Ammonium 2-amino-4-(hydroxymethylphosphinyl)butyrate

Endpoint summary

Description of key information

Repeated dose toxicity, oral route of exposure

Rat

There are several oral repeated dose studies (of variable durations) in the rat available.  

In an oral 28-day study (range-finding study for the long-term studies), Glufosinat ammonium (GA) was administered with the diet to Wistar rats at concentrations of 0, 50, 500, 2500 or 5000 ppm for 28 days. The control animals received plain diet.

No deaths occurred throughout the study and no signs or symptoms of systemic toxicity were observed. Reduced bw gain was noted for males and females receiving the test substance at concentrations of 500, 2500 and 5000 ppm (not dose-related). The food consumption was slightly reduced in all animals of the 2500 and 5000 ppm group during the first week of treatment. The assessment of hematological, biochemical and urinalysis data revealed no treatment-related

changes. A statistically significant increase in relative kidney weights was seen in males of the 2500 ppm group and in all treated females (not dose-related). Gross pathological examination revealed slight reddening observed in the gastric mucosa of four males of the 5000 ppm group, and several dark red areas in the gastric mucosa of one male of the 50 ppm group. These changes were possibly treatment-related. There were no microscopic changes considered to be treatment-related. Although certain parameters were affected at all dose levels, no clear dose response was observed. Due to the absence of clinical signs and microscopic pathology the NOAEL for male and female rats was set at ≥5000 ppm (534 and 557 mg/kg bw/d for males and females, respectively).

 

In a 90-day feeding study performed similar to OECD guideline No. 408 with minor deviations, Glufosinate-ammonium was administered by admixture with the diet to Fischer rats at concentrations of 0, 8, 64, 500 or 4000 ppm. The control animals received the diet without GA. 20 animals/sex/dose level were sacrificed at the end of the 13-week study period and then 10 animals /sex/dose level were maintained for a 4-week post-dose recovery period. One male rat fed 4000 ppm died on day 71 of administration. No treatment-related clinical signs of toxicity were seen in any dosage group. Decreased bodyweight gain was observed for males and females fed 4000 ppm through the second or third week of administration, after which bw gain was comparable to or greater than that of control animals. Decreased food consumption was observed for males and females fed 4000 ppm from the first through third week of administration. A decrease in water intake was observed for males and females of the highest dosage group during the first week of treatment. Water intake of the males fed the highest dose showed a tendency to increase from weeks 7 to 13. The ophthalmologic examinations did not show treatment-related findings. Hematological investigation revealed no changes considered to be related to GA-intake.

In the serum biochemical examination, females of the 4000 ppm group showed statistically significant decreased lactate dehydrogenase (LDH) activity on day 47. This decrease was not seen in samples taken at the end of the dosing period. Other differences in the results of biochemical parameters were considered incidental and of normal biological variation. Urinalysis at the termination of administration revealed a tendency towards lower pH for both sexes administered 4000 ppm as compared to controls. This tendency was also noted in samples taken from animals late in the recovery period. The specific gravity at the termination of administration tended to increase in females of the highest dosage group.

Statistically significant absolute and/or relative kidney weight increases were observed for males fed 500 ppm and up and females fed 4000 ppm. The kidney weight increases continued even after the recovery period in the 500 ppm and 4000 ppm treated males. No treatment-related macroscopic or microscopic changes were observed.

The NOEL for male rats was 64 ppm (4.1 mg/kg bw/d) based on increased absolute and relative kidney weights at 500 ppm and 4000 ppm. In addition, decreased bw gain, decreased food consumption, decreased water intake and urinalysis changes were noted at 4000 ppm. The NOEL for female rats was 500 ppm (39 mg/kg bw/d). Due to the absence of clinical signs and pathological changes the NOAEL for male and female rats was set at 4000 ppm (equivalent to 263 mg/kg bw/d in males and 311 mg/kg bw/d in females).

 

In a second 90-day feeding study, Glufosinate-ammonium was administered by admixture with the diet to Wistar rats (10/sex/dose level) at concentrations of 0, 7500, 10000 or 20000 ppm for 13 weeks. The control animals received plain diet. A functional observational battery (modified Irwin screen test) was performed at pre-test, and at weeks 1, 2, 3, 4, 8 and 13 of treatment.

2 female animals of the highest dosage group died on day 6 or 8, respectively, of treatment. At histopathological examination, the cause of death could not be detected. However, these deaths were considered to be treatment-related. Treatment-related clinical signs were noted in both male and female animals of the 20000 ppm dosage group. The clinical symptoms noted were: sedation, lateral recumbency, hunched posture, dyspnoea, ruffled fur, emaciation, spasm (females only) and lacrimation (females only). Except for ruffled fur, which was observed until week 4 of treatment, all other signs were noted only in the first two weeks of treatment. Slight bw loss was noted for both male and female animals of the 20000 ppm dosage group during the first week of treatment. Decreased bw gain was noted for all treated animals during the first part of treatment. Thereafter the bw gain was comparable to or greater than that of control animals. Reduced food consumption was noted for all treated animals during the first part of treatment. The ophthalmologic examinations did not show treatment-related findings.

Statistically significant changes observed in hematology parameters suggest a compensated erythropoietic response due to a more rapid turnover of circulating erythrocytes.

The noted statistically significant changes in clinical biochemistry were considered to be of no toxicological relevance. The slight increase in the total bilirubin concentrations may be an effect related to an increased rate of free bilirubin production from circulating erythrocytes as a result of increased hemolysis.

The general behavior of all treated rats during the modified Irwin screen test was statistically significantly altered in a dose-dependent manner. Animals receiving 7500 ppm showed miosis and a slight decrease in exploratory activity, alertness and/or startle response (occasionally observed, particularly in the early stages of treatment). Animals receiving 10 000 ppm showed similar signs as well as increased body tone, increased pain response and fearfulness. With the exception of week 4, the frequency of signs decreased as the study progressed. In addition, diarrhea, increased vocalization and apathy were recorded for animals of the 20 000 ppm group. In week 1, isolated animals of the 10000 and 20000 ppm groups showed rearing during which convulsive twitches and perfuse salivation were observed. The miosis, perfuse salivation, diarrhea (increased intestinal movement) and increase in body tone indicate a stimulation or overactivity of the parasympathetic nervous system.

Macroscopic or microscopic examination did not reveal any abnormal treatment-related findings. In the two females, which died during the study, thymic atrophy was noted. This finding was considered to be non-specific and not treatment-related.

No NOEL for male and female rats could be determined in this study. Reduced bw gain, reduced food consumption, minor changes in hematological and biochemical parameters and behavioral alterations observed in the functional observation battery were noted for all treated animals. In addition, deaths and clinical signs of toxicity were observed in animals of the highest dose group. Also, no NOAEL for male and female rats could be determined in this study, as behavioral alterations were observed for all treated animals.

There are two long-term studies with GA available: a combined chronic and carcinogenicity study and a 2-year carcinogenicity study.

In the first rat combined chronic and oncogenicity study, Wistar rats were exposed for 130 weeks at dose levels of 40, 140 and 500 ppm. 10 animals/sex/dose were sacrificed after 52 weeks of treatment and 20/sex/dose after 104 weeks. The remaining 50/sex/dose were assigned to the oncogenicity phase of the study and designated for sacrifice at 130 weeks for evaluation of carcinogenicity following lifetime exposure to the test substance. No treatment-related clinical signs of toxicity were observed in any dose group. Statistically significant increased bw gain was noted for males of the 2 highest dosage groups from the chronic toxicity phase of the study, and for females of the 40 and 500 ppm dosage groups from the oncogenicity phase of the study. Transient increased food consumption values were noted during the treatment period in all treated groups of the oncogenicity animals, and in treated groups of chronic toxicity males. Ophthalmologic examinations and hearing tests did not show treatment-related findings. Urinalysis data revealed no changes considered to be related to the compound administration. Hematological investigations showed statistically significant decreased hemoglobin (HB) concentration and hematocrit (HCT) values in both sexes of the highest dosage group after 52 weeks only. In addition, statistically significant decreased erythrocyte count (RBC) was noted for females of the highest dosage group after 52 weeks. Mean corpuscular haemoglobin concentration (MCHC) was statistically significant decreased for these female animals after 52, 78 and 104 weeks of treatment. The assessment of biochemical data indicated no changes of toxicological significance for standard biochemical parameters. No treatment-related macroscopic changes were noted. There were no signs of oncogenic potential of GA in this study. No NOEL for female rats was determined in this study. Changes in biochemical parameters (increased kidney GS activity) were noted for all treated females. In addition, changes in hematological parameters (decreased hemoglobin concentration, decreased hematocrit values, decreased erythrocyte count, decreased mean corpuscular hemoglobin concentrations) (500 ppm group), changes in biochemical parameters (inhibition of liver GS activity (140 and 500 ppm)), inhibition of brain GS activity (500 ppm), decreased concentrations of glutathione in the liver (140 and 500 ppm), decreased concentrations of GSH in blood (140 and 500 ppm), increased ammonia levels in urine and brain (500 ppm), and increased kidney weights (500 ppm) were noted for female rats. NOEL for male rats was 40 ppm (approximately 2 mg/kg bw/d) based on changes in biochemical parameters (inhibition of liver GS activity (140 and 500 ppm), increased kidney GS activity (140 and 500 ppm), decreased GSSG levels in the liver (140 and 500 ppm), decreased GSH levels in blood (500 ppm), increased GSSG concentrations in blood (500 ppm), changes in hematological parameters (decreased hemoglobin concentration, decreased hematocrit values) (500 ppm group), and increased kidney weights (140 and 500 ppm) noted for males rats. In the absence of clinical signs or correlative pathological changes, observed hematological and biochemical effects, and increased kidney weights were not considered to be adverse. NOAEL for male and female rats was considered to be > 500 ppm (24.4 and 28.7 mg/kg bw/d for males and females, respectively).

In a second oncogenicity study performed later, rats were administered at 1000, 5000 and 10000 ppm. No treatment related mortalities or clinical signs of toxicity were observed. Statistically significant decreased bw was noted for males and females of the 5000 and 10 000 ppm groups during the first weeks of treatment. Relative food consumption was decreased in animals of the 1000, 5000 and 10 000 ppm groups during the first weeks of treatment. When compared to controls higher relative food consumption was noted for males of the 5000 and 10 000 ppm groups during weeks 3 and 4 of the study, and for females of the 10 000 ppm group during weeks 3 and 4 of the study. Higher relative food consumption was also noted for males of the 10 000 ppm groups from week 21 until the end of the study. Hematological investigations revealed no treatment-related effects on differential white cell count. Statistically significant increased weight of the kidneys was noted for all treated male and female animals. Gross pathology showed lower incidence of pituitary nodules for males in the treated groups when compared to the controls. This finding was confirmed microscopically by a statistically significant negative trend with respect to dose for pituitary adenoma of the pars distalis in males of the 5000 and 10 000 ppm groups. Microscopic evaluations showed no increase in any type of neoplasm in any dose group. Statistically significant increase in retinal atrophy characterised by partial or complete loss of the outer nuclear layer of one or both eyes was noted for males and females of the 10 000 ppm group and for females of the 5000 ppm group. In kidneys, a statistically significant decrease of multifocal corticomedullary mineralisation was noted for all treated females, and there was a statistically significant decrease in caliceal mineralisation in females of the 5000 and 10 000 ppm.

No indications of any oncogenic potential of GA were observed at doses up to 10000 ppm (equivalent to 466 and 579 mg/kg bw/d for males and females, respectively). No NOEL for male and female rats was determined in this study. Increased kidney weights were noted for all treated animals. In addition, statistically significant increase in retinal atrophy was noted for females of the 5000 and 10 000 ppm groups and for males of the 10000 ppm group. Reduced bw was also noted for animals of the 5000 and 10 000 ppm groups. Two commonly occurring age-related changes in rats appear to have been reduced by treatment: the incidence of pituitary adenoma in males and mineralisation of the kidneys in females at all doses. The etiology of these effects is not known. The NOAEL was considered to be 5000 ppm (228.9 mg/kg bw/d) and 1000 ppm (57.1 mg/kg bw/d) for males and females, respectively based on the effects on the retina.

 

Mouse

In a 13-week feeding study in mice with GA, the test substance was administered by admixture with the diet to NMRI mice (10/sex/dose level) at concentrations of 0, 80, 320 or 1280 ppm for 90 days. The control animals received the diet without the test compound.

No treatment-related mortalities or clinical signs of toxicity were noted in any animal. One female animal of the lowest dosage (80 ppm) group died during blood sampling on the last test day. The mean bw gain of all treated mice was similar to that of the control group. No dose-related difference in the mean food consumption of any group was noted. Hearing tests and ophthalmologic examinations did not show treatment-related findings. Some statistical differences observed in the results of the hematological parameters were considered to be incidental and of normal biological variation. Statistically significant higher aspartate aminotransferase (ASAT/GOT) activity was noted for males of the highest dosage group. Statistically significant higher alkaline phosphatase (ALP) activity was noted for females of the highest dosage group. In addition, statistically significant increased potassium concentrations were noted for males receiving the test substance at concentrations of 320 and 1280 ppm. Statistically significant increased liver to bw ratios was noted for males of the highest dosage group. Macroscopic or microscopic examination did not reveal any abnormal treatment-related findings. The NOEL for male mice was 80 ppm (17 mg/kg bw/d) based on changes in hematological and biochemical parameters (reduced total leukocyte count (WBC) and increased potassium concentration) noted in animals fed 320 and 1280 ppm. In addition to that, higher aspartate aminotransferase (ASAT/GOT) activity and increased liver weight was noted for males of the highest dosage group. The NOEL for females was 80 ppm (19 mg/kg bw/d) based on reduced erythrocyte count (RBC) and hematocrit (HCT) noted in animals fed 320 and 1280 ppm, and changes in biochemical parameters (higher alkaline phosphatase (ALP) activity) in the highest dosage group. NOAEL for male and female mice was set at 1280 ppm (equivalent to 278 and 288 mg/kg bw/d for male and female animals, respectively). The changes in hematological and biochemical parameters in males and females, and the increased liver to bw ratio in males were considered of no toxicological significance since no correlative histopathological changes or clinical signs were observed. Further on, they were not reproduced in another 90-day toxicity study in mice conducted at higher dose levels or in a subsequent long-term toxicity study.

There is a second 90-day study in mice available, where the animals were exposed to higher concentrations of Glufosinate-ammonium. GA was administered by admixture with the diet to NMRI mice (10/sex/dose level) at concentrations of 0, 1750, 3500 or 7000 ppm for 90 days. As all animals in the top dose group died within 8 days, only the low and mid dose animal terminal findings were reported (concentrations corresponded to a dose rate of 0, 274, 561 mg/kg bw/d for males, and 0, 356, 644 mg/kg bw/d for females).

All animals at the highest dosage (7000 ppm) group died within 8 days of treatment. At 3500 ppm, a mortality rate of 50% was noted during the study: one male died on day 45 of treatment, the other deaths (4 males and 5 females) were observed between days 6 and 11 of treatment. At 1750 ppm, one female (10%) died on day 8 of treatment. In addition, one male of the 1750 ppm group died at scheduled necropsy after blood sampling (this death was considered to be accidentally and not test article related). Clinical signs of toxicity were noted in all dose groups. At 3500 and 7000 ppm, ruffled fur, sedation, spasm, ventral recumbency or hunched posture, dyspnoea, apathy, ataxia and emaciation were noted. At 1750 ppm, ruffled fur was evident in all animals and sedation and emaciation in some females. Bodyweight loss and reduced food consumption was noted during the first week of treatment for animals of the 1750 and 3500 ppm groups. Hematological investigations revealed no changes considered to be related to the compound administration. In the biochemical examinations, statistically significant increased alkaline phosphatase (ALP) activity was noted for males of the 3500 ppm group and statistically significant decreased potassium concentration was noted for males of the 1750 ppm and 3500 ppm groups. Statistically significant reduced mean liver weight was noted for males of the 3500 ppm group. Macroscopic or microscopic examination did not reveal any abnormal treatment-related findings.

No NOEL/NOAEL was established for male and female mice in this study. Death (one female), clinical signs of toxicity, reduced bw loss, reduced food consumption and minor changes in biochemical parameters (decreased potassium concentrations in males) were noted for animals fed 1750 ppm (274 mg/kg bw/d for males, and 356 mg/kg bw/d for females). Additionally, increased alkaline phosphatase (ALP) activity and reduced mean liver weight was noted for males of the 3500 ppm group. At 3500 ppm, a mortality rate of 50% was noted during the study. At the highest dosage level (7000 ppm) all animals died. Due to the high mortality rate among the mid- and high-dose animals, the study is of limited value for hazard assessment.

In a 2-year oncogenicity study in mice, Glufosinate-ammonium was administered by admixture with the diet to NMRI mice (60/sex/dose) at concentrations of 0 (controls), 20, 80 or 160 (males only) or 320 (females only) ppm. 10/sex/dose were designed for interim sacrifice after 52 weeks of treatment (chronic toxicity groups); 50/sex/dose were designed for terminal sacrifice after 104 weeks (2 years) of treatment (oncogenicity groups). Special investigations were carried out on glutathione levels in whole blood and liver tissue after 104 weeks of treatment. NOEL/NOAEL for male and NOEL for female mice was 80 ppm (equivalent to 11 and 16 mg/kg bw/d for males and females, respectively).

A more detailed description of the results can be found in the IUCLID chapter for carcinogenicity.

 

Dog

There are three repeated-dose oral toxicity studies in dogs available.

In the 90-day study, Glufosinate-ammonium (GA) was administered by admixture with the diet to beagle dogs (4/sex/dose level) at concentrations of 0, 4, 8, 16, 64 or 256 ppm for 90 days. The concentrations corresponded to a dose rate of 0, 0.13, 0.26, 0.57, 2.06 and 7.98 mg/kg bw/d for males, and 0, 0.13, 0.25, 0.49, 1.97 and 7.63 mg/kg bw/d for females. A liver-function test (BSP- bromsulfophthalein method) was carried out on all dogs of the 2 high-dose groups and the control group during week 13; a kidney-function test (PSP phenolsulfonephthalein method) was conducted on the 2 high-dose groups and the control group during week 12. Electrocardiography was performed in all dogs of the control and the highest dosage group at week 13.

No deaths occurred during the study. No treatment-related clinical signs of toxicity were seen in any dosage group. Statistically significant reduced bw was noted for female animals of the highest dosage group over the second half of the study. Food consumption was slightly lower for males and females of the highest dosage group. The ophthalmologic examinations did not show treatment-related findings. The assessment of hematology and urinalysis revealed no changes considered to be related to the compound administration. In the biochemical examinations, statistically significant decreased plasma inorganic phosphate levels were noted for males of the highest dosage group and for females of the 4, 16 and 64 ppm groups in week 7 only. At study termination, decreased plasma bilirubin levels were noted for males of all dose groups (statistically significant for males of the 64 and 256 ppm groups). The finding showed no dose response and was not accompanied by any changes in total-bilirubin levels and is therefore considered not to be of toxicological significance. Plasma PSP concentrations were reduced in males and females of the two high-dosage groups (statistically significant for females of the 64 ppm group only). The values of the controls (mean 1.8 males; mean 2.1 females) were relatively high as compared to historical control values with dogs of the same strain and age (according to the author historical control values range between 0.84-1.48). Since an increase rather than a decrease in PSP concentration is the reflection of impaired renal function and since no treatment related pathological changes were observed in the kidneys, it is concluded that these findings are of no toxicological significance. The mean results of the BSP-retention test were comparable for all groups and all individual data were essentially normal. There were no changes in the electrocardiograms that could be related to the feeding of the test substance. Macroscopic or microscopic examination did not reveal any abnormal treatment-related findings. No statistically significant differences in organ weights were observed between test groups and the control.

The NOEL for male dogs was 64 ppm (2.06 mg/kg bw/d) based on reduced food consumption noted for males fed 256 ppm. NOEL for female dogs was 64 ppm (1.97 mg/kg bw/d) based on reduced bw and reduced food consumption noted for females fed 256 ppm. NOAEL for male and female dogs was 256 ppm (the highest dose, equivalent to 7.98 mg/kg bw/d for males and 7.63 mg/kg bw/d for females).

In a 12-month oral toxicity study, Glufosinate-ammonium (GA) was administered to beagle dogs with the diet for 1 year (8/sex/dose). 4 animals/sex/dose levels were assigned for interim sacrifice after 6 months and 1 year of treatment, respectively. Electrocardiograms (ECG) of each animal were recorded at pre-test and at 6 or 12 months prior to sacrifice. The bromosulfophthalein (BSP) clearance test for measuring liver function and the phenolsulfonphthalein (PSP) clearance test for measuring kidney function were performed after 6 and 12 months of treatment. The nominal dose levels are 0, 2. 5 or 8.5 mg/kg bw/d (achieved dose levels were 0, 1.8. 4.5 or 8.4 mg/kg bw/d). In the first 10 to 17 days of the study, high dose males received the test substance in doses between 10.6 and 13.6 mg/kg bw/d, and high dose females between 15.4 and 16.0 mg/kg bw/d. Adjustments in dietary concentration were progressively made on day 11 and onwards to correct the situation. One male and one female from the highest dosage group died on days 14 and 9, respectively. The following clinical signs of toxicity were noted immediately after test article consumption in the 2 high dose animals which died and on day 9-10 for another high dose female that survived: trismus salivation and hyperactivity followed by somnolence and hypoactivity as well as stereotypic stiff gait, tremor, ataxia, whining, urinating, tonic-clonic spasm, paddling movements, opisthotonus and lateral recumbency. No other animals exhibited any unusual behavior attributed to treatment. The deaths of the animals were caused by heart and circulatory failure due to marked myocardial necrosis in one dog, and to slight myocardial necrosis and severed necrotising aspiration pneumonia in the other dog. These animals received an increased dose of the test article during the first 10-17 days of treatment. The mean bw gain of the high dose group males was reduced during the first six months of treatment, when compared with the control group. Statistically significant reduced food consumption was noted for the high dose group males during the first week of treatment. Otherwise, the mean food consumption of treated male and female dogs was comparable to that of the controls. Hearing tests and ophthalmologic examinations did not show treatment-related findings. Urinalysis data revealed no changes considered to be related to the compound administration. The assessment of hematological and clinical biochemistry data indicated no changes of toxicological significance after 1, 3, 6 and 12 months of treatment.

The mean results of the PSP and BSP-clearance tests were comparable for all groups. No treatment related abnormalities in ECG parameters were noted for surviving animals. There were no changes in absolute and relative organ weights considered to be related to GA exposure. No treatment-related macroscopic or microscopic findings were noted in any of the dogs killed after 6 or 12 months of treatment. Macroscopic findings in the male that died spontaneously were increased fluid in the pericardium and subendocardial hemorrhages in the right heart. In the female that died spontaneously, the main gross findings were numerous grey-green foci in the lung; the lung was not collapsed. Microscopic findings in both dogs that died were multifocal myocardial necrosis. In addition, marked aspiration of diet material with beginning inflammatory reaction in the lungs and bronchial epithelium was noted in the female dog that died during the study. A small acute ulcer in stomach was also noted in this animal. The surviving animal of the high dose that also showed signs of neurotoxicity recovered completely.

The NOAEL of the study has been set at 8.5 mg/kg bw/day (nominal), which was tolerated without any signs of intoxication.

There is an additional 28-day toxicity study in beagle dogs available, which was conducted specifically in order to explore the mode of action and toxicokinetic of glufosinate-ammonium (GA). This summary focusses mainly on the toxicity endpoints.

GA was administered orally (gelatine capsule) at a dose of 0, 1 or 8 mg/kg bw/day to beagle dogs (6/sex/group) for 18 days. From day 19 to 28, the animals received the radiolabelled test substance (GA-14C). The control animals received gelatine capsules containing distilled water. One dog per sex and per group was sacrificed at day 18, 19 and 28 of the treatment period and at day 1, 2 and 4 post-treatment (recovery), respectively. Animals were observed twice daily for mortality and clinical signs. Food consumption was measured daily. Body weight was recorded weekly. Eye examinations were performed pre-test and on days 14 and 25 of treatment. Males and females in all control and high dose groups were subjected to neurobehavioral assessments before dosing and on days 1, 2, 3, 4, 8, 11, 15, 18, 22 and 25. Males and females from the low dosage group were subjected to neurobehavioral assessments before dosing and on days 4, 11, 18 and 25. Blood and urine samples were taken for hematology, clinical biochemistry and urinalysis, which included measurement of catecholamines, before dosing and on days 11, 28 and 4 days post dosing. Tissues taken at necropsy were liver, heart, kidney, spinal cord, and 4 brain regions (cortex, midbrain, cerebellum and brain stem). Tissues were weighed and examined. A range of other tissues was taken at necropsy, fixed but not examined. A special biochemical examination of glutamine synthetase activity and free amino acid levels in different tissues was performed. Brain cholinesterase levels were measured in the cerebellum only. After 18 to 28 days of oral administration by capsule at 8 mg/kg/day, glufosinate ammonium induced no mortality or clinical signs, except a slight increase in gait activity in males; a lower body weight gain in the males at the end of the first week and in females during the whole study in parallel to a slight food consumption decrease. Glutamine synthetase activity and glutamine and taurine levels were also decreased at this dose level. A few changes were observed at 1 mg/kg/day but usually in only one sex and in only a few animals among each group: decrease in arginine level in female spinal cord (2 animals out of 6), in phosphoethanolamine in male cerebellum (3 animals out of 6), in glutamine in male heart without dose-effect relationship, in taurine in female cerebellum (1 animal out of 6) and female cortex. The dose of 8 mg/kg bw/day can be considered as a LOAEL with regard to the decreased glutamine synthetase activity in the brain. The dose level of 1 mg/kg bw/day can be considered as NOAEL.

 

Repeated dose toxicity, inhalation route of exposure

Rat

There are two subacute inhalation studies in the rat available. In the first study, Wistar rats (5/sex) were exposed nose-only to dust particulate aerosol atmospheres of glufosinate ammonium (GA) at concentrations of 0, 0.012, 0.025 or 0.050 mg/l air, 6 hrs/d, 5 d/week (28 exposures over 40 d). The control animals received air only. 1 day after the last exposure 10/sex/group were killed and dissected. The remaining rats were killed and dissected after a 29-day recovery period. 2 females of the highest dosage group died during the 2^ndexposure, and one male of the highest dosage group died after the 2^ndand another after the 17^thexposure. Clinical signs observed in the rats at the highest dosage level were squatting position, staggering gait, tremors, salivation, contracted flanks, narrowed eye opening, piloerection hyperactivity, aggressiveness, tonoclonic convulsions and hematuria. Staggering gait, squatting position, tremors, hyperactivity, aggressiveness, tonoclonic convulsions and hematuria were observed in animals of the 0.025 mg/l group. There were no behavioral abnormalities in rats in any of the groups during the recovery period. Statistically significant increased bw gain was noted for males and females of the highest dosage group, and for females in the 0.025 mg/l group. Slight increased food consumption was noted for males and females of the highest dosage group. Relative water consumption was increased during periods of the study for males and females of the 2 highest dosage groups. Hematological investigations and biochemical examinations revealed no changes considered to be related to the compound administration. The few statistically significant changes observed were within the range of normal biological variability for the strain of the test species and in many cases nonconcentration-related. There were no changes in relative organ weights considered to be related to the compound administration. No treatment-related macroscopic or microscopic findings were noted in any of the rats surviving the duration of the study. One male that died showed aspiration pneumonia. The other 3 animals that died showed cell atrophy in thymus and bone marrow and contraction of the spleen. In addition, blood congestion and focal or single hyperkeratosis in the fore-stomach was noted in two of these animals.

NOEL/NOAEL for male and female rats was 0.012 mg/l air based on clinical signs of toxicity (neurotoxic symptoms) and increased bw gain (females only) noted in animals receiving 0.025 mg/l air. In addition to that, mortalities and increased food consumption were noted in animals of the highest (0.05 mg/l air) dosage group.

During a second 28-day inhalation study in CD rats, the systemic toxic potential of technical liquid Glufosinate ammonium (50% aqueous solution) to rats by nose-only inhalation administration for 6 hours a day, 5 days a week, was assessed over a period of 4 weeks. Two groups, each comprising five male and five female rats received Glufosinate ammonium at target levels of 0.05 or 0.1 mg GA/L. These concentrations are equivalent to 0.109 and 0.205 mg/L of the technical liquid, respectively. A similarly constituted Control group was exposed to clean air only. During the study, clinical condition, detailed physical examination, bodyweight, food consumption, ophthalmic examination, hematology, blood chemistry, organ weight, and macropathology investigations were undertaken. A Group 3 female rat was sacrificed on humane grounds on Day 3 of the study due to adverse clinical signs including piloerection, unsteady gait, partially closed eyelids, hunched posture and pallor. There were no treatment related clinical signs observed in male animals exposed at 0.109 mg/L. In females exposed at 0.109 mg/L there were occasional incidences of aggression, salivation and vocalisation in one animal; underactivity and hunched posture in a second animal; and repetitive head movements in a third. In view of the low incidence and transient nature of these signs and their absence in males, these observations are considered treatment-related but not adverse. Treatment related clinical signs were observed in all animals given 0.205 mg/L, including irritable, nervous and underactive behaviour. Repetitive movements of the head were seen intermittently in a proportion of male and female animals. Male rats were observed to have several bite marks following 2 exposures to the test compound and these animals were housed singly from Day 3 of exposures. These clinical signs represent a clear response to treatment at this dose. There were no treatment-related effects on bodyweight gain, food consumption or ophthalmic findings considered to be attributed to the administration of Glufosinate ammonium. A dose related reduction in mean urea levels in all treated groups was seen in Week 4, but this was only statistically significant for females in Group 3. Increased kidney weights were seen in treated males receiving 0.109 and 0.205 mg/L.

The no observed adverse effect level (NOAEL) was considered to be 0.109 mg/L of the technical liquid (corresponding to 0.05 mg GA/L) but, due to the minor transient clinical signs seen at this level a no observed effect level (NOEL) could not be established.

Considering toxicity after repeated inhalation exposure to GA , the 28-day study with the technical liquid is regarded as key study, as GA is only handled in aqueous solution. This is reflected in the legal classification (CLP Annex VI), concluded on by the ECB in 2007. All the data on repeated dose toxicity available in this REACh dossier has been available to ECB for their classification decision.

 

Repeated dose toxicity, dermal route of exposure

Rat

Wistar rats (6/sex/group) were dermally exposed to glufosinate-ammonium (GA) for 6 hrs/d, 5 d/week (21 applications over 30 d) at dose levels of 0, 100, 300 or 1000 mg/kg bw/d. The control animals received the vehicle only. Additionally, 5 animals/sex/group were treated by a similar regimen at dose levels of 0 or 1000 mg/kg bw/d followed by a 14-d recovery period. No deaths occurred during the study. One male animal of the highest dosage group refused its food almost entirely from the beginning of the second week of treatment and had to be removed from the study in a cachectic condition on day 16; encrusted blood was also observed around the eyes and the mouth/nose of this animal. Clinical signs observed in rats at the highest dosage level were timid or aggressive behavior, increased motor excitation, piloerection or squatting position. One male animal of the 300 mg/kg bw group showed aggressive behavior, squatting position, piloerection and convulsive jumping and rolling spasms at the end of the treatment period. No dermal irritation was noted in any group. Bw gain, food- and water consumption data showed no substance-related changes in any of the dose groups. Hematological examinations and urinalysis revealed no changes considered to be related to the compound administration. Biochemical examinations showed a statistically significant increase in uric acid in the serum in the males of the 300 and 1000 mg/kg bw groups. There were no changes in absolute or relative organ weights considered to be related to GA-exposure. Macroscopic or microscopic examinations did not reveal any abnormal GA-related findings. The male animal of the highest dosage group, which was killed in a moribund condition on day 16, showed contracted spleen.

NOEL/NOAEL for male rats was 100 mg/kg bw/d based on clinical signs of toxicity (neurotoxic symptoms) and minor changes in biochemical parameters (increase in uric acid in the serum) noted in male animals of the 300 and 1000 mg/kg bw/d groups. NOEL/NOAEL for female rats was 300 mg/kg bw/d based on clinical signs of toxicity (neurotoxic symptoms) noted in female animals of the 1000 mg/kg bw/d groups.

Summary

In conclusion, the main treatment-related findings observed after glufosinate ammonium administration through oral, inhalation or dermal routes, are neurological signs. The most sensitive species is dog, followed by mouse (oral route of exposure).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jun 2006 - Feb 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (28-Day (Subacute) Inhalation Toxicity Study

Version / remarks:

May 1981

Deviations:

yes

Remarks:

no histopathological examination, temperatures ranged from 18-23°C

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

50% aqueous solution

Species:

rat

Strain:

other: Crl:CD(SD)IGS BR

Details on species / strain selection:

The rat was chosen as the test species because of its acceptance as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The Crl:CD® (SD)IGS BR strain was used because of the historical control data available in this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, Kent, England
- Total number: 45 males and 45 females, 23 each were allocated to the study
- Age at study initiation: control and low dose: 61-74 days; high dose: 75-88 days
- Weight at study initiation: males (control and low dose): 337-376 g; females (control and low dose): 197-252 g; males (high dose): 385-471 g; females (high dose): 211-276 g
- Fasting period before study: overnight before blood sampling and during exposure
- Housing: groups of five
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 25 days (control and low dose); 39 days (high dose)

DETAILS OF FOOD AND WATER QUALITY: Routinely checked for chemical and microbiological contaminants

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40-70
- Air changes (per hr): monitored
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 08 June 2006 To: 14 Aug 2006

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

ca. 0.26 - ca. 2.71 µm

Remarks on MMAD:

Group 2: MMAD (µm) was between 0.26 and 2.71. Inhalable fraction % <7 µm: 92; GSD 2.15
Group 3: MMAD (µm) was between 0.26 and 2.57. Inhalable fraction % <7 µm: 91; GSD 2.29

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The inhalation system comprised a snout-only inhalation exposure chamber, restraining tubes a concentric jet atomiser, a glass elutriator and a syringe pump. The snout-only inhalation chamber was a modular apparatus of aluminium alloy construction comprising a base unit, a variable number of animal exposure sections and a top section incorporating a central aerosol inlet surrounded by a tangential air inlet.
- Method of holding animals in test chamber: Restraining tube
- Source and rate of air: not specified
- Method of conditioning air: Air supply and extract were monitored using in-line flowmeters.
- System of generating particulates/aerosols: A polypropylene syringe of Test Article was positioned onto a syringe driver, which delivered Test Article to the inlet of a concentric jet atomiser via a polythene needle.
- Temperature, humidity, pressure in air chamber: Temperature was recorded manually, every 30 minutes during exposure; a small negative pressure
- Air flow rate: 1 l/min
- Air change rate: not specified
- Method of particle size determination: Marple Personal Cascade Impactor
- Treatment of exhaust air: not specified

TEST ATMOSPHERE
- Brief description of analytical method used: gravimetry
- Samples taken from breathing zone: yes

OTHER:
All animals (including spares) were subjected to restraint procedures and exposed to air only (‘Sham dosing’) for 5 or 6 days, in order to accustom animals to the restraining procedure

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber concentrations
The achieved chamber concentrations of Glufosinate and total particulate were calculated from the mass of Glufosinate and total material deposited on the open face filter sample and the volume of chamber atmosphere sampled

Duration of treatment / exposure:

4 w

Frequency of treatment:

6 h/d, 5 d/w

Dose / conc.:

0.05 mg/L air

Remarks:

0.056 mg/l (actual dose exposed) equivalent to 0.109 mg/l of the technical liquid

Dose / conc.:

0.1 mg/L air

Remarks:

0.105 mg/l (actual dose exposed) equivalent to 0.205 mg/l of the technical liquid

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on an inhalation study performed previsouly with the solid form.
- Rationale for administration route: The inhalation route of administration was chosen to simulate the conditions of potential human exposure.
- Rationale for animal assignment (if not random): random
- Fasting period before blood sampling for clinical biochemistry:
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups:
- Section schedule rationale (if not random):
- Other:

Positive control:

not included

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: On the days of exposure (pre-exposure, during exposure, up to 2 h post-exposure, as late as possible in the working day)
- Time schedule for general health: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: One week pre-exposure, on exposure starting date, weekly throughout treatment, and before necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean weekly diet consumption calculated as g food/rat/week: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-exposure and during week 4 of treatment
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during week 4 of treatment (before dosing)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: Haematocrit (Hct), Haemoglobin concentration (Hb), Erythrocyte count (RBC), Reticulocytes (Retic), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Mean cell volume (MCV), Total white cell count (WBC), Differential WBC count, Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC), Platelet count (Plt). Additional blood samples were taken to examine prothrombin time (PT) and activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during week 4 of treatment (before dosing)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Total bilirubin (Bili), Urea
Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Total protein (Total Prot). Electrophoretic protein fractions; (Alb), alpha1 globulin (a1), alpha2 globulin (a2), beta globulin (Beta), gamma globulin (Gamma) were analysed with agarose gel, albumin

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes / No / Not specified
- Time schedule for analysis:
- Dose groups that were examined:
- Number of animals:
- Parameters checked in table [number] were examined.
LUNG BURDEN: Yes / No / Not specified
- Time schedule for analysis:
- Dose groups that were examined:
- Number of animals:
- Parameters checked in table [number] were examined.

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all animals
- a full macroscopic examination of the tissues was performed
- The following organs were weighed: adrenals, heart, kidneys, liver, lungs including bronchi, spleen, testes

HISTOPATHOLOGY: No

Other examinations:

Tissue glutamine synthetase: These samples were the subject of a separate study (not reported herein) to investigate possible effects on glutamine synthetase activity.

Statistics:

- Bartlett's test: body weight, blood chemistry and hematology, organ weights
- Fisher's exact test, Mantel test, Dunnett's test, Bartlett's test, William's test, square-root transformation, Shirley's test: body weight, organ weight, and clinical pathology data

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- 0.205 mg/l: Treatment-related clinical signs following exposure to the test substance were noted in males and females. The number and incidence of these signs was greatest post exposure on Day 2 and pre exposure on Day 3. Irritable – slight to moderate (5/5 males, 1/5 females); nervous – slight to moderate (3/5 males, 2/5 females) and underactive – slight to moderate (2/5 males, 3/5 females) behaviour was observed intermittently pre, during and postdose in the majority of animals. Repetitive sideways movements of the head – slight to marked (1/5 males, 2/5 females) were observed following 2 exposures to the test substance. Other signs recorded for animals included salivation, piloerection, vocalising, eyes partially closed, fast/or shallow breathing and standing on hind limbs. Ungroomed coats in all males. All males were noted to have several bite marks at the first check on Day 3. Following exposure on Day 3, males were housed individually.
- 0.109 mg/l: In females: eyes partially closed (3/5), fast breathing (1/5), underactive – slight (1/5), vocalising (1/5), salivation (1/5), aggressive (1/5), hunched posture (1/5) and pallor – slight (1/5). Vocalising, salivation and aggression were all recorded in the same animal. These signs most commonly resolved by the final check on Day 3. A single female displayed repetitive sideways movements of the head on Day 3 and 4 of exposures. Ungroomed coats in 2/5 females

Mortality:

mortality observed, non-treatment-related

Description (incidence):

- 0.205 mg/l: One female was killed on humane grounds on Day 3 of exposure due to clinical signs of, piloerection, unsteady gait, partially closed
eyelids, hunched posture and pallor.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean bodyweight gain for male exposed rats was similar to or less than Control for 0.109 mg/l and 0.205 mg/l, respectively. Mean bodyweight gain for females exposed rats was statistically significantly more than Control. In view of the inconsistent responses for the 2 sexes and the lack of dose relationship it is considered that the differences in weight gain are fortuitous.

Food consumption and compound intake (if feeding study):

no effects observed

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):

- 0.205 mg/l: Mean haematocrit, haemoglobin, mean cell haemoglobin, mean cell haemoglobin concentration and mean cell volume were all slightly higher for males and females. Although the degree of elevation was slight (typically 1.03 to 1.09Xcontrol), statistical significance was achieved for most parameters at this dose level and in both sexes. Large unstained cell counts were statistically significantly higher in males and females. However, these changes were very small and are not considered to constitute an adverse effect.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Mean urea levels were lower in all treated groups of males and females (by up 0.79x control) with a dose related trend apparent. Statistical significance was attained for females given 0.205 mg/L. The toxicological significance of this finding is unknown.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

- 0.205 mg/l: Increased aggression was evident in males following exposure on Day 2. Irritable – slight to moderate (5/5 males, 1/5 females); nervous – slight to moderate (3/5 males, 2/5 females) and underactive – slight to moderate (2/5 males, 3/5 females) behaviour was observed intermittently pre, during and postdose in the majority of animals. Repetitive sideways movements of the head – slight to marked (1/5 males, 2/5 females) were observed following 2 exposures to the test substance. Vocalising and standing on hind limbs.
- 0.109 mg/l: In females: underactive – slight (1/5), vocalising (1/5), aggressive (1/5), hunched posture (1/5) and pallor – slight (1/5). Vocalising and aggression were all recorded in the same animal. These signs most commonly resolved by the final check on Day 3. A single female displayed repetitive sideways movements of the head on Day 3 and 4 of exposures.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- 0.205 mg/l: In females, dose related increase in absolute and relative lung weights and statistically significant increase in absolute lung weight in males, statistically significant increase in absolute and relative kidney weights in males
- 0.109 mg/l: In females, dose related increase in absolute and relative lung weights, statistically significant increase in absolute lung weight in males, statistically significant increase in absolute and relative kidney weights in males

However, in the absence of a correlation between the sexes, and the absence of microscopic examination to corroborate this finding, the toxicological significance of increased kidney weights is unknown. A dose related increase in lung weights of both male and females rats was noted at termination and is considered to be related to exposure to the test material. In the absence of microscopic examination the aetiology of this increase in weight is unknown.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Dose descriptor:

NOAEL

Effect level:

0.109 mg/L air

Based on:

test mat. (total fraction)

Remarks:

technical liquid

Sex:

male/female

Basis for effect level:

clinical signs

Critical effects observed:

yes

Lowest effective dose / conc.:

0.205 mg/L air

System:

central nervous system

Organ:

brain

Treatment related:

yes

Dose response relationship:

yes

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Oct 1983 - May 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 82-2 (Repeated Dose Dermal Toxicity -21/28 Days)

Version / remarks:

Nov 1982

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Hoe: WISKf (SPF71)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hoechst AG, Pharma Forschung Toxikologie, Kastengrund, Germany
- Age at study initiation: about 8-10 weeks
- Weight at study initiation: males: 143-154 g; females: 144-154 g
- Housing: single
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 8/9 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 55 +/- 10
- Air changes (per hr): 16
- Photoperiod (hrs dark / hrs light): 12/12 (illumination: 7 am to 7 pm)

IN-LIFE DATES: From: 17.10.1983 To: 08/09.12.1983

Type of coverage:

occlusive

Vehicle:

other: physiological saline

Details on exposure:

TEST SITE
- Area of exposure: nape (shaved)
- % coverage: 10
- Type of wrap if used: bandage (aluminium foil, Fixomull-Strech adhesive plaster)
- Time intervals for shavings or clipplings: at least once weekly

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Yes, washed
- Time after start of exposure: Exposure for 6 h. After removal of bandage, treated skin was washed.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 1.3 ml/kg bw
- Concentration (if solution): 7.5, 22.5, 75.0% solution (w/v)
- Constant volume or concentration used: yes
- For solids, paste formed: no, solution in deionized water

VEHICLE
- Justification for use and choice of vehicle (if other than water): deionized water

USE OF RESTRAINERS FOR PREVENTING INGESTION: not specified

Duration of treatment / exposure:

30 days (21 applications)

Frequency of treatment:

Monday-Friday, 6 h/d

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

6 (5 for recovery groups; control and high dose)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
- Rationale for animal assignment (if not random): random
- Fasting period before blood sampling for clinical biochemistry:
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random):
- Other:

Positive control:

not included

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Twice weekly

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule: Twice weekly

WATER CONSUMPTION: Yes
- Time schedule for examinations: Once weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the study
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: All animals
- Parameters checked: hemoglobin, erythrocytes, leucocytes, hematocrit, reticulocytes, differential blood count*, thrombocytes, coagulation time, erythrocyte sedimentation rate, thromboplastin time, activated partial thromboplastin time, methemoglobin*. MCV, MCH, and MCHC were also calculated. (* no evaluation for 100 and 300 mg/kg bw/day groups, since the findings for 1000 mg/kg bw/day were normal)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the study
- Animals fasted: No
- How many animals: All animals
- Parameters checked: sodium, potassium, inorg. phosphorus, uric acid, total bilirubin, direct bilirubin, creatinine, serum glucose, urea nitrogen, calcium, chloride, SGOT, SGPT, AP, LDH, cholesterol, total lipids, total protein, serum electrophoresis
- In view of the results at the end of the treatment period, the only parameters examined at the end of the recovery period were chloride and uric acid in the males and sodium, potassium and chloride in the females.

URINALYSIS: Yes
- Time schedule for collection of urine: At the end of the study (In the night from Day 28 to 29)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Appearance, color, protein, glucose, hemoglobin, bilirubin, pH, sediment. No urinalysis was carried out at the end of the recovery period, since there were no substance-related changes

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Autopsy included skin, orifices, eyes, teeth, oral mucosa, and internal organs
- Organs weighed: heart, lungs, liver, kidneys, spleen, brain, testes (without epididymes)/ovaries, adrenals, pituitary, thyroid

HISTOPATHOLOGY: Yes
heart, urinary, bladder, pancreas, lungs, testes, adrenals, liver, epididymides, thymus, kidneys, prostate, pituitary, spleen, seminal vesicles, brain, stomach, ovaries, eye with optic nerve, jejunum, uterus, bone marrow (femoral), colon, thyroid, treated skin areas, untreated skin areas

Statistics:

Dunnett's test, Sidak test, Nemenyi/Dunnett, Nemenyi/Sidak

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- 1000 mg/kg bw/day: 4 males and 2 females showed piloerection. One male animal refused its food almost entirely from the beginning of the
second week of treatment and had to be removed from the study in a cachectic condition on day 16; encrusted blood was also observed around the eyes and the mouth/nose of this animal.
- 300 mg/kg bw/day: one male animal showed notably aggressive behaviour, squatting position, pilo-erection and convulsive jumping and rolling spasms at the end of the treatment period.

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

On days 5 and 6 of the study, slight signs of irritation appeared on the treated skin areas of the females. During the further course of the study no
signs of dermal irritation were observed in either the females or the males.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

- 1000 mg/kg bw/day: One male was humanely killed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- 300 mg/kg bw/day: decreased APTT in males, increased APTT in females. Decreased leucocytes in males

Since these changes were not dose-related and occurred only to a minimal extent and without further toxicological correlates, they must be considered as non-substance-related and of no toxicological relevance.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- 1000 mg/kg bw/day: In males, a slight increase in uric acid and potassium; Decreases in sodium in females and chloride in both sexes
- 300 mg/kg bw/day: In males, a slight increase in uric acid and alpha3 globulin; decrease in sodium in females
- 100 mg/kg bw/day: Increase in gamma1 globulin in females

Effects cannot be considered as toxicologically relevant (independent of dose level and only to a minimum extent). Uric acid levels were still within range of normal biological variation and were thus considered not toxicologically relevant.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

- 1000 mg/kg bw/day: 4 males and 2 females showed either timid or aggressive behavior, increased motor excitation (especially followign tactile stimuli), or squatting position
- 300 mg/kg bw/day: one male showed notably aggressive behavior, squatting position, convulsive jumping, and rolling spasms

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

clinical signs

Remarks on result:

other: NOEL comparable to NOAEL

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day

System:

central nervous system

Organ:

brain

Treatment related:

yes

Dose response relationship:

yes

Table 1: Clinical findings

Dose group (mg/kg bw/day)	Sex	Day of treatment	Symptoms
control	Male	1-30	No symptoms
	Female	1-30	No symptoms*
100	Male	1-30	No symptoms
	Female	1-30	No symptoms
300	Male
	Animal No 19	10-15 10-19	aggressive behavior easily startled
	Animal No 23	24-30 27-30	Aggressive behavior, ‘kangaroo’ position, piloerection Convulsive jumpings
	Female	1-30	No symptoms
1000	Male
	Animal No 24	7-16 8-16 13-16	Right upper rodent tooth broken, ‘kangaroo’ position, crusted eyes, blood crusted nose Narrowed eye opening Piloerection, hyporeflexia, decreased respiratory rate, abdominal position
	Animal No 26	16-23 20-23 23	Aggressive behavior Easily startled piloerection
	Animal No 27	10-15 10-26	Aggressive behavior Easily startled
	Animal No 28	20-26	Lesion on the left foreleg
	Animal No 30	7-12 7-26	Aggressive behavior Hyperactivity following tactile stimulus
	Animal No 33	10-16 10-22	Aggressive behavior Easily startled
	Female
	Animal No 64	6-15	Hyperactivity following tactile stimulus
	Animal No 68	12-19 12-32	‘kangaroo’ position piloerection

* Four females showed lesions; however, they were due to removing the occlusive bandage by test animal

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Additional information

For additional information please refer to the chapter "specific investigations - neurotoxicity".

Justification for classification or non-classification

Following repeated administration, the most prominent effects consist of neurological signs. Therefore, in accordance with Regulation (EC) 1272/2008Annex VI, classification for target organ toxicity after repeated exposure is warranted (STOT RE Cat.2; H373).