5-trifluoromethyl-2-pyridyloxy)phenoxy)propionate

Endpoint summary

Description of key information

16 week rat feeding study NOEL: 0.065 mg/kg/day in males and 0.2 mg/kg/day in females; GLP study; Reliability = 2

1 year rat feeding study NOEL: 0.065 mg/kg/day in both males; GLP study; Reliability = 2

21-day rat dermal study NOAEL > 50 mg/kg/day; OECD 410; Reliability = 1

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

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Additional documentation provided in IUCLID assesment reports (Chapter 13) supports the read across approach

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

no guideline followed

Principles of method if other than guideline:

The test substance was fed in the diet to primary or recovery rats (10/sex/group). Primary groups were fed for 16 weeks at dose levels of 0 (control), 0.065, 0.2, or 2 mg/kg bw/day. Recovery groups were fed at 0 or 2 mg/kg bw/day for 16 weeks and untreated feed for a further 4 weeks. Parameters evaluated were general health status; body weights and weight gains; hematology; clinical chemistry; urinalysis (primary group only), including microscopic examination of pooled samples/sex/group sediment; final fasted body weight; absolute and relative-to-body-weight organ weights and gross and histopathologic findings in scheduled tissues and gross lesions from all animals.

GLP compliance:

yes

Specific details on test material used for the study:

XRD-535
Lot # AGR-258981
Purity: 99.4%

Species:

rat

Strain:

Fischer 344

Details on species / strain selection:

Fischer-344 rats were selected because of their general acceptance and suitability for toxicity testing.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, NewYork
- Age at study initiation: 6 weeks
- Fasting period before study: No
- Diet: Feed (Purina Certified Rodent Chow #5002) ad libitum
- Water: Muncipal water ad libitum
- Acclimation period: 14 days

Route of administration:

oral: feed

Details on route of administration:

The probable routes of human exposure to the test material are via ingestion of foodstuffs that might contain low residues of the test material or from accidental ingestion or dermal contact during manufacture or use. Thus, formulation with feed was the desired method of delivery to assess the potential systemic toxicity of the test material following oral administration.

Vehicle:

other: diet

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet: Weekly (based on group mean body weights and feed consumption values so as to maintain the desired dose levels on a mg/kg/day basis)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Results of analyses for homogeneity in samples taken on Study Day 8 of the female 0.2 mg/kg/day diet were not entirely satisfactory. In view of this, analysis for homogeneity was repeated on samples of both male and female 0.2 mg/kg/day diets mixed the next week (Study Day 15); results of this more extensive analysis showed homogeneous distribution of the test material in the feed, and the Study Day 8 results were judged to reflect sampling variability. Other analyses showed that the test substance was stable in the #5002 feed for at least 7 days, the period of time over which the animals were fed. Samples of premix, control feed, and diets mixed on Study Days 8, 64, 99, and 106 were analyzed to verify target concentrations. With one exception, results were accepted as sufficiently comparable to target concentrations for meaningful interpretation of the data. The concentration check on Study Day 106 showed that test material had been omitted from the diets fed to the female 0.065 and 0.2 mg/kg/day groups for the 16th (final) week of the study. However, analysis of retained samples of the female treated diets prepared the previous week (Study Day 99) allowed the conclusion that this inadvertent deviation from the study plan affected only a small portion of the dosing regimen and did not materially interfere with the study objectives.

Duration of treatment / exposure:

16 weeks

Frequency of treatment:

Daily

Dose / conc.:

0.065 mg/kg bw/day (nominal)

Remarks:

Primary group

Dose / conc.:

0.2 mg/kg bw/day (nominal)

Remarks:

Primary group

Dose / conc.:

2 mg/kg bw/day (nominal)

Remarks:

Primary group

Dose / conc.:

2 mg/kg bw/day (nominal)

Remarks:

Recovery group

No. of animals per sex per dose:

Primary group (4 groups): 10 per sex per dose
Recovery group (2 groups): 10 per sex per dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: On the basis of work with related material, administration of the test material at 2 mg/kg/day for 16 weeks was expected to induce unequivocal hepatotoxicity. It was thought likely that the intermediate dose level of 0.2 mg/kg/day would provide evidence of dose response and the lowest dose level, 0.065 mg/kg/day, would induce no toxicologically significant effects.
- Fasting period before blood sampling for clinical biochemistry: Yes
- Post-exposure recovery period in satellite groups: Yes; two additional groups of 10 rats/sex/group (recovery groups) were fed either 0 or 2 mg/kg/day and then kept for a recovery period of four additional weeks, during which no test material was added to the diet.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly once

FOOD CONSUMPTION AND COMPOUND INTAKE :
- Food consumption for each animal determined and mean daily diet consumption calculated: Yes
- Compound intake calculated: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: On receipt of animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: On Study Days 112-113 (primary males and females) and Study Day 140 (recovery males and females)
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- Parameters checked: Packed cell volume, hemoglobin, red and white blood cells, and platelet counts, complete blood smear examinations (differential WBC counts on 100 cells and an assessment of RBC, WBC, and platelet morphology)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On Study Days 112-113 (primary males and females) and Study Day 140 (recovery males and females)
- Animals fasted: Yes
- Parameters checked: Alanine transaminase, aspartate transaminase, alkaline phosphatase, bilirubin, calcium, phosphorus, cholesterol, creatinine, glucose, total protein, albumin, triglycerides, urea nitrogen, chloride, potassium, and sodium.

URINALYSIS: Yes
- Time schedule for collection of urine: Study Day 108 from the primary group males and females.
- Parameters checked: pH, protein, occult blood, glucose, ketones, bilirubin, urobilinogen and urine specific gravity

Sacrifice and pathology:

GROSS PATHOLOGY: All animals were necropsied after overnight fasting. Each animal was examined externally and internally for gross pathologic alterations. At the time of the primary and recovery group necropsies, the final fasted body weight of each rat was recorded, and the weights of the adrenal glands, brain, liver, kidneys, heart, thymus, and testes or ovaries were recorded. Organ weights relative to final fasted body weights were afterwards calculated.

HISTOPATHOLOGY: Yes
Representative sections of all the below tissues, including gross lesions, from the primary group high dose and control animals were examined histopathologically.
Adrenal glands, Aorta, Bone, Bone marrow, Brain, Cecum, Cervix, Coagulating gland, Colon, Duodenum, Epididymis, Esophagus, Eyes, Gross lesions, Heart, Harderian/lacrimal gland, Ileum, Jejunum, Kidneys, Larynx, Liver, Lungs, Manmary gland, Mediastinal lymph node, Mediastinal tissue, Mesenteric lymph node, Mesenteric tissue, Nasal tissue, Oral tissues, Ovaries, Oviduct, Pancreas, Parathyroid gland, Peripheral nerves, (sciatic), Pituitary gland, Prostate, Rectum, Salivary gland, Seminal vesicle, Skeletal muscle, Skin, Spinal cord, Spleen, Stomach, Testes, Thymus, Thyroid gland, Tongue, Trachea, Urinary bladder, Uterus, Vagina. In addition, sections of liver, kidneys, and lungs from the primary group intermediate and low dose animals were examined, and, from the recovery group high dose and control animals, gross lesions, lungs, liver, kidneys, and adrenal glands were examined.

Statistics:

Body weights, organ weights, clinical chemistry data, hematology data, and urinary specific gravity data were evaluated by Bartlett's test for equality of variances. Based on the outcome of Bartlett's test, exploratory data analysis was performed by a parametric or nonparametric analysis of variance (ANOVA), followed respectively by Dunnett's test or the Wilcoxon Rank-Sum test with a Bonferroni correction for multiple comparisons. Outliers were identified by a sequential, statistical test, but routinely excluded only from feed consumption data.

Clinical signs:

no effects observed

Description (incidence and severity):

The daily cageside observations and the weekly clinical observations elicited no positive findings, and the general health status of the animals throughout the study was judged to be unaffected by treatment.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The high dose level females (fed 2 mg/kg bw/day) ate more than controls throughout the study. In these same females, body weight and weight gain values were, for the most part, higher than control values and, in several instances, the differences from control were statistically significant. Body weight gain values of the female low and intermediate dose level groups also tended to be higher than control, but with no concomitant increases in feed consumption. In view of the variability in the data, it could not be concluded that administration of the test material induced increased feed consumption and/or body weight gain.

Food consumption and compound intake (if feeding study):

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The male high dose (2 mg/kg bw/day) level group showed small decreases in packed cell volume, hemoglobin, and red blood cell count after both 16 weeks of treatment and 4 weeks of recovery. These differences were statistically significant and appear to be associated with administration of the test material. It is doubtful, however, that these differences are toxicologically significant. The differences from control were small (7-8% after 16 weeks and 2-3% after recovery) and, clearly, have no adverse consequences to the rats since they are well within the normal ranges for erythron values for the species.
Refer any other information on results incl. tables

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Alkaline phosphatase activity was statistically significantly increased at the highest dose level (2 mg/kg bw/day) in both sexes after 16 weeks of treatment, more in males than in females. The elevation of this serum enzyme is consistent with the liver toxicity noted at this dose level.
Cholesterol was decreased, relative to control, in the primary group males and females fed at 2 mg/kg bw/day and also in the primary group males given 0.2 mg/kg bw/day.
Potassium was statistically significantly increased in the primary group animals, both males and females, fed at 2 mg/kg bw/day. This difference was also rapidly reversed, with no increase seen again in the high dose level animals allowed to recover for 4 weeks.
Refer any other information on results incl. tables

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute and relative liver weight means were increased in both sexes fed at 2 mg/kg bw/day. Relative liver weight increases over control also occurred in primary group males fed 0.2 mg/kg bw/day for 16 weeks and in males (only) of the recovery group. The small size of the liver weight increases and the absence of histopathologic liver lesions in the males after 4 weeks on untreated feed were interpreted as signs of recovery.
Absolute and relative weights of the testes were slightly decreased (5-7%) in both the primary and recovery group males fed at the highest dose level (2 mg/kg bw/day). While these minor testicular weight differences appear to be related to the treatment regimen, it is highly unlikely that they would be associated with any adverse reproductive consequences since the histologic appearance of the testes was completely normal.
Refer any other information on results incl. tables

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No gross lesions attributable to administration were found. The cystic ovarian bursas noted in some recovery group females and the hepatic hernias found in a high dose level group female at 16 weeks and in a high dose level group male at 20 weeks are common, incidental, spontaneous findings in Fischer-344 rats.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Lesions attributable to administration of the test material were found only in the primary group, only in males, and only at the highest dose level (2 mg/kg bw/day). These lesions consisted of an Increase in eosinopnilic staining properties of hepatocytes and a slight Increase in the size of centrilobular hepatocytes.
The hepatic alterations seen in the high dose level males after 16 weeks of treatment were not present in liver sections from recovery group rats of either sex. No histopathologic lesions attributable to treatment were present in the livers of the male rats fed at the low and intermediate dose levels of 0.065 or 0.2 mg/kg bw/day or the female rats fed at any dose level (0.065, 0.2, or 2 mg/kg bw/day).
Incidental lesions, present in liver sections from both male and female primary and recovery group rats, were mononuclear cell foci, microfoci of necrosis often accompanied by inflammatory cells, and bile duct hyperplasia. None of these alterations had a significant impact on the physiologic status of the involved rat liver. Other lesions seen in scattered organs-lacrimal gland mononuclear cell aggregates, suppurative inflammation in the lacrimal gland, colonic nematodiasis, myocardial degeneration, pulmonary alveolar histiocytosis, gastric erosion, and a dermal abscess-were considered to be incidental and spontaneous due primarily to the lack of dose response.

Key result

Dose descriptor:

NOEL

Effect level:

0.065 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOEL

Effect level:

0.2 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

0.065 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Table 1: Haematology data (significant observations)

	Dose (mg/kg/day)	PCV (%) (Mean/SD)	Hgb (g/dL) (Mean/SD)	RBC (10E6/mmE3 blood) (Mean/SD)
Primary group males	0	41.3 0.7	15.1 0.1	8.71 0.09
Primary group males	0.065	41.0 0.8	15.0 0.3	8 62 0.17
Primary group males	0.2	41.0 0.5	14.8 0.3	8.55* 0 11
Primary group males	2.0	38.2* 0.8	14.1* 0.2	8.04* 0.17
Recovery group males	0	40.5 0.7	14.8 0.3	8.46 0.18
Recovery group males	2.0	39.6* 1.0	14.3* 0.5	8.21* 0.17
Primary group females	0	40.0 1.0	14.5 0.4	7.75 0.17
Primary group females	0.065	38.9 0.9	14.1 0.3	7.65 0 19
Primary group females	0.2	39.0 1.0	14.1 0.4	7.66 0.20
Primary group females	2.0	40.0 1.1	14.3 0.3	7.77 0 18
Recovery group females	0	39.2 0.9	14.1 0.4	7.58 0.13
Recovery group females	2.0	39.4 1.0	14.4 0.4	7.67 0.17

*Statistically different from control by Dunnett's test, alpha-0.05, two-sided

Table 2: Clinical chemistry (significant observations)

	Dose  (mg/kg/day)	ALP (U/L) (Mean/SD)	Cholesterol (mg/dL) (Mean/SD)	Potassium (meq/L) (Mean/SD)
Primary group males	0	69.4 6.5	61.1 8.0	4.40 0.19
Primary group males	0.065	68.3 7.1	55.6 4.0	4.34 0.17
Primary group males	0.2	64.9 6.0	50.5^$ 2.0	4.53 0.11
Primary group males	2.0	105.1* 14.4	45.8^$ 4.7	4.68* 0.21
Recovery group males	0	75.5 6.7	77.0 29.1	4.32 0.19
Recovery group males	2.0	70.5 6.5	83.0^$ 6.1	4.39 0.17
Primary group females	0	49.9 5 6	109.3 11.8	4.19 0.34
Primary group females	0.065	50.1 6.4	110.8 17.2	4.24 0.23
Primary group females	0.2	45.3 3.7	116.6 11.7	4.31 0.23
Primary group females	2.0	61.5* 10.4	95.6 13.5	4.53* 0.23
Recovery group females	0	42 9 5.1	142.1 11.4	4.07 0.19
Recovery group females	2.0	46.8 6.6	138.6 10.7	4.09 0.22

*Statistically different from control by Dunnett's test, alpha-0.05, two-sided

$Statistically different from control by Wilcoxon's test, alpha-0.05, two-sided

 

Table 3: Organ weights (significant observations)

	Dose  (mg/kg/day)	Absolute liver weight (g) (Mean/SD)	Relative liver weight (g) (Mean/SD)	Absolute testes weight (g) (Mean/SD)	Relative testes weight (g) (Mean/SD)
Primary group males	0	8.508 0.772	2.661 0.122	3.106 0.114	0.974 0.036
Primary group males	0.065	8.436 0.789	2.653 0.092	3.048 0.092	0.964 0.073
Primary group males	0.2	8.708 0.412	2.830* 0.061	3.021 0.057	0.983 0.039
Primary group males	2.0	12.492* 0.642	3.922* 0.132	2.889* 0.106	0.908* 0.044
Recovery group males	0	8.831 0.456	2.600 0.071	3.170 0.106	0.935 0.045
Recovery group males	2.0	9.228 0.476	2.734* 0.099	2.992* 0.100	0.887* 0.028
Primary group females	0	4.846 0.263	2.697 0.132	N/A	N/A
Primary group females	0.065	4.715 0.216	2.684 0.068	N/A	N/A
Primary group females	0.2	4.762 0.190	2.731 0.097	N/A	N/A
Primary group females	2.0	5 453* 0.289	2.998* 0.129	N/A	N/A
Recovery group females	0	4.720 0.267	2.672 0.111	N/A	N/A
Recovery group females	2.0	4.942 0.226	2.751 0.074	N/A	N/A

*Statistically different from control by Dunnett's test, alpha-0.05, two-sided

Conclusions:

16 week oral NOEL (Rat, male): 0.065 mg/kg bw/day
16 week oral NOEL (Rat, female): 0.2 mg/kg bw/day

Executive summary:

The test substance was fed in the diet to primary or recovery rats (10/sex/group). Primary groups were fed the test substance for 16 weeks at dose levels of 0 (control), 0.065, 0.2, or 2 mg/kg bw/day. Recovery groups were fed the test substance at 0 or 2 mg/kg bw/day for 16 weeks and untreated feed for a further 4 weeks.

Parameters evaluated were general health status; body weights and weight gains; hematology (PCV, Hgb, RBC and WBC, platelets, and WBC differential); clinical chemistry (glucose, BUN, creatinine, total protein, albumin, globulin, total bilirubin, ALP, ALT, AST, cholesterol, triglycerides, Na, K, Cl, Ca, and P); urinalysis (primary group only), including microscopic examination of pooled samples/sex/group sediment; final fasted body weight; absolute and relative-to-body-weight organ weights (adrenal glands, brain, heart, kidneys, liver, testes or ovaries, and thymus gland); and gross and histopathologic findings in scheduled tissues and gross lesions from all animals. Histopathologic examination was confined, in primary intermediate and low dose animals, to liver, kidneys, and lungs and, in the recovery animals, to liver, kidneys, lungs, and adrenal glands.

Administration of the test substance resulted primarily in liver changes, more in males than in females, confined almost entirely to animals given the highest dose (2 mg/kg bw/day). Liver involvement was evidenced by very slight/slight hypertrophy and increased eosinophilic staining of centrilobular hepatocytes in males, but not females, fed at 2 mg/kg bw/day; by increased liver weights in both males and females given 2 mg/kg bw/day and in males of the intermediate (0.2 mg/kg bw/day) group; and by increased ALP in males and females fed at 2 mg/kg bw/day. The liver changes appeared to regress quickly. The histopathologic alterations and the increased ALP that occurred after 16 weeks of treatment were not found in the animals allowed to recover for 4 weeks. While relative liver weights were again increased in the recovery group males, the increase over control in the recovery males (5%) vs the primary group males (47%) was comparatively minor.

In addition to the liver effects, the data suggested other, non-target-organ effects of the treatment regimen, particularly at the high dose; however, none of these effects had any immediate adverse consequences for the test animals and would not be expected to result in any long-term consequences for the Involved organ systems. In both primary and recovery group males fed 2 mg/kg bw/day, administration of the test substance was also associated with small decreases in absolute and relative testes weights and small decreases in RBC parameters. No corresponding testicular or bone marrow changes were found histopathologically. In primary group males fed 0.2 and males and females fed 2 mg/kg bw/day, treatment appeared to be additionally associated with decreased cholesterol; increased serum K was found in both sexes treated at 2 mg/kg bw/day. The small cholesterol and potassium differences were not judged to be toxicologically significant.

Under the conditions of this study, the liver was identified as the target organ, and males were more susceptible to the hepatic effects than females. These effects were generally reversible in nature. While the liver weight effects in males at the highest dose were accompanied by histopathologic changes and serum enzyme alterations, at the 0.2 mg/kg bw/day level the only evidence of liver involvement was a slight increase in relative liver weight. The no-observed-effect level (NOEL) for subchronic oral administration of the test substance to rats was 0.065 mg/kg bw/day in males and 0.2 mg/kg bw/day in females.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Additional documentation provided in IUCLID assesment reports (Chapter 13) supports the read across approach

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOEL

Effect level:

0.065 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOEL

Effect level:

0.2 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

0.065 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Conclusions:

16 week oral NOEL (Rat, male): 0.065 mg/kg bw/day
16 week oral NOEL (Rat, female): 0.2 mg/kg bw/day

Executive summary:

The test substance was fed in the diet to primary or recovery rats (10/sex/group). Primary groups were fed the test substance for 16 weeks at dose levels of 0 (control), 0.065, 0.2, or 2 mg/kg bw/day. Recovery groups were fed the test substance at 0 or 2 mg/kg bw/day for 16 weeks and untreated feed for a further 4 weeks.

Parameters evaluated were general health status; body weights and weight gains; hematology (PCV, Hgb, RBC and WBC, platelets, and WBC differential); clinical chemistry (glucose, BUN, creatinine, total protein, albumin, globulin, total bilirubin, ALP, ALT, AST, cholesterol, triglycerides, Na, K, Cl, Ca, and P); urinalysis (primary group only), including microscopic examination of pooled samples/sex/group sediment; final fasted body weight; absolute and relative-to-body-weight organ weights (adrenal glands, brain, heart, kidneys, liver, testes or ovaries, and thymus gland); and gross and histopathologic findings in scheduled tissues and gross lesions from all animals. Histopathologic examination was confined, in primary intermediate and low dose animals, to liver, kidneys, and lungs and, in the recovery animals, to liver, kidneys, lungs, and adrenal glands.

Administration of the test substance resulted primarily in liver changes, more in males than in females, confined almost entirely to animals given the highest dose (2 mg/kg bw/day). Liver involvement was evidenced by very slight/slight hypertrophy and increased eosinophilic staining of centrilobular hepatocytes in males, but not females, fed at 2 mg/kg bw/day; by increased liver weights in both males and females given 2 mg/kg bw/day and in males of the intermediate (0.2 mg/kg bw/day) group; and by increased ALP in males and females fed at 2 mg/kg bw/day. The liver changes appeared to regress quickly. The histopathologic alterations and the increased ALP that occurred after 16 weeks of treatment were not found in the animals allowed to recover for 4 weeks. While relative liver weights were again increased in the recovery group males, the increase over control in the recovery males (5%) vs the primary group males (47%) was comparatively minor.

In addition to the liver effects, the data suggested other, non-target-organ effects of the treatment regimen, particularly at the high dose; however, none of these effects had any immediate adverse consequences for the test animals and would not be expected to result in any long-term consequences for the Involved organ systems. In both primary and recovery group males fed 2 mg/kg bw/day, administration of the test substance was also associated with small decreases in absolute and relative testes weights and small decreases in RBC parameters. No corresponding testicular or bone marrow changes were found histopathologically. In primary group males fed 0.2 and males and females fed 2 mg/kg bw/day, treatment appeared to be additionally associated with decreased cholesterol; increased serum K was found in both sexes treated at 2 mg/kg bw/day. The small cholesterol and potassium differences were not judged to be toxicologically significant.

Under the conditions of this study, the liver was identified as the target organ, and males were more susceptible to the hepatic effects than females. These effects were generally reversible in nature. While the liver weight effects in males at the highest dose were accompanied by histopathologic changes and serum enzyme alterations, at the 0.2 mg/kg bw/day level the only evidence of liver involvement was a slight increase in relative liver weight. The no-observed-effect level (NOEL) for subchronic oral administration of the test substance to rats was 0.065 mg/kg bw/day in males and 0.2 mg/kg bw/day in females.

Reference 3

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Additional documentation provided in IUCLID assesment reports (Chapter 13) supports the read across approach

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

no guideline followed

Principles of method if other than guideline:

Groups of ninety 5-week old male and female rats were provided test substance in the diet at targeted dose levels of 0 (control), 0.01, 0.03, 0.065 or 0.1 and 0 (control), 0.01, 0.03, 0.065 or 1.0 mg/kg body weight/day, respectively; of these, twenty rats/sex/dose level were sacrificed at 6 months and 1 year.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

DOWCO 453
Lot #: AGR 187381
Purity: 99.7%

Species:

rat

Strain:

other: CDF Fischer 344

Details on species / strain selection:

This widely used strain of rat was selected because of the available historical data, reliable commercial source and general acceptance for use in toxicity studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Michigan
- Age: 4 weeks
- Housing: The animals were housed (2/cage) in suspended stainless steel cages with wire mesh floors
- Diet: Purina Certified Rodent Chow #5002, ad libitum
- Water: Municipal tap water, ad libitum
- Acclimation period: 7days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 40-60
- Air changes (per hr): 10-12
- Photoperiod (hrs dark / hrs light): 12 hrs/12 hrs

Route of administration:

oral: feed

Details on route of administration:

Possible routes of human exposure to the test substance include ingestion of produce from crops which have been treated with the compound or accidental ingestion during application or manufacture. Thus, formulation with feed was considered the best method of administration to evaluate the effects of repeated oral exposure to the test substance in rats.

Vehicle:

other: diet

Details on oral exposure:

DIET PREPARATION
Treated diets were prepared according to the Standard Operating Procedures of the Subchronic/Chronic Toxicity Area at weekly intervals through the first 3 months of the study and biweekly thereafter through the 1-year interim sacrifice by serially diluting the 5E-3 or 5E-4% premixes with Purina Certified Laboratory Rodent Chow #5002 using Hobart paddle-type mixers.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Assay of the diets for the test substance concentrations using an electron capture/gas chromatography technique was conducted on all test diet concentrations for weeks 1, 15, 38 and 51.

Duration of treatment / exposure:

1 Year

Frequency of treatment:

Daily

Dose / conc.:

0.01 mg/kg bw/day (nominal)

Remarks:

Male/Female

Dose / conc.:

0.03 mg/kg bw/day (nominal)

Remarks:

Male/Female

Dose / conc.:

0.065 mg/kg bw/day (nominal)

Remarks:

Male/Female

Dose / conc.:

0.1 mg/kg bw/day (nominal)

Remarks:

Male

Dose / conc.:

1 mg/kg bw/day (nominal)

Remarks:

Female

No. of animals per sex per dose:

20

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Twice daily

BODY WEIGHT:
- Time schedule for examinations: Weekly for first 3 months and once per month thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Food consumption was determined weekly for all rats during the first 3 months and over a period of one week each month for the remainder of the study.

HAEMATOLOGY: Yes / No / Not specified
- Time schedule for collection of blood: At 6 month and 1-year interim sacrifices
- Animals fasted: Yes
- How many animals: 20/sex/dose

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: At 6 month and 1-year interim sacrifices
- Animals fasted: Yes
- How many animals: 20/sex/dose

URINALYSIS:
- Time schedule for collection of urine: At 6 month and 1-year interim sacrifices
- Urine was obtained via spontaneous urination or compression of the urinary bladder
- Animals fasted: Yes

Sacrifice and pathology:

GROSS PATHOLOGY: At the termination of the 6 month and 1-year interim studies, all rats were fasted overnight, weighed, anesthetized with methoxyflurane and the tracheas clamped. They were killed by decapitation and examined for gross pathological alterations by a veterinary pathologist.

HISTOPATHOLOGY: A complete set of tissues from each rat was collected and preserved in neutral, phosphate buffered 10% formalin. Lesions of a possible tumorous nature occuring in rats sacrificed at 1-year were also histopathologically examined.

Statistics:

Body weight, clinical biochemistry, hematology (excluding red cell indices and differential white cell counts), urine specific gravity, organ weights, and organ to fasted body weight ratio data were evaluated by Bartlett's test for equality of variances. Based upon the outcome of Bartlett's test, a parametric or nonparametric analysis of variance (ANOVA) was performed followed by Dunnett's test or the Wilcoxon Rank-Sum test with Bonferroni's correction if the ANOVA was significant. Outliers were identified by a sequential outlier test and excluded from the analysis when judged scientifically correct.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Routine observation of the rats on a daily basis did not reveal overt signs of toxicity or alterations in demeanor in any of the test groups through the 1-year interim sacrifice.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were statistically identified differences in the male body weights at various times, however, the differences were not the result of treatment since they were not consistent or dose-related and were within the normal range of historical control values.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The mean food consumption was unaffected by treatment

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The only differences noted were very slight but statistically identified decreases of questionable clinical significance in RBC, Hgb concentration or PCV at various dose levels and sacrifices. In some cases statistical changes that were seen at the 6-month sacrifice were not present at 1 year.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Slight statistically identified increases in SGPT activity were noted at both the 6 month and 1-year sacrifices in males fed diets containing 0.065 and 0.1 mg/kg body weight/day of the test substance. In addition, statistically identified decreases, which were not considered clinically significant, in serum globulin levels were noted in the high dose males at 6 months and the high dose females at 1-year. The various other serum biochemical parameters were considered normal for rats of this age, strain and sex.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

At 6 months and 1-year, urinalyses were normal. However, a slight statistically identified decrease in specific gravity was observed in the high dose females at 1 year. Similar values were obtained upon repeat analyses of specific gravity for these females approximately 1 week later. In order to determine whether these observations were treatment-related or had occurred only in this subset of animals, urinalyses were conducted on all control and top dose females scheduled for the 2-year sacrifice approximately 3 weeks after the 1-year interim sacrifice. The results from these analyses indicated no difference between the control and top dose, hence, the slight decrease in specific gravity noted in females sacrificed at 1-year was not judged to be toxicologically significant.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At 6 months, increases in liver weights attributed to the intake of the test substance were confined to the relative liver weights of the males from the top dose and both the absolute and relative liver weights of females of the top dose as well as relative liver weight of the females of the 0.065 mg/kg body weight/day group. At 1 year, an increase in liver weight was not present in the top dose males, however, statistically identified increases in relative l1ver weights were noted in females provided diets containing 0.03 or 1.0 mg/kg body weight/day. The increase seen in females provided 0.03 mg/kg body weight/day was not treatment related. The statistically identified changes in absolute or relative kidney weights seen in males and females at either the 6 month or 1-year interim sacrifices were also not treatment related.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Grossly observed slightly dark or greenish discoloration of the renal cortex was identified in the top dose of each sex at 6 months and in the top two doses in males and females at 1 year. The discoloration at 6 months was histopathologically characterized by a very slight increase in the quantity of pigment in the proximal convoluted tubules of the top dose females but not in the males. The pigment was brown to yellowish-green. somewhat refractile and localized to cytoplasmic granules within the epithelial cells of the proximal convoluted tubules. These granules were variable in size (approximately 0.2-7µ in diameter), shape (irregular to spherical) and in distribution between epithelial cells and proximal convoluted tubules. At 1 year, an increase in pigment granules (similar to that at 6 months) was observed histopathologically in the top dose males and females. Histochemical characterization of the granules from the control and treated rats demonstrated the smaller, spherical brown granules stained positive for iron. while larger more irregular greenish-yellow granules were weakly PAS positive and had an orange-yellow autofluorescence. The observed increase in pigment in the kidneys of the high dose animals sacrificed at 1 year appeared to be primarily associated with greenish-yellow auto fluorescent granules. Neither of the granule types were acid fast or stained positively for fat.

All other grosspathologic observations made from the 6 month and 1-year interim sacrifices were not treatment related but were normal variations of a spontaneous nature or were associated with necropsy techniques.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related changes were confined to the liver and kidney at both interim sacrifices. Livers from the high dose males and females were histopathologically characterized by a very slight increase in hepatocyte size and eosinophilic staining of the cytoplasm (altered tinctorial properties) in the centrilobular region. Similar observations were noted at 1 year only in the females at the top dose. There was no histopathologic evidence of injury to hepatocytes at either the 6 month or 1-year interim sacrifices.

All other histopathologic observations made from the 6 month and 1-year interim sacrifices were not treatment related but were normal variations of a spontaneous nature or were associated with necropsy techniques.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No evidence of treatment-related changes of a neoplastic or nonneoplastic nature.

Key result

Dose descriptor:

NOEL

Effect level:

0.065 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

0.1 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

kidney

liver

Treatment related:

yes

Table-1: Summary of clinical biochemistry data - 6 month sacrifice

Dose level (mg/kg/day)	SGPT (mU/mL)	Glob (g/dL)
Male
0	68 ± 57	2.8 ± 0.2
0.065	68 ± 11^a	2.6 ± 0.2
0.1	67 ± 13^a	2.6 ± 0.3^b

a Statistically identified difference from control mean using Dunnett's test, alpha = 0.05

b Statistically identified difference from control mean using Wilcoxon's test, alpha = 0.05

Table-2: Summary of clinical biochemistry data - 12 month sacrifice

Dose level (mg/kg/day)	SGPT (mU/mL)	Glob (g/dL)
Male
0	70 ± 10	2.8 ± 0.2
0.065	84 ± 19^a	2.8 ± 0.2
0.1	95 ± 22^a	2.8 ± 0.2

Dose level (mg/kg/day)	Glob (g/dL)
Female
0	2.8 ± 0.2
0.1	2.6 ± 0.1^b

a Statistically identified difference from control mean using Wilcoxon's test, alpha = 0.05

b Statistically identified difference from control mean using Dunnett's test, alpha = 0.05

Table-3: Summary of terminal body and organ weight data - 6 month sacrifice

Dose level (mg/kg/day)	Fasted body weight (g)	Liver
Males		g	g/100g
0	333.3 ± 43.7	8.138 ± 1.233	2.439 ± 0.125
0.1	338.7±37.4	8.633 ± 0.947	2.551 ± 0.107^a

Dose level (mg/kg/day)	Fasted body weight (g)	Liver		Kidneys
Females		g	g/100 g	g	g/100 g
0	175.2 ± 10.8	4.546 ± 0.324	2.595 ± 0.111	1.339 ± 0.095	0.764 ± 0.032
0.1	176.9 ± 13.6	4.729 ± 0.401	2.673 ± 0.089	1.398 ± 0.118	0.790 ± 0.032^a
0.065	175.7 ± 9.7	4.782 ± 0.316	2.721 ± 0.108^a	1.385 ± 0.091	0.788 ± 0.031
1.0	175.3 ± 7.0	4.834 ± 0.258	2.759 ± 0.148^a	1.311 ± 0.072	0.748 ± 0.033

a Statistically identified difference from control mean using Dunnett's test, alpha = 0.05

Table-4: Summary of terminal body and organ weight data - 12 month sacrifice

Dose level (mg/kg/day)	Fasted body weight (g)	Liver
Males		g	g/100 g
0	394.3 ± 42.4	9.735 ± 1.114	2.470 ± 0.123
0.1	401.7 ± 34.8	9.878 ± 1.006	2.459 ± 0.122

Dose level (mg/kg/day)	Fasted body weight (g)	Liver		Kidneys
Females		g	g/100 g	g	g/100 g
0	219.4 ± 27.4	5.397 ± 0.442	2.474 ± 0.167	1.592 ± 0.145	0.729 ± 0.048
0.01	205.1 ± 18.7	5.129 ± 0.361	0.506 ± 0.109	1.517 ± 0.107	0.741 ± 0.031
0.03	210.3 ± 24.4	5.441 ± 0.686	2.588 ± 0.165^a	1.560 ± 0.120	0.745 ± 0.047
1.0	211.0 ± 10.4	5.576 ± 0.267	2.645 ± 0.103^a	1.485 ± 0.065^a	0.704 ± 0.026

a Statistically identified difference from control mean using Dunnett's test, alpha = 0.05

Table-5: Histopathologic observations (liver) - 6 month sacrifice

Sex	Male		Female
Dose (mg/kg/day)	0	0.1	0	0.1
No. of rats examined	10	10	10	10
Liver (No. of tissues examined)	10	10	10	10
Increased size of hepatocytes often accompanied by altered tinctorial properties, centrilobular (very slight)	0	7	0	6
Aggregates of mononuclear (predominately lymphoid) cells (very slight)	9	9	9	8
Aggregates of mononuclear (predominately lymphoid) cells adjacent to degenerative or necrotic cells (very slight)	2	1	3	4
Aggregates of reticuloendothelial cells (very slight)	2	1	4	4
Architecture altered secondary to diaphragmatic hernia, focal	0	1	0	0
Focus of altered cells, hepatocellular	4	0	3	1
Granulomas (very slight)	0	0	1	0
Hyperplasia, bile ducts (very slight)	7	2	0	3
Necrosis, hepatocellular, focal or multifocal	1	0	0	0
Vacuolization consistent with fatty change, hepatocellular (very slight)	0	2	0	0
Aggregates of RE cells frequently adjacent to degerative or necrotic hepatocytes (very slight)	1	0	0	1
Inflammation – subacute to chronic, capsure (very slight to slight)	1	0	0	0

Table-6: Histopathology observations (liver) - 12 month sacrifice

Sex	Male		Female
Dose (mg/kg/day)	0	0.1	0	0.1
No. of rats examined	10	10	10	10
Liver (No. of tissues examined)	10	10	10	10
Increased size of hepatocytes often accompanied by altered tinctorial properties, centrilobular (very slight)	0	0	0	6
Aggregate(s) of mononuclear (predominately lymphoid) cells (Very slight to slight)	10	10	10	9
Architecture altered secondary to diaphragmatic hernia, focal	0	0	1	1
Degeneration with or without inflammation, hepatocellular, multifocal (very slight)	0	0	0	0
Extramedullary haematopoiesis (very slight)	0	0	0	0
Focus of altered cells, hepat0cellular (1)	3	5	1	2
Foci of altered cells, hepatocellular (2-5)	1	1	4	5
Foci of altered cells, hepatocellular (6-15)	0	0	2	2
Hyperplasia, bile ducts (very slight to slight)	10	10	4	4
Necrosis, hepatocellular, focal or multifocal (very slight to slight)	2	1	0	0
Necrosis, hepatocellular, focal (Severe)	0	0	0	0
Vacuolization consistent with fatty change, hepatocellular (very slight)	9	9	4	2
Inflammation - subacute to chronic, capsule, focal (very slight)	0	0	1	0
Inflammation - subacute to chronic, portal, multifocal (very slight to slight)	1	0	0	0

Table-7: Histopathology observations (Kidney) - 6 month sacrifice

Sex	Male		Female
Dose (mg/kg/day)	0	0.1	0	0.1
No. of rats examined	10	10	10	10
Kidney (No. of tissues examined)	10	10	10	10
Pigment, convoluted tubule – proximal, multifocal (very slight)	10	10	10	0
Increased pigment, convoluted tubule – proximal, multifocal (very slight)	0	0	0	10
Aggregates of mononuclear (predominately lymphoid) cells, interstitium (very slight)	10	9	3	2
Atrophy, tubules (very slight to slight)	8	8	3	0
Casts, tubules (very slight)	6	5	3	0
Mineralization, tubules (very slight)	1	1	7	3

Table-8: Histopathology observations (Kidney) - 12 month sacrifice

Sex	Male		Female
Dose (mg/kg/day)	0	0.1	0	0.1
No. of rats examined	10	10	10	10
Kidney (No. of tissues examined)	10	10	10	10
Pigment, convoluted tubule – proximal, multifocal (very slight)	0	0	10	0
Pigment, convoluted tubule – proximal, multifocal (slight)	10	0	0	0
Increased pigment, convoluted tubule – proximal, multifocal (very slight)	0	10	0	10
Aggregates of mononuclear (predominately lymphoid) cells, interstitium (very slight)	10	10	8	7
Atrophy, tubules (very slight to slight)	10	10	5	6
Casts, tubules (very slight to slight)	9	10	3	3
Mineralization, tubules (very slight)	0	0	3	6

Conclusions:

Rat NOEL: 0.065 mg/kg bw/day

Executive summary:

Groups of ninety 5-week old male and female rats were provided test substance in the diet at targeted dose levels of 0 (control), 0.01, 0.03, 0.065 or 0.1 and 0 (control), 0.01, 0.03, 0.065 or 1.0 mg/kg body weight/day, respectively; of these, twenty rats/sex/dose level were sacrificed at 6 months and 1 year.

Treatment related effects of toxicological significance were confined to the liver and kidneys of male rats ingesting 0.1 mg/kg body weight/day and female rats ingesting 1.0 mg/kg body weight/day. These included increases in the absolute and/or relative liver weights of males and females provided the high dose with concomitant minor histopathologic changes characterized by an increase in hepatocyte size and altered tinctorial properties. A grossly visible increase in kidney pigment was seen in both sexes fed the high dose for 6 months and in both sexes fed the two highest doses for 1 year. Histological examination revealed this pigment to be in the epithelial cells of the proximal convoluted tubules of the control and treated animals. However, it was only histologically differentiated from the control in the high dose females at 6 months and in the high dose males and females at 1 year. These kidney and liver changes were slight in degree and did not appear to adversely affect the animals.

In this study the liver and kidney were identified as organs affected by the test substance. At the 1-year interim sacrifice, the no-observable-effect level (NOEL) was 0.065 mg/kg body weight/day for both sexes.

Reference 4

Endpoint:

chronic toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Additional documentation provided in IUCLID assesment reports (Chapter 13) supports the read across approach

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOEL

Effect level:

0.065 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

0.1 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

kidney

liver

Treatment related:

yes

Conclusions:

Rat NOEL: 0.065 mg/kg bw/day

Executive summary:

Groups of ninety 5-week old male and female rats were provided test substance in the diet at targeted dose levels of 0 (control), 0.01, 0.03, 0.065 or 0.1 and 0 (control), 0.01, 0.03, 0.065 or 1.0 mg/kg body weight/day, respectively; of these, twenty rats/sex/dose level were sacrificed at 6 months and 1 year.

Treatment related effects of toxicological significance were confined to the liver and kidneys of male rats ingesting 0.1 mg/kg body weight/day and female rats ingesting 1.0 mg/kg body weight/day. These included increases in the absolute and/or relative liver weights of males and females provided the high dose with concomitant minor histopathologic changes characterized by an increase in hepatocyte size and altered tinctorial properties. A grossly visible increase in kidney pigment was seen in both sexes fed the high dose for 6 months and in both sexes fed the two highest doses for 1 year. Histological examination revealed this pigment to be in the epithelial cells of the proximal convoluted tubules of the control and treated animals. However, it was only histologically differentiated from the control in the high dose females at 6 months and in the high dose males and females at 1 year. These kidney and liver changes were slight in degree and did not appear to adversely affect the animals.

In this study the liver and kidney were identified as organs affected by the test substance. At the 1-year interim sacrifice, the no-observable-effect level (NOEL) was 0.065 mg/kg body weight/day for both sexes.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

0.065 mg/kg bw/day

Study duration:

subchronic

Experimental exposure time per week (hours/week):

168

Species:

rat

Quality of whole database:

16 weeks and 1 year data is available on rats

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

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3200 (Repeated Dose Dermal Toxicity -21/28 Days)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Haloxyfop-R-Methyl Ester
Lot # OC30150101 (TSN102229)
Purity: 96.7% R + 1.09% S

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat (Rattus norvegicus) was selected as a test system because it is a readily available laboratory rodent species. It has been historically shown to be a suitable model for repeated dose toxicity studies. The OECD and other regulatory authorities also recommend it. The results of the study are believed to be of value in predicting the toxicity of the test item to man.

Sex:

male/female

Type of coverage:

semiocclusive

Vehicle:

corn oil

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Triplicate samples from each concentration and corn oil (Vehicle) were sent for homogeneity and active ingredient concentration analysis on day 1. The results of A.I. concentration and homogeneity from the samples collected on day 1 (pre-dosing) revealed mean percent recovery of 107, 110 and 108 at the concentrations of 2 (low dose), 10 (mid dose) and 50 (high dose) mg/mL, respectively. The homogeneity and active ingredient content of the test substance in corn oil was within the allowable limit of ± 15% of nominal concentration.

Duration of treatment / exposure:

6 h

Frequency of treatment:

Daily

Dose / conc.:

2 mg/kg bw/day

Remarks:

G2 (Low dose)

Dose / conc.:

10 mg/kg bw/day

Remarks:

G3 (Mid dose)

Dose / conc.:

50 mg/kg bw/day

Remarks:

G4 (High dose)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Clinical signs:

no effects observed

Description (incidence and severity):

No abnormality was observed in any animal during the detailed physical examination of animals.

Dermal irritation:

no effects observed

Description (incidence and severity):

No local skin reaction was observed in any treatment group during the study period.

Mortality:

no mortality observed

Description (incidence):

No mortality or morbidity was observed in any group throughout the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No statistically significant changes in mean body weight and body weight change were observed in any treatment group when compared to the control group throughout the study period.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant increase (27.49%) in mean food consumption was observed in the female rats of the high dose group (G4-50 mg/kg b. wt.) during day 1-8 when compared with the control group (G1-0 mg/kg b. wt.) which was not considered as a treatment-related finding as this was not observed during subsequent periods of the study.
No other differences in the mean food consumption were observed for male and female rats of treatment groups compared to control group during the study.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ophthalmological changes were observed in male and female rats of any group.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant increase (23.71 %) in platelet count of male rats from the mid dose group (G3) and decrease (17.50 %) in female rats of the high dose group (G4) was observed when compared with the control group. These variations observed in platelet counts were either inconsistent or not dose-dependent and, hence, were not considered as treatment-related findings. All other haematological parameters among different dose groups were comparable with the control group.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant increase in albumin level (4.89 %) and ALB:GLB ratio (33.53 %), and a reduction (21.46 %) in globulin level of male rats from the high dose group (G4) were observed when compared with the control group. These changes were within historical control data of testing laboratory (albumin: 4.33-5.11 g/dL, globulin: 1.24-3.34 g/dL and ALB:GLB ratio:1.38 - 3.99) hence, all these variations were not considered as treatment-related findings. All other clinical chemistry parameters among different dose groups were comparable with the control group.

Urinalysis findings:

no effects observed

Description (incidence and severity):

All the urine analysis parameters in both males and females from the treatment (G2 to G4) groups were comparable with the control (G1) group.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The terminal body weight, absolute organ weight and relative organ weight in all the animals belonging to all the treated groups (G2 to G4) were comparable with the control group (G1). However, although not statistically significant, there was slight increase (8.26% and 6.80% in males and females, respectively) in absolute liver weight of the animals belonging to the high dose group (G4). This change was of slight magnitude without an evidence of any histopathological finding and thus, was considered not related to the treatment with the test item.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

External and internal examination of terminally sacrificed animals did not reveal any lesions of pathological significance.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Microscopic examination did not reveal any treatment related lesion of pathological significance.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Conclusions:

21-Day Dermal Rat NOAEL (male/female): ≥50 mg/kg b. wt./day

Executive summary:

The study was conducted to determine the systemic toxicity potential of the test substance in Wistar rats following 21 days of repeated dermal exposure. This study was performed as per the requirements of guidelines of European Commission for repeated dose (28 Days) toxicity (Dermal), January 1997, guideline of OECD N° 410 (May 12, 1981) and guideline of US EPA OPPTS 870.3200 (August 1998).

A total of 20 male and 20 female Wistar rats were randomly allocated into four groups. Each group comprised 5 male and 5 female rats. Not less than 10 percent of the body surface area was clipped free of hair 24 h prior to the first dermal application of the test substance and weekly thereafter. One group (G1) served as the control and was treated with corn oil at a dose volume the same as that used for the high dose group. The other groups were treated at the dose levels of 2 mg/kg b. wt./day (G2), 10 mg/kg b. wt./day (G3) and 50 mg/kg b. wt./day (G4) of the test substance through dermal application. The test substance was applied over the clipped area by a single dermal application and was held in contact with the skin using porous gauze dressing and a non-irritating tape throughout the 6 h of exposure every day to prevent any loss of the test substance and also to ensure that the animals did not lick or ingest it. At the end of the exposure period, the residual test substance was removed using cotton moistened with corn oil. The fixed dose volume of 1 mL/kg b. wt. was used to dose each of the animals. The dose formulations were prepared by dissolving the test substance in corn oil. The dose formulation analysis was performed on day 1.

Each rat was observed for visible signs of reaction to treatment and for morbidity and mortality twice daily during the treatment period. All animals were observed for local skin reaction once daily as per protocol. The detailed clinical observations were performed before commencement of study and at weekly intervals until terminal sacrifice. Body weight was recorded on day 1 (prior to treatment) and at weekly intervals thereafter during the treatment period. Food consumption was calculated at weekly intervals until terminal sacrifice.

Ophthalmological examination was performed on all animals prior to commencement of treatment and prior to terminal sacrifice. Haematology, clinical chemistry investigation and urinalysis were performed on day 22 for all animals following an overnight fast.

All the terminally sacrificed rats were subjected to a detailed gross pathological examination. Absolute organ weights were recorded and relative organ weights were calculated for the organs viz., adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, uterus, thyroid and parathyroid and thymus for all rats. Histopathological examination was carried out for the organs and tissues of G1 (control) and G4 (high dose group). As microscopic findings were observed in the livers of high dose group animals, histopathological examination was extended to the low and mid dose groups.

No mortality was observed during the study period. No clinical signs were observed in any animal from any of the groups.

No significant changes were observed in mean body weight and percent body weight change of male and female rats treated with the test substance in the low dose, mid dose and high dose groups when compared with the control group throughout the study period.

No treatment-related changes were observed in mean food consumption for treatment groups when compared with the control group.

No treatment-related alterations were observed in clinical pathology estimations for treatment groups when compared with the control group.

No significant changes were observed in mean terminal body weights, absolute organ weights and relative organ weights of animals from low dose, mid dose and high dose groups when compared with the control group.

External and internal examination of terminally sacrificed animals did not reveal any lesions of pathological significance. Microscopic examination did not reveal any treatment related lesion of pathological significance.

Based on the results of this study, it is concluded that the test substance did not produce any local as well as systemic adverse, treatment-related effects upto the dose level of 50 mg/kg b. wt./day after repeated dermal application for 21 days in Wistar rats. Hence, the NOAEL for the test substance is considered as ≥50 mg/kg b. wt./day under the conditions and procedures followed in the present study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Only one study was available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3200 (Repeated Dose Dermal Toxicity -21/28 Days)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Haloxyfop-R-Methyl Ester
Lot # OC30150101 (TSN102229)
Purity: 96.7% R + 1.09% S

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat (Rattus norvegicus) was selected as a test system because it is a readily available laboratory rodent species. It has been historically shown to be a suitable model for repeated dose toxicity studies. The OECD and other regulatory authorities also recommend it. The results of the study are believed to be of value in predicting the toxicity of the test item to man.

Sex:

male/female

Type of coverage:

semiocclusive

Vehicle:

corn oil

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Triplicate samples from each concentration and corn oil (Vehicle) were sent for homogeneity and active ingredient concentration analysis on day 1. The results of A.I. concentration and homogeneity from the samples collected on day 1 (pre-dosing) revealed mean percent recovery of 107, 110 and 108 at the concentrations of 2 (low dose), 10 (mid dose) and 50 (high dose) mg/mL, respectively. The homogeneity and active ingredient content of the test substance in corn oil was within the allowable limit of ± 15% of nominal concentration.

Duration of treatment / exposure:

6 h

Frequency of treatment:

Daily

Dose / conc.:

2 mg/kg bw/day

Remarks:

G2 (Low dose)

Dose / conc.:

10 mg/kg bw/day

Remarks:

G3 (Mid dose)

Dose / conc.:

50 mg/kg bw/day

Remarks:

G4 (High dose)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Clinical signs:

no effects observed

Description (incidence and severity):

No abnormality was observed in any animal during the detailed physical examination of animals.

Dermal irritation:

no effects observed

Description (incidence and severity):

No local skin reaction was observed in any treatment group during the study period.

Mortality:

no mortality observed

Description (incidence):

No mortality or morbidity was observed in any group throughout the study period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No statistically significant changes in mean body weight and body weight change were observed in any treatment group when compared to the control group throughout the study period.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant increase (27.49%) in mean food consumption was observed in the female rats of the high dose group (G4-50 mg/kg b. wt.) during day 1-8 when compared with the control group (G1-0 mg/kg b. wt.) which was not considered as a treatment-related finding as this was not observed during subsequent periods of the study.
No other differences in the mean food consumption were observed for male and female rats of treatment groups compared to control group during the study.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No ophthalmological changes were observed in male and female rats of any group.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant increase (23.71 %) in platelet count of male rats from the mid dose group (G3) and decrease (17.50 %) in female rats of the high dose group (G4) was observed when compared with the control group. These variations observed in platelet counts were either inconsistent or not dose-dependent and, hence, were not considered as treatment-related findings. All other haematological parameters among different dose groups were comparable with the control group.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant increase in albumin level (4.89 %) and ALB:GLB ratio (33.53 %), and a reduction (21.46 %) in globulin level of male rats from the high dose group (G4) were observed when compared with the control group. These changes were within historical control data of testing laboratory (albumin: 4.33-5.11 g/dL, globulin: 1.24-3.34 g/dL and ALB:GLB ratio:1.38 - 3.99) hence, all these variations were not considered as treatment-related findings. All other clinical chemistry parameters among different dose groups were comparable with the control group.

Urinalysis findings:

no effects observed

Description (incidence and severity):

All the urine analysis parameters in both males and females from the treatment (G2 to G4) groups were comparable with the control (G1) group.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The terminal body weight, absolute organ weight and relative organ weight in all the animals belonging to all the treated groups (G2 to G4) were comparable with the control group (G1). However, although not statistically significant, there was slight increase (8.26% and 6.80% in males and females, respectively) in absolute liver weight of the animals belonging to the high dose group (G4). This change was of slight magnitude without an evidence of any histopathological finding and thus, was considered not related to the treatment with the test item.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

External and internal examination of terminally sacrificed animals did not reveal any lesions of pathological significance.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Microscopic examination did not reveal any treatment related lesion of pathological significance.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Conclusions:

21-Day Dermal Rat NOAEL (male/female): ≥50 mg/kg b. wt./day

Executive summary:

The study was conducted to determine the systemic toxicity potential of the test substance in Wistar rats following 21 days of repeated dermal exposure. This study was performed as per the requirements of guidelines of European Commission for repeated dose (28 Days) toxicity (Dermal), January 1997, guideline of OECD N° 410 (May 12, 1981) and guideline of US EPA OPPTS 870.3200 (August 1998).

A total of 20 male and 20 female Wistar rats were randomly allocated into four groups. Each group comprised 5 male and 5 female rats. Not less than 10 percent of the body surface area was clipped free of hair 24 h prior to the first dermal application of the test substance and weekly thereafter. One group (G1) served as the control and was treated with corn oil at a dose volume the same as that used for the high dose group. The other groups were treated at the dose levels of 2 mg/kg b. wt./day (G2), 10 mg/kg b. wt./day (G3) and 50 mg/kg b. wt./day (G4) of the test substance through dermal application. The test substance was applied over the clipped area by a single dermal application and was held in contact with the skin using porous gauze dressing and a non-irritating tape throughout the 6 h of exposure every day to prevent any loss of the test substance and also to ensure that the animals did not lick or ingest it. At the end of the exposure period, the residual test substance was removed using cotton moistened with corn oil. The fixed dose volume of 1 mL/kg b. wt. was used to dose each of the animals. The dose formulations were prepared by dissolving the test substance in corn oil. The dose formulation analysis was performed on day 1.

Each rat was observed for visible signs of reaction to treatment and for morbidity and mortality twice daily during the treatment period. All animals were observed for local skin reaction once daily as per protocol. The detailed clinical observations were performed before commencement of study and at weekly intervals until terminal sacrifice. Body weight was recorded on day 1 (prior to treatment) and at weekly intervals thereafter during the treatment period. Food consumption was calculated at weekly intervals until terminal sacrifice.

Ophthalmological examination was performed on all animals prior to commencement of treatment and prior to terminal sacrifice. Haematology, clinical chemistry investigation and urinalysis were performed on day 22 for all animals following an overnight fast.

All the terminally sacrificed rats were subjected to a detailed gross pathological examination. Absolute organ weights were recorded and relative organ weights were calculated for the organs viz., adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, uterus, thyroid and parathyroid and thymus for all rats. Histopathological examination was carried out for the organs and tissues of G1 (control) and G4 (high dose group). As microscopic findings were observed in the livers of high dose group animals, histopathological examination was extended to the low and mid dose groups.

No mortality was observed during the study period. No clinical signs were observed in any animal from any of the groups.

No significant changes were observed in mean body weight and percent body weight change of male and female rats treated with the test substance in the low dose, mid dose and high dose groups when compared with the control group throughout the study period.

No treatment-related changes were observed in mean food consumption for treatment groups when compared with the control group.

No treatment-related alterations were observed in clinical pathology estimations for treatment groups when compared with the control group.

No significant changes were observed in mean terminal body weights, absolute organ weights and relative organ weights of animals from low dose, mid dose and high dose groups when compared with the control group.

External and internal examination of terminally sacrificed animals did not reveal any lesions of pathological significance. Microscopic examination did not reveal any treatment related lesion of pathological significance.

Based on the results of this study, it is concluded that the test substance did not produce any local as well as systemic adverse, treatment-related effects upto the dose level of 50 mg/kg b. wt./day after repeated dermal application for 21 days in Wistar rats. Hence, the NOAEL for the test substance is considered as ≥50 mg/kg b. wt./day under the conditions and procedures followed in the present study.

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Haloxyfop R ester was fed in the diet to primary or recovery rats (10/sex/group). Primary groups were fed for 16 weeks at dose levels of 0 (control), 0.065, 0.2, or 2 mg/kg bw/day. Recovery groups were fed at 0 or 2 mg/kg bw/day for 16 weeks and untreated feed for a further 4 weeks. Under the conditions of this study, the liver was identified as the target organ, and males were more susceptible to the hepatic effects than females. These effects were generally reversible in nature. While the liver weight effects in males at the highest dose were accompanied by histopathologic changes and serum enzyme alterations, at the 0.2 mg/kg bw/day level the only evidence of liver involvement was a slight increase in relative liver weight. The no-observed-effect level (NOEL) for subchronic oral administration of the substance to rats was 0.065 mg/kg bw/day in males and 0.2 mg/kg bw/day in females.

Another study with groups of ninety 5-week old male and female rats were provided Haloxyfop acid in the diet at targeted dose levels of 0 (control), 0.01, 0.03, 0.065 or 0.1 and 0 (control), 0.01, 0.03, 0.065 or 1.0 mg/kg body weight/day, respectively; of these, twenty rats/sex/dose level were sacrificed at 6 months and 1 year. Treatment related effects of toxicological significance were confined to the liver and kidneys of male rats ingesting 0.1 mg/kg body weight/day and female rats ingesting 1.0 mg/kg body weight/day. These included increases in the absolute and/or relative liver weights of males and females provided the high dose with concomitant minor histopathologic changes characterized by an increase in hepatocyte size and altered tinctorial properties. A grossly visible increase in kidney pigment was seen in both sexes fed the high dose for 6 months and in both sexes fed the two highest doses for 1 year. Histological examination revealed this pigment to be in the epithelial cells of the proximal convoluted tubules of the control and treated animals. However, it was only histologically differentiated from the control in the high dose females at 6 months and in the high dose males and females at 1 year. These kidney and liver changes were slight in degree and did not appear to adversely affect the animals. In this study the liver and kidney were identified as organs affected by the test substance. At the 1-year interim sacrifice, the no-observable-effect level (NOEL) was 0.065 mg/kg body weight/day for both sexes.

A dermal rat study was conducted to determine the systemic toxicity potential of the test substance following 21 days of repeated exposure. The study was performed as per the requirements of guidelines of European Commission for repeated dose (28 Days) toxicity (Dermal), January 1997, guideline of OECD N° 410 (May 12, 1981) and guideline of US EPA OPPTS 870.3200 (August 1998). One group (G1) served as the control and was treated with corn oil at a dose volume the same as that used for the high dose group. The other groups were treated at the dose levels of 2 mg/kg b. wt./day (G2), 10 mg/kg b. wt./day (G3) and 50 mg/kg b. wt./day (G4) of the test substance through dermal application. Based on the results of the study, it is concluded that the test substance did not produce any local as well as systemic adverse, treatment-related effects upto the dose level of 50 mg/kg b. wt./day after repeated dermal application for 21 days in Wistar rats. Hence, the NOAEL for the test substance is considered as ≥50 mg/kg b. wt./day under the conditions and procedures followed in the present study.

Justification for classification or non-classification

Under the criteria of CLP Regulation [EC] No. 1272/2008, STOT RE may be assigned on the basis of a substance demonstrating evidence of significant or severe specific organ toxicity in a 90-day oral study at or below a guidance value of 100 mg/kg bw/day (basis of Category 2). This guidance value is adjusted in accordance with the Haber’s rule for studies of different durations. ‘Significant’ toxicity is taken to mean changes that clearly indicate functional disturbance or morphological changes that are toxicologically relevant. ‘Severe’ toxicity is considered to be more profound or serious and indicates changes that are of a considerably adverse nature with a significant impact on health. 

There is no evidence for any adverse findings or serious target organ toxicity in 16 week repeat dosing studies in rats that meet the criteria of CLP Regulation [EC] No. 1272/2008 for STOT RE. In the study where Haloxyfop R ester at dosages of 0.065, 0.2 and 2 mg/kg bw/day indicated the liver as the only target organ in the Fischer 344 rat. No effects occurred at 0.065 mg/kg bw/day for males and 0.065 and 0.2 mg/kg bw/day for females. The effects in these feeding studies were changes of alkaline phosphatase, potassium levels and cholesterol levels at 2 mg/kg bw/day, in both sexes. Cholesterol levels were also decreased at 0.2 mg/kg bw/day in males. All parameters returned to normal after a 4-week recovery phase. Liver weights were increased for both sexes at the top dose level, and also slightly at 0.2 mg/kg bw/day for males. Histopathological changes were only seen in males at 2 mg/kg bw/day, consisting of slight increases in centrilobular hepatocyte size and eosinophilia, but these were not evident after the recovery phase. The test substance did not induce any adverse effects when tested by the dermal route in short-term repeat exposure studies.  Therefore, the test substance is not classified for STOT RE according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.