Registration Dossier
Registration Dossier
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EC number: 259-627-5 | CAS number: 55406-53-6
Endpoint summary
Administrative data
Description of key information
Data on subchronic and chronic toxicity via oral route in rats, rabbits and mice indicate that a NOAEL of 20 mg/kg bw/d can be applied for the test item.
Based on observations in a subchronic toxicity study applying dermal exposure to rats, NOEL was determined to be 50 mg/kg bw/d, whereas NOAEL was considered to be 200 mg/kg bw/d.
In a subchronic inhalation toxicity study a NOAEC of 1.16 mg/m^3 was determined in rats.
Key value for chemical safety assessment
Repeated dose toxicity: via oral route - systemic effects
Link to relevant study records
- Endpoint:
- chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 1989-03-21
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- other: U.S. EPA Pesticide Assessment Guidelines F, Subdivision 83-5
- Deviations:
- no
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
- Deviations:
- yes
- Remarks:
- please refer to "principles of method if other than guideline"
- Principles of method if other than guideline:
- Deviations:
- individual body weight of the animals designated for the interim necropsy after one year are not given
- hamatology, clinical chemistry, and urinalysis were not performed after 18 months. Only 10 instead of 20 animals per sex and group were bleed - GLP compliance:
- yes
- Limit test:
- no
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: P-2848-8603-P100 - Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River (U.K.) Limited, Manston, Kent, U.K.
- Age at study initiation: ca. 6 weeks
- Weight at study initiation: 191 - 195 g (males); 132 - 137 g (females)
- Housing: group housing: 5 males or 5 females per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 14 days
ENVIRONMENTAL CONDITIONS
- Temperature: 20 +/- 2 °C
- Humidity: 55 +/- 5 %
- Air changes: ca. 14 per hr
- Photoperiod: 12 / 12 hrs dark / hrs light - Route of administration:
- oral: feed
- Vehicle:
- unchanged (no vehicle)
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS: Diets were prepared by direct admixture of the test material to the diet and mixing for 20 min on a Winkworth Change Drum Mixer.
DIET PREPARATION
- Rate of preparation of diet: twice each week - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- A 100 g sample of diet from each group/sex was retained immediately after each diet preparation.
In addition, samples from all diets were analysed during Weeks 1, 3, 5, 7, 9, 12, 15, 18, 22, 23, 26, 27, 32, 33, 39, 45, 46, 51, 52, 57, 60, 64, 71, 77, 83, 91, 92 and 97 under a separate project (IRI Project No. 335018). - Duration of treatment / exposure:
- Interim kill animals: 13, 26, 51 weeks
Complete study: 104 weeks - Frequency of treatment:
- daily
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Dose / conc.:
- 20 mg/kg bw/day (nominal)
- Dose / conc.:
- 40 mg/kg bw/day (nominal)
- Dose / conc.:
- 80 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- Complete study: 50
- Control animals:
- yes, plain diet
- Details on study design:
- - Dose selection rationale: preliminary sub-chronic toxicity study
- Rationale for animal assignment: random - Positive control:
- not applicable
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily examination for reaction to treatment; detailed clinical examination and palpation once each week
BODY WEIGHT: Yes
- Time schedule for examinations: weekly until week 13; and at 4 weely intervals after week 16
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, per group of n = 5; twice a week until week 13 and 4 weekly intervals thereafter
FOOD EFFICIENCY: No
WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: water consumption was monitored by visual inspection throughout the treatment period
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pre-treatment and at interim kill
- Dose groups that were examined: control and high-dose group (80 mg/kg bw/d)
HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 13, 26 and 51 (interim kill group) and week 100
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: 10 per sex per dose
- Parameters examined: Haematocrit, haemoglobin concentration, erythrocyte count, total and differential leukocyte count, platelet count, Hepato Quick test
- Differential blood count: parameters: neutrohpiles, lymphocytes, monocytes, and eosinophiles
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 13, 26 and 51 (interim kill group) and week 100
- Animals fasted: Not specified
- How many animals: 10 per sex and dose
- Parameters examined: Blood urea nitrogen, glucose, AST, ALT, AP, sodium, potassium, calcium, chloride, total protein, albumin, albumin-globulin ratio, creatinine, inorganic phosphorus, bilirubin total, cholesterol, plasma RBC and brain cholinesterase activity using the methods of Ellman or Pilz
URINALYSIS: Yes
- Time schedule for collection of urine: week 13, 26 and 51 (interim kill group) and week 100
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters examined: Volume, pH, specific gravity, protein, glucose, ketones, blood pigments, bilirubin, urobilinogen; microscopic examination of the spun deposit
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes; all organs taken from control and high dose animals, as well as premature decedents from low- and mid- dose - Statistics:
- Organ weight and body weight data were statistically analysed for homogeneity of variance using the F-max test. If the group variances appeared homogeneous a parametric ANOVA was used and pairwise comparisons made yia Student's t-test using Fisher's F-protected LSD. If the variances were non-homogeneous log or square root transformations were used in an attempt to stabilise the variances. If the variances remained non-homogeneous, then a non-parametric test such as a Kruskal-Wallis ANOVA was used.
Histopathology data were analysed using Fisher's Exact Probability test. - Clinical signs:
- no effects observed
- Description (incidence and severity):
- There were no clinical signs that could be attributed to dosing with the test item. Those observations recorded, such as perigenital swellings, encrustations around eyes and nose, staining on the body, are considered to be normal for rats of this age and strain.
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- There were 142 unscheduled deaths distributed as throughout the dose groups. There was no statistical evidence of any differentrial mortality between any of the dose groups receiving test item and controls nor was there any common factor evident.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Males
There was a dose related reduction in body weight gain across all the dose groups which received test item; slight in the Low dose (11%); moderate in the Intermediate dose (14%) and marked in the High dose (29%). These reductions, in terms of absolute body weight, attained statistical significance (P < 0.05 – P < 0.001) from Week ca. 1 onwards.
Females
There was a moderate reduction in body weight gain seen in the Intermediate dose group (11%) and a marked reduction in the High dose group (24%). These changes in body weight attained statistical significance (P<0.05 - P<0.001) from Week ca. 1 onwards in terms of absolute body weight.
Body weight gain in the Low dose was similar to that of Controls. - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Males
There were slight reductions in total food consumed in all dose groups which received test item; 5% Low dose; 6% Intermediate dose and 10%, High dose group. Only the latter is considered to be outside the normal intergroup variability of food consumption.
Females
There were slight reductions in total food consumed in the Intermediate and High dose groups (6% and 4% respectively), but these are considered to be within the bounds of normality. Total food consumed in the Low dose was similar to that of Controls. - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- no effects observed
- Ophthalmological findings:
- no effects observed
- Haematological findings:
- no effects observed
- Description (incidence and severity):
- Males
Week 13: The High dose group showed reductions compared to the Controls in haemoglobin concentration (7 %, P < 0.01), RBC counts (6 %, P < 0.01) and haematocrit (5 %, P < 0.05). The Low and Intermediate dose groups tended to have values close to the Controls. MCHC was also reduced in the Intermediate (3 %, P < 0.001) and High (3 %, P < 0.01) dose groups.
Week 26: The reductions in RBC parameters seen at Week 13 were not evident. The only instances of statistical significance were equivocal reductions in lymphocyte counts in the Intermediate (23 %, P < 0.05) and High (20 %, P < 0.05) dose groups, but the low magnitude of effect suggests this is of no importance biologically.
Week 51: As in Week 26 lymphocyte counts showed equivocal reductions in the Intermediate (20 %, P < 0.05) and High (17 %, not significant) dose groups, again not considered to be of biological importance.
Week 100 (Carcinogenicity Study Animals): All groups receiving test item showed decreased MCH values compared to Controls, but this is thought to be due to high Control values as there was no effect on the other RBC parameters. Platelet counts were increased compared to Controls in all groups receiving test item, but this is considered to be a chance effect due to lack of a relationship to dose level and high individual variability. Low dose neutrophil counts were decreased but this again did not follow a dose relationship.
Females
Week 13: The only instance of statistical significance was a slight increase in lymphocyte levels (36 %, P < 0.05) in the Low dose group, but this is considered a chance effect due to the small magnitude of effect and lack of an obvious pattern related to dose administered.
Week 26: Only the Low dose group Hepato Quick time showed a statistically significant effect (8 %, P < 0.05), but this is considered a chance effect due to the lack of effect in the other dose groups.
Week 51: On this occasion all groups receiving test item showed decreased MCH and MCV values compared to Controls, but this is thought to be due to high Control values as there was no effect on the other RBC parameters and there had been no difference at the earlier timepoints.
Week 100 (Carcinogenicity Study Animals): There was no evidence of any treatment related effects. Those intergroup differences that did occur showed no dose relationship.
In conclusion, haematological parameters were not considered to be changed by test item treatment. Because dose-response relationship could not be shown, effects were not consistent throughout the treatement period and/or were of low magnitude. Therefore, changes observed were considered within normal biological variation. - Clinical biochemistry findings:
- no effects observed
- Description (incidence and severity):
- Males
Week 13: The Intermediate and High dose groups ALT levels were lower than Controls with the reduction in the High dose group achieving statistical significance (22 %, P < 0.01). All groups receiving test item showed higher cholesterol levels than Controls, with the Low (16 %, P < 0.05) and High (21 %, P < 0.01) dose groups achieving statistical significance. Low dose group AP levels were lower (22 %, P < 0.01) than Controls, but is considered a chance effect as the other groups receiving test item showed no difference. There was a reduction (21 %, not statistically significant) in red blood cell cholinesterase activity in High dose rats.
Week 26: The Intermediate and High dose groups showed lower AST (20%, P<0.01 and 23%, P<0.001 respectively) and ALT (17%, P<0.05 and 24%, P<0.01 respctively) than Controls. As at Week 13 all groups receiving test item showed higher cholesterol levels than Controls, but only the High dose group achieved statistical significance (25%, P<0.001). Again, there was a reduction (20%, not statistically significant) in red blood cell cholinesterase activity in High dose rats. The High dose group showed increased creatinine levels (7%, P<0.05) compared to Controls. The statistical significances achieved by the Low and Intermediate dose group albumin-globulin levels (P<0.01 in both cases) are considered to be chance effects as the High dose group was the same as Controls and there was no notable effect on the other protein parameters.
Week 51: Sodium levels were decreased in all groups receiving test item with the Low and High dose groups achieving statistical significance (3%, P<0.01). All groups receiving test item showed lower phosphate levels than Controls; Low dose -15%, P<0.01; Intermediate dose -9%, P<0.05; High dose -12%, P<0.01, Calcium levels were also slightly reduced in the High dose group only (5%, P<0.01). The High dose group once more showed reduced (32%, P<0.01) red blood cell cholinesterase levels compared to Control.
Week 100 (Carcinogenicity Study Animals): All groups receiving test item showed lower plasma cholinesterase levels than Controls, but as the dose response was inverted and a high degree of individual variation was present, this was considered to be a chance effect. RBC and brain cholinesterase levels showed no intergroup differences.
The only other intergroup difference to show statistical significance was a reduction in High dose calcium levels (6%, P<0.01). Although there was evidence of a dose relationship and this parameter had shown a slight reduction at Week 51, the effect is not considered to be of clinical importance.
Females
Week 13: All groups receiving test item showed lower plasma cholinesterase levels than Controls. This achieved statistical significance in the Intermediate (23%, P<0.05) and High (38%, P<0.001) dose groups.
Week 26: On this occasion only the High dose group showed a reduction (35%, P<0.05) in plasma cholinesterase levels. Other instances of statistical significance are considered to be chance effects.
Week 51: The Intermediate and High dose groups showed reductions (29%, P<0.05 and 39%, P<0.01, respectively) in plasma cholinesterase levels. These groups also showed reductions (15%, P<0.01 in both cases) in blood urea nitrogen levels.
Week 100 (Carcinogenicity Study Animals): As at previous timepoints the High dose group showed lower plasma cholinesterase levels than Controls (14%, P<0.05). RBC and brain cholinesterase levels showed no intergroup differences.
In conclusion, changes in clinical chemistry parameters were not considered to be of toxicological relevance, because they were not observed throughout the treatment period and/or were observed without a dose-response relationship. Furthermore, changes were of low magnitude and are therefore considered to be of normal biological variation. - Urinalysis findings:
- no effects observed
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Description (incidence and severity):
- There were no notable intergroup differences in either sex.
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- The only notable intergroup differences that could be correlated with histopathological findings were associated with the stomachs of Intermediate dose males and High dose males and females, where the incidences of depressed and raised foci were increased.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Week 52 Sacrifice:
Stomach:
Acanthosis was present as a significant treatment related change in the High and Intermediate groups of both sexes although it was also present in 2 Control and 4 Low dose males. Hyperkeratosis, present in a number of groups was only significant in the Intermediate dose group males. Submucosal inflammation was a treatment related change in female rats of the High dose group. Other changes, that did not achieve significance, but were possibly related to treatment, by pathological association, were ulceration, submucosal oedema and inflammation of the keratin layer itself. With the exception of acanthosis, the gastric lesions in the Low dose males were seen as only single incidences and never were 2 lesions seen in the same animal. These findings were not seen in the Low dose females.
All other changes observed were not related to treatment and were those neoplastic and non-neoplastic changes that are commonplace in laboratory rats of this age and strain.
Premature Decedents: The premature decedent High dose male (in which the gastric section was examined) had the apparent treatment related lesions of acanthosis and keratin layer inflammation. There were no severe lesions in the other 2 premature decedents.
Week 104 Sacrifice:
There were a number of treatment related findings. The major effects were seen in the keratinized stomach and salivary glands of both sexes. In the keratinized stomach of the Intermediate and High dose groups of both sexes, there were increased incidences of submucosal inflammation, acanthosis, hyperkeratosis, and basal cell proliferation. Significant increases in the incidences of submucosal oedema were seen only in the male Intermediate and High dose groups. Inflammation of the keratin layer also affected the male High dose group. There were also increased incidences of ulceration in the male High dose group and in the female Intermediate and High dose groups. Lesions associated with ulceration were increased in the female Intermediate and High dose groups. Salivary gland lobular degeneration was increased in both the male and female Intermediate and High dose groups, but in addition, the male High dose group also showed increased incidences of fibroplasia.
Other findings that were increased in incidence in the male Intermediate and High dose groups included pulmonary foamy macrophage aggregates and thyroid colloid basophilia.
Other findings which were increased in incidence in females included compression of the brain thalamus in the Low and Intermediate dose groups, and internal hydrocephalus in the Intermediate dose group. The lack of these effects in the High dose groups eliminated these changes as being truly related to treatment and are consequently believed to result from chance incidences.
In the males there were 2 findings that were reduced with treatment; vacuolation of the lumbar spinal cord in the High dose group and basophilic cell foci of the liver in all dose groups which received test item.
Other changes which occurred with greater frequency in the male High dose group than in the Control group included cardiac chronic active myocarditis and pancreatic lobular degeneration. These changes were statistically significant only in the terminal kill animals, and ceased to be statistically significant when combined with decedents and those killed prematurely. - Histopathological findings: neoplastic:
- no effects observed
- Description (incidence and severity):
- Week 52 Sacrifice:
There were occasional neoplasms present. The incidence of these indicated that there was not a treatment related change.
Week 104 Sacrifice: There were no neoplasms that showed a treatment related increase.
In females, the incidence of mammary fibroadenomas was increased in the Low dose group and the incidence of pituitary adenomas was increased in the Intermediate dose group. These increases are regarded as spurious. A consideration of the overall tumour incidence in the Control and High dose groups did not indicate a treatment related increase in either sex. - Other effects:
- no effects observed
- Description (incidence and severity):
- Cholinesterase activity:
At termination, plasma cholinesterase activity was statistically significantly decreased in all treated males, which is considered as a result of high plasma cholinesterase activity in concurrent control. Reduced plasma cholinesterase activity was observed in females at High-Dose group and occasionally in Mid-dose group. Whereas RBC cholinesterase was reduced at only one single time point and thus, a dose-response relationship is lacking. Furthermoe, no difference in brain cholinesterase is activity was observed. - Details on results:
- Analysis of formulated diets:
Generally, all diets were within acceptable limits of accuracy and homogeneity (+/- 15 %). Only occasional diets fell outwith this limit; analyses of these diets were repeated at subsequent mixes and proved to be satisfactory showing that there was no continuing problem with mixing. - Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 40 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 20 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- Key result
- Critical effects observed:
- no
- Conclusions:
- Dosing Sprague-Dawley rats with test item via the diet for 104 weeks produced reductions in body weight gain at 20, 40 and 80 mg/kg bw/day in males and at 40 and 80 mg/kg bw/day in females. Both males and females receiving 40 or 80 mg/kg bw/day showed increased incidences of lesions in the keratinised stomach (submucosal oedema and inflammation) and submaxillary salivary glands (lobular degeneration).
There was no evidence of carcinogenic potential in either sex. A no observable effect level in terms of major structural effect can be set out at 20 mg/kg bw/day in both sexes. - Executive summary:
Groups of 50 male and 50 female Sprague-Dawley rats were offered diet ad libitum formulated with test item at concentrations calculated to give dose levels of 20, 40 and 80 mg/kg bw/day. A further group of 50 males and 50 females were offered untreated diet to act as Controls. During Weeks 53/54, 79 and 104 a blood smear was prepared via tailsnip from all surviving animals and a differential blood count performed on Control and High dose group animals. During Week 100 blood samples were taken from 10 males and 10 females from each group in order that a haematology and clinical chemistry screen could be carried out in order to put in context results from earlier time points on the concurrent toxicity study.
After 104 weeks continual dosing all surviving animals were killed by carbon dioxide asphyxiation followed by exsaguination and a detailed necropsy, with a selection of organs being placed in fixative. All organs taken were examined histopathologically from all Control and High dose animals, as well as premature decedents from the Low and Intermediate dose groups. In addition liver, kidneys, lungs, stomach and salivary glands were examined from all survivors in the Low and Intermediate dose groups.
Regarding mortality, there were no notable intergroup differences observed. There were no clinical signs thought to be attributable to dosing with the test item. There was a reduction in body weight gain in all male dose groups which receive test item ranging from slight in the Low dose group to marked in the High dose group. There was also a moderate reduction in body weight gain in the female Intermediate dose group and a marked reduction in the female High dose group. A slight reduction in food consumption in the male dose group receiving 80 mg/kg bw/day was found. There were no visual intergroup differences observed regarding water consumption.
There were no notable intergroup differences at any time in regards to differential blood counts nor in regards to organ weights in either sex. Intermediate dose males and High dose males and females showed increased incidences of depressed and raised foci in the stomach. Effects of treatment were present in the salivary glands and stomach of Intermediate and High dose groups in both sexes. In the submaxillary salivary gland lobular degeneration was evident in both groups, both sexes. In the keratinized stomach of both groups, in both sexes, notable intergroup differences were seen for submucosal oedema, submucosal inflammation, acanthosis, hyperkeratosis, inflammation in the keratinized layer, ulceration, basal cell proliferation and 'lesions associated with ulcer'.
Accroding to a subsequent pathological review and interpretation of selected thyroid and forestomach lesions, lesions reported are not thought to pose a potential carcinogenic risk to humans.
There were no lesions reported in the thyroid which were thought to be related to administration of the test item. The increased incidences of colloid basophilia of the thyroid reported in mid and high dose male rats represented several artifacts which happened to be reported more commonly in these treatment groups.
Administration of the test item was associated with non-neoplasitc proliferative and inflammatory lesions of the forestomach at 40 and 80 mg/kg bw/d in both male and female rats. These lesions are not thought to pose a potential carcinogenic risk to humans.- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 1983-08-25 to 1984-05-11
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
- Deviations:
- yes
- Remarks:
- please refer to "principles of method if other than guideline"
- Principles of method if other than guideline:
- Heamatological parameter for blood clotting as well as triglycerides and cholesterol determiniation was not performed.
- GLP compliance:
- yes
- Limit test:
- no
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: YY-97-III
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature - Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Breeding Laboratory, Kingston, NY
- Age at study initiation: 28 d
- Weight at study initiation: 217 - 280 (males); 140 - 191 (females)
- Diet: ad libitum
- Water: ad libitum
ENVIRONMENTAL CONDITIONS
- Temperature: 23.3 +/- 5°C
- Humidity: was monitored daily
- Air changes: 6.8 per hr
- Photoperiod: 12/12 hrs dark / hrs light - Route of administration:
- oral: gavage
- Vehicle:
- corn oil
- Details on oral exposure:
- DIET PREPARATION
- Rate of preparation of diet: Concentrations were prepared weekly at 0, 4, 10 and 25 mg/mL in corn oil. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Each dose level of the first, sixth, ninth and thirteenth lot of dose formulation was analyzed by high performance liquid chromatography (HPLC) prior to dose administration. The results of these analyses demonstrated that the mixing procedure yielded the required dose concentrations for this study (all analytical results were within 10% of the target dose concentrations).
- Duration of treatment / exposure:
- 90 d
- Frequency of treatment:
- 5 days per week
- Dose / conc.:
- 0 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 20 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 50 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 125 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: preliminary dose-range-finder study
- Rationale for animal assignment: random
- Post-exposure recovery period in satellite groups: 28 d - Positive control:
- not applicable
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
BODY WEIGHT: Yes
- Time schedule for examinations: weekly
FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each group determined and reported as weekly total: Yes
FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No
WATER CONSUMPTION AND COMPOUND INTAKE: No
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to treatment and at termination
- Dose groups that were examined: all
HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 13
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: all
- Parameters examined: haematocrit, haemoglobin concentration, erythrocyte count, total and differential leukocyte count, platelet count
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 13
- Animals fasted: No
- How many animals: all
- Parameters examined: glucose, blood urea nitrogen, cholinesterase (method not indicated), creatinine, total bilirubin, protein, albumin, chloride, sodium, potassium, calcium, phosphorus, alanine aminotransferase, aspartate aminotransferase, gamma glutamyl transpeptidase
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes - Statistics:
- Means and standard deviations were calculated on body weights, food intake (by cage, means only), hematology parameters, clinical chemistry parameters, organ weights and organ to body weight ratios. One-way analysis of variance was conducted using a TI-59 programmable calculator on parameters suspected of differing from controls. In the case of body weights a number of intervals were suspected of being significantly different and three intervals (weeks 4, 8 and 12) were selected for analysis to confirm the suspected body weight difference.
One-way analysis of variance was also conducted on the initial body weights to confirm the absence of any differences among groups.
Parameters showing significance at p <0.05 through a one-way analysis of variance were further analyzed using the Least Significant Difference test. - Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Animals generally remained normal throughout the study. The test material was irritating to the animals as evidenced by a transient dose related behavior immediately following treatment. This behavior consisted of burrowing in the hard wood chip bedding and was often accompanied by salivation. The behavior was generally noted in levels II, III and satellite animals and was absent in the low dose and control animals.
- Mortality:
- no mortality observed
- Description (incidence):
- There were no deaths due to treatment with the test material. Two animals died due to technical error. This error was due to improper gavage. Male rat # 8 (vehicle control) was found dead on September 27, 1983. Male rat # 37 (level II) was found dead on September 20, 1983.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- A decrease in body weight gain was observed for male animals receiving 125 mg/kg through week 13. The satellite animals, of both sexes, showed a marked increase in body weight gain after treatment with the test material was stopped.
Statistical analysis of these data at weeks 4, 8 and 12 revealed significant decreases in body weight gain (p< 0.05) for male rats at each of these intervals for satellite animals when compared to controls. Level III males were also significantly (p< 0.05) lower than controls at week 12. Female body weights were not different statistically (p<0.05) between high dose (level III or satellite) and the control group at these intervals. - Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- No consistent changes in food intake was found when treated animals are compared to controls. For both male and female satellite animals, a marked increase in food intake was noted after treatment was stopped. In female rats, food intake appeared to decrease after the initial increase and, by week 17, were comparable to values seen between weeks 1-13. This initial increase in food intake could be due to withdrawal of the test material or removal of the effect of corn oil administration on food intake.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Haematological findings:
- no effects observed
- Description (incidence and severity):
- No differences were observed in any parameters due to treatment.
- Clinical biochemistry findings:
- no effects observed
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Analysis of mean organ weight data show that the apparant increase in mean liver weights was statistically significant (p <0.05) in males of group III and in females in groups II and III. The mean liver to body weight ratios were statistically greater than controls (p < 0.05) for females and males of groups II and III. Additionally, the mean female satellite liver to body weight ratio, while lower than group III, was still significantly (p<0.05) greater than that for the vehicle control.
It is noteworthy that the liver weights decreased after cessation of treatment. - Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- Gross lesions were noted for the two animals which died on study. These lesions reflected the sequaelae of improper gavage and subsequent death. Animals surviving treatment and killed at termination of their respective groups were generally normal when necropsied.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Rats treated with 125 mg/kg bw/d had hepatocyte enlargement which was reflected in the greater liver weights. The hepatocytes appeared normal and the increased size was attributed to the induction of drug metabolizing enzymes. Hepatocytes from satellite animals did not show enlargement, correlating with the reduced liver weights of these animals. Cardiomyopathy was elevated in satellite male rats when compared to vehicle control animals. This lesion is often associated with aging in male rats and was not attributed to treatment with the test material.
The forestomach of some rats in all groups showed a very low incidence of edema, acanthosis, hyperkeratosis and inflammation except for level I females, which had a higher incidence. These changes reflected irritation to the forestomach and were judged the result of treatment with the vehicle and test material. Satellite animals did not show these signs of irritancy; this recovery supports the conclusion that this irritancy was transient in nature.
Other lesions noted were incidental, relating to those commonly seen in rats of this strain and age. - Histopathological findings: neoplastic:
- not examined
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- 20 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- clinical signs
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 50 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- clinical signs
- Key result
- Critical effects observed:
- no
- Conclusions:
- Treatment of rats with the test material at dose levels of 0, 20, 50 and 125 mg/kg bw/d by oral gavage, five times per week for 13 weeks produced evidence of toxicity as noted by decreased body weight gain for high dose males. Physiologic response to treatment also resulted in increased liver weights and was reflected by hepatocyte enlargement, due probably to enzyme induction. Satellite animals showed marked increase in body weight gain after treatment was stopped and showed no evidence of forestomach irritation or hepatocyte enlargement. These data suggest that the test material induces only transient changes in the rat.
The maximum test dose for a chronic toxicity study is suggested as 125 mg/kg. - Executive summary:
Graded doses (0, 20, 50 and 125 mg/kg bw/d of the test material were formulated in corn oil and administered by oral gavage five times per week to Sprague Dawley rats for thirteen weeks. A separate satellite group received 125 mg/kg bw/d thirteen weeks and was held without treatment for 28 days after the remaining groups were terminated. Each group consisted of 10 males and 10 females.
The animals were observed for mortality, clinical signs, body weight, food intake, ophthalmologic changes and changes to hematology and clinical chemistry parameters. Each animal was necropsied and selected organs were weighed. Complete microscopic evaluation of the tissues was conducted for all control animals and those receiving 125 mg/kg bw/d (group III and Satellite). Selected tissues, target organs and gross lesions were examined for the animals treated with 20 and 50 mg/kg bw/d.
The test material did not cause deaths among the test animals. Clinically, most animals remained normal throughout the test period. However, there was behavioral evidence of a dose-related irritancy of the test material. This irritancy was transient and the animals returned to normal behavior shortly after treatment. Body weight gain was generally lowered in male rats while food intake was not consistently affected in the treated animals. The decrease in body weight was not dramatic and showed a marked recovery when the satellite animals were removed from treatment. Food intake values also increased in satellite animals at this time. There were no changes to any of the hematology or clinical chemistry parameters measured. Ophthalmologic changes were not evident. The livers of animals treated with 125 mg/kg bw/d were larger than control livers. The livers of satellite animals showed a marked weight reduction toward normal values after treatment was stopped. Microscopic evaluation showed evidence of mild irritation of the stomachs in animals treated with the test material as well as those treated with the vehicle alone. Slight physiologic hepatocyte enlargement correlated with the increased liver mass noted. Satellite animals showed normal liver and stomach histology after the recovery period of 28 days.
- Endpoint:
- sub-chronic toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 1996-03-21 to 1997-02-13
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)
- Version / remarks:
- 1981
- Deviations:
- no
- Principles of method if other than guideline:
- Deviations to current OECD 409: One of the four groups consisted of 6 males and 4 females (recommended 5 animals per sex). Urinalysis was not conducted. Gall bladder ovaries, uterus, thymus, spleen, brain, and heart were not weighed. Prostate was not examined by histopathology
- GLP compliance:
- yes
- Limit test:
- no
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 95068239 - Species:
- rabbit
- Strain:
- New Zealand White
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: HRP Inc. Denver, PA 17517
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 4 months
- Weight at study initiation: 2.2 - 2.5 kg (males); 2.1 - 2.4 kg (females)
- Housing: individually
- Diet: 125 g feed/day
- Water: ad libitum
- Acclimation period: 23 d
ENVIRONMENTAL CONDITIONS
- Temperature: 16- 21 °C
- Humidity: 28 - 74 %
- Photoperiod: 12/12 hrs dark / hrs light - Route of administration:
- oral: feed
- Vehicle:
- unchanged (no vehicle)
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The concentration of test item in the diet was analytically verified for the two highest dose groups (83.2 to 85.9% of nominal). The mean analytical concentration of the low dose was 54.1% of nominal. Test material intake was calculated on the basis of measured test item concentration in the diet and not on the basis of nominal concentration.
- Duration of treatment / exposure:
- 90 d
- Frequency of treatment:
- daily
- Dose / conc.:
- 0 ppm
- Dose / conc.:
- 500 ppm
- Remarks:
- equivalent to approx. 13 mg/kg bw/d
- Dose / conc.:
- 2 000 ppm
- Remarks:
- equivalent to approx. 75 mg/kg bw/d
- Dose / conc.:
- 4 000 ppm
- Remarks:
- equivalent to approx. 150 mg/kg bw/d
- No. of animals per sex per dose:
- 5; except of the low dose group with 6 males and 4 females
- Control animals:
- yes, plain diet
- Details on study design:
- - Dose selection rationale: preliminary dose-range finder study
- Rationale for animal assignment: random - Positive control:
- not applicable
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily
BODY WEIGHT: Yes
- Time schedule for examinations: weekly
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 EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No
WATER CONSUMPTION AND COMPOUND INTAKE: No
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pretest and at termination
- Dose groups that were examined: all
HAEMATOLOGY: Yes
- Time schedule for collection of blood: pretest and at day 45 at 90 (study termination)
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: all surviving
- Parameters examined: haemoglobin concentration, haematocrit, erythrocyte count, reticulocyte count, platelet count, MCV, MCH, MCHC, Prothrombin time, activated partial thromboplastin time, total and differential leukocyte count, erythrocyte morphology
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: pretest and at day 45 at 90 (study termination)
- Animals fasted: No
- How many animals: all surviving
- Parameters examined: AST, ALT, AP, BUN, fasting glucose, total protein, albumin, globulin, A/G ratio, creatinine, total bilirubin, sodium, potassium, chloride, calcium, inorganic phosphate, gamma-glutamyl transferase
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes - Statistics:
- Body weight, body weight change from Day 1, food consumption, hematology and clinical chemistry parameters, terminal organ and body weights and organ/body weight and organ/brain weight ratios were analyzed. Mean values of all dose groups were compared to control at each time interval either by Dunnett’s, by Kruskal-Wallis, or by Dunn’s rank test as appropriate.
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- No adverse effect of treatment with test item was evident from the detailed physical examinations. Observations seen among the treated animals occurred sporadically and at low incidence during the study and were not considered indicative of a treatment-related response.
- Mortality:
- no mortality observed
- Description (incidence):
- No treatment-related mortality occurred during the study. One high-dose male was sacrificed moribund on Day 45 shortly after being bled for the scheduled hematology/clinical chemistry assessment. Morbidity was attributed to handling-related stress during the blood collection procedure and not treatment-related. No other mortality occurred among the treated groups.
One control female died on Day 90 after being bled for the terminal hematology/clinical chemistry assessment. Death was attributed to handling-related stress during the blood collection procedure. No other mortality was seen in the control group. - Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- No adverse effect of treatment at dietary levels of 500 and 2000 ppm was evident from body weight data. Mean weekly body weights for the low- and mid-dose animals were comparable to control data throughout the study. At the 4000 ppm dietary level, an adverse effect of treatment was evident from body weight data. High-dose animals experienced a slight weight loss after one week of treatment in comparison to a slight weight gain seen in the control group. Mean weekly body weights for the high-dose animals continued to be reduced in comparison to control data for the remainder of the study and many of these differences were statistically significant. Mean weight gain over the entire 13 week study period for the high-dose group was also lower than control data.
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- No adverse effect of treatment at dietary levels of 500 and 2000 ppm was evident from food consumption data. At 500 ppm, the test diets were readily consumed from the start of study and food consumption data for this group were comparable to control throughout the 13-week period. At the 2000 ppm dietary level, it took approximately one week for animals to adjust to the test diets. Daily food consumption during this week for this group was slightly lower than control and some of these differences were statistically significant; however, for the remainder of the study, food consumption for the 2000 ppm group was considered comparable to control data.
In the 4000 ppm group, food consumption was depressed early in the study as the animals adjusted to the test diets. Mean food consumption for the high-dose animals was markedly depressed during the first week of study as animals were eating approximately 30% of their daily allotment of feed. There was a gradual increase in food consumption for this group for the remainder of the study and by Week 7 in the males and Week 8 in the females, high-dose animals were eating at approximately the same level as the control animals. - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Description (incidence and severity):
- No adverse effect of treatment with test item was evident from the ophthalmoscopic examinations.
- Haematological findings:
- no effects observed
- Description (incidence and severity):
- No adverse effect of treatment at a dietary level up to and including 4000 ppm was indicated from hematology data. A few statistically significant changes were seen in one or more parameters between the control and a test group at Day 45 and study termination but no consistent response was seen among the test groups in comparison to control data to indicated an adverse effect of treatment.
- Clinical biochemistry findings:
- no effects observed
- Description (incidence and severity):
- No adverse effect of treatment at dietary levels of 500 and 2000 ppm was evident from clinical chemistry data. The few statistically significant changes seen in one or more parameters at Day 45 and termination between the control and one or both of these treated groups were not considered toxicologically significant.
At the high-dose level, mean gamma-glutamyl transferase (GGT) levels for the females were slightly increased in comparison to control at both Day 45 and termination and these differences were statistically significant. GGT levels were also slightly elevated in comparison to control for the high-dose males at termination but these differences were not statistically significant. The elevated GGT levels seen in the high-dose group were considered indicative of a treatment-related response and consistent with the elevated liver weights and microscopic changes (i.e., hepatocellular hypertrophy) seen at this dose level.
The few remaining statistically significant changes seen in one or more of the clinical chemistry parameters at Day 45 and termination between the control and high-dose group were not considered toxicologically significant. Statistically significant reductions in glucose levels in the high-dose animals at Day 45 in comparison to control data were considered related to the decreased food consumption seen in these animals during this portion of the study. - Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- No adverse effect of treatment at a dietary level of 500 ppm was evident from organ weight data. At the 2000 and 4000 ppm dietary levels, the only effect of treatment seen from organ weight data was an increase in liver weights in the females. Mean liver weights for the mid- and high-dose females were approximately 17% and 24%, respectively, heavier than control and this difference in the high-dose group was statistically significant. Mean liver to body weight and liver to brain weight ratios for the mid-and high-dose females were also elevated in comparison to control data and many of these differences were also statistically significant. An increase in liver weight accompanied by elevated liver enzyme levels and microscopic changes as seen in this study at the high-dose level, is not an uncommon response for animals exposed to xenobiotics and may represent an adaptive response to treatment. The spectrum of events that can produce this liver enlargement involve microscopic changes (hypertrophy) accompanied by accumulations of increased amounts of granular eosinophilic cytoplasm resulting from proliferation of the smooth endoplasmic reticulum and other subcellular organelles containing xenobiotic metabolizing enzymes (e.g., GGT).
A statistically significant increase in the kidney to body weight ratio in comparison to control data was seen in the high-dose females; however, in the absence of any histopathological changes in the kidneys for these animals, this was not considered indicative of a toxicologically significant change.
Other organ weight data for the mid- and high-dose animals were considered comparable to control and not adversely affected by treatment. - Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- Red discoloration of the liver was noted in the high-dose males (4/5 animals affected) and females (1/5 animals affected). No microscopic correlate was observed for this change and therefore, it was not considered indicative of a treatment-related response.
There were no macroscopic postmortem changes considered treatment-related. - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- Treatment-related microscopic changes were confined to the livers of Group III (2000 ppm) and Group IV (4000 ppm) male and female rabbits. The most prominent change, hepatocellular enlargement ("Hepatocytes: Cell Swelling)" was present in the livers of rabbits from Groups III (2/5 males; 4/5 females) and IV (5/5 males; 5/5 females). Affected hepatocytes (primarily centrilobular) were slightly to moderately enlarged and had an increased amount of finely granular cytoplasm which often had brownish tinctorial characteristics ("Hepatocytes: Brown Pigment"). Hepatic sinusoidal macrophages also contained a similar brown pigment in Group IV male rabbits. Periportal fibrosis was also noted in the livers from two Group IV female rabbits. Similar findings were not seen in the livers of control or low-dose animals. There were no other microscopic findings considered treatment-related. The changes showed either a similar incidence among the control and the treated groups, or otherwise occurred sporadically.
- Histopathological findings: neoplastic:
- not examined
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- 13 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- histopathology: non-neoplastic
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 75 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- histopathology: non-neoplastic
- Key result
- Critical effects observed:
- no
- Conclusions:
- Test item was administered to male and female New Zealand white rabbits at nominal dietary concentrations of 0, 500, 2000 and 4000 ppm over a 13-Week period. Treatment produced hepatic changes at the two higher concentration levels. These effects consisted of slightly elevated GGT enzyme levels in the high-dose group only, increased liver weights in the females at the two higher dietary levels and microscopic changes consisting of centrilobular hepatocellular enlargement (cell swelling) and brown pigment in the cytoplasm of hepatocytes and sinusoidal macrophages. Therefore the no-observed-effect level (NOEL) was determined to be 500 ppm dietary level, equivalent to a dose level of approximately 13 mg/kg bw/d.
- Executive summary:
This study was designed to assess the sub-chronic toxicity of the test item when administered orally, via the diet, to 30 New Zealand White rabbits. The test material was mixed with the granulated rabbit diet and the mixtures pelletized. Diets at targeted concentration levels of 500, 2000 and 4000 ppm were prepared and animals (five/sex/group) fed these diets for a period of 90 days. Control animals (five/sex) received untreated standard rabbit diet that was granulated and re-pelleted.
Physical observations, ophthalmoscopic examinations, body weight, food consumption, hematology and clinical chemistry evaluations were performed on all animals pretest and at selected intervals during the treatment period.
Following treatment, all surviving animals were sacrificed, selected organs were weighed and organ/body weight and organ/brain weight ratios calculated. Complete macroscopic postmortem examinations were conducted on all animals. Selected tissues were taken from all animals and histopathological evaluation performed for tissues from the control and high-dose animals. Due to findings in the high-dose animals, the livers from the low- and mid-dose animals were also evaluated histopathologically.
Analysis of the first batch of diets confirmed that the preparation procedure used for this study produced homogeneous mixtures.Additional analyses performed either during a two-week pilot study (95-2395) or in this study confirmed that the pelleted diets at the concentration levels used in this study were stable when stored frozen for at least 33 days.The mean analytical concentrations for the diets used on study were 271, 1663 and 3436 ppm and the average dose levels received by the animals consuming these diets over the entire study were approximately 13, 75 and 150 mg/kg bw/day, respectively.
No adverse effect of treatment at the 500 or 2000 ppm dietary levels was evident from body weight or food consumption data. At the 4000 ppm level, animals adjusted more gradually to the test diet, ate less during the early weeks of the study and gained less weight. Treatment produced hepatic changes at the two higher concentration levels. These effects consisted of elevated gamma-glutamyl transferase levels in the high-dose group only, increased liver weights absolute and relative to either brain or terminal body weight, in the females at the two higher dietary levels and microscopic changes in the liver of males and females at the mid- and high-dose levels consisting of swelling of the hepatocytes and brown pigment in the cytoplasm of hepatocytes and sinusoidal macrophages. On the basis of these data, the no-observed-effect level (NOEL) for this study was the 500 ppm dietary level. This was equivalent to a dose level of approximately 13 mg/kg bw/day.
Referenceopen allclose all
Table 1. Two-year Combined Chronic/Carcinogenicity Study in Rats
Parameter |
0 mg/kg bw/d |
20 mg/kg bw/d |
40 mg/kg bw/d |
80 mg/kg bw/d |
dose-response |
||||||
m |
f |
m |
f |
m |
f |
m |
f |
m |
f |
|
|
Number of animals in group |
50 |
50 |
50 |
50 |
50 |
50 |
50 |
50 |
|
|
|
Mortality |
22 |
17 |
20 |
21 |
24 |
10 |
14 |
14 |
- |
- |
|
clinical signs |
ne |
ne |
ne |
ne |
ne |
ne |
ne |
ne |
- |
- |
|
body weight gain [% of control] |
|
|
89 |
99 |
86 |
89 |
71 |
76 |
+ |
+ |
|
total food consumption |
ne |
ne |
ne |
ne |
ne |
ne |
ne |
ne |
|
|
|
clinical chemistry |
ne |
ne |
ne |
ne |
ne |
ne |
ne |
ne |
|
|
|
haematology |
ne |
ne |
ne |
ne |
ne |
ne |
ne |
ne |
|
|
|
urinalysis |
ne |
ne |
ne |
ne |
ne |
ne |
ne |
ne |
|
|
|
Liver |
|
|
|
|
|
|
|
|
|
|
|
adjusted weight |
|
|
|
|
|
|
|
|
|
|
|
interim kill |
19.12 |
10.32 |
18.28 |
10.93 |
19.58 |
12.30*** |
20.60** |
11.72** |
+ |
+ |
|
terminal kill |
18.67 |
14.72 |
20.41 |
12.93 |
20.11 |
14.67 |
18.43 |
13.46 |
- |
- |
|
gross pathology |
|
|
|
|
|
|
|
|
|
|
|
interim kill |
ne |
ne |
ne |
ne |
ne |
ne |
ne |
ne |
- |
- |
|
terminal kill |
|
|
|
|
|
|
|
|
|
|
|
dark red foci |
5 |
11 |
3 |
13 |
8 |
17 |
10 |
19 |
+ |
+ |
|
histopathology |
|
|
|
|
|
|
|
|
|
|
|
interim kill |
ne |
ne |
ne |
ne |
ne |
ne |
ne |
ne |
- |
- |
|
terminal kill |
|
|
|
|
|
|
|
|
|
|
|
basophilic cell focus/ area |
12/50a) |
24/50 |
2**/50 |
17/49 |
2**/50 |
22/50 |
3*/50 |
18/50 |
+ |
- |
|
Submaxillary salivary glands |
|
|
|
|
|
|
|
|
|
|
|
terminal kill |
|
|
|
|
|
|
|
|
|
|
|
lobular degeneration |
1/49 |
1/50 |
3/50 |
4/49 |
16***/50 |
20***/50 |
26***/49 |
26***/50 |
+ |
+ |
|
fibroplasia |
0/49 |
0/50 |
1/50 |
0/49 |
1/50 |
2/50 |
6*/49 |
0/50 |
- |
- |
|
Thyroid 1terminal kill |
|
|
|
|
|
|
|
|
|
|
|
colloid basophilia |
0/49 |
1/50 |
0/22 |
2/21 |
5**/29 |
1/10 |
6*/49 |
2/50 |
+ |
- |
|
Thyroid 2terminal kill |
|
|
|
|
|
|
|
|
|
|
|
colloid basophilia |
0/48 |
1/48 |
0/23 |
2/21 |
2/24 |
1/10 |
6*/48 |
2/49 |
+ |
- |
|
Lungsterminal kill |
|
|
|
|
|
|
|
|
|
|
|
foamy macrophage aggregates |
3/50 |
3/50 |
8/49 |
5/50 |
14**/50 |
6/50 |
18***/50 |
9/50 |
+ |
- |
|
Stomach |
|
|
|
|
|
|
|
|
|
|
|
gross pathology |
|
|
|
|
|
|
|
|
|
|
|
interim kill |
|
|
|
|
|
|
|
|
|
|
|
erosions |
0 |
0 |
0 |
0 |
1 |
0 |
1 |
4 |
- |
+ |
|
terminal kill |
|
|
|
|
|
|
|
|
|
|
|
mass |
0 |
0 |
0 |
0 |
2 |
0 |
2 |
0 |
+ |
- |
|
depressed foci |
4 |
0 |
8 |
4 |
17 |
7 |
21 |
15 |
+ |
+ |
|
Stomach keratinsed/Forestomach |
|
|
|
|
|
|
|
|
|
|
|
interim kill |
|
|
|
|
|
|
|
|
|
|
|
submucosal oedema |
0/15 |
0/15 |
1/15 |
0/15 |
1/14 |
2/15 |
0/14 |
2/15 |
- |
- |
|
submucosal inflammation |
0/15 |
0/15 |
1/15 |
0/15 |
1/14 |
2/15 |
1/14 |
8/15 |
- |
+ |
|
acanthosis |
2/15 |
0/15 |
4/15 |
0/15 |
12/14 |
4/15 |
8/14 |
8/15 |
+ |
+ |
|
inflammation of keratin layer |
0/15 |
0/15 |
1/15 |
0/15 |
1/14 |
2/15 |
1/14 |
3/15 |
- |
+ |
|
hyperkeratosis |
0/15 |
0/15 |
1/15 |
0/15 |
5/14 |
3/15 |
1/14 |
1/15 |
- |
- |
|
ulceration |
0/15 |
0/15 |
0/15 |
0/15 |
1/14 |
0/15 |
0/14 |
3/15 |
- |
- |
|
basal cell proliferation |
0/15 |
0/15 |
0/15 |
0/15 |
0/14 |
2/15 |
1/14 |
0/15 |
- |
- |
|
terminal kill |
|
|
|
|
|
|
|
|
|
|
|
submucosal oedema |
4/49 |
2/50 |
7/49 |
1/49 |
15**/48 |
7/50 |
19***/49 |
6/50 |
+ |
- |
|
submucosal inflammation |
5/49 |
1/50 |
9/49 |
3/49 |
26***/48 |
8*/50 |
28***/49 |
20***/50 |
+ |
+ |
|
acanthosis |
13/49 |
8/50 |
13/49 |
7/49 |
37***/48 |
28***/50 |
43***/49 |
38***/50 |
+ |
+ |
|
inflammation of keratin layer |
0/49 |
0/50 |
0/49 |
0/49 |
5*/48 |
5/50 |
10**/49 |
2/50 |
+ |
- |
|
hyperkeratosis |
1/49 |
0/50 |
5/49 |
3/49 |
24***/48 |
13***/50 |
29***/49 |
21***/50 |
+ |
+ |
|
ulceration |
3/49 |
0/50 |
5/49 |
3/49 |
10*/48 |
6*/50 |
16**/49 |
12***/50 |
+ |
+ |
|
basal cell proliferation |
4/49 |
3/50 |
7/49 |
1/49 |
28***/48 |
11*/50 |
45***/49 |
39***/50 |
+ |
+ |
|
lesions associated with ulcer |
5/49 |
0/50 |
4/49 |
3/49 |
9/48 |
6*/50 |
13/49 |
17***/50 |
+ |
+ |
|
Significance from control: * p < 0.05; ** p < 0.01; *** p < 0.001
a) number of animals with findings/number of animals examined
Abbreviations:
ne no effects
ni not indicated
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEL
- 20 mg/kg bw/day
- Study duration:
- chronic
- Species:
- rat
- Quality of whole database:
- GLP and guideline study.
Repeated dose toxicity: inhalation - systemic effects
Link to relevant study records
- Endpoint:
- sub-chronic toxicity: inhalation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1994-11-11
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
- Version / remarks:
- 1981
- Deviations:
- yes
- Remarks:
- adrenals were not weighed
- GLP compliance:
- yes
- Limit test:
- no
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 2DR-293-TSI - Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River (UK) Ltd
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation: Group mean weight first delivery: males: 268 - 273 g; females: 188 - 193 g; second delivery: males: 312 g; females: 219 g
- Housing: individually
- Diet: ad libitum
- Water:ad libitum
DETAILS OF FOOD AND WATER QUALITY: There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test substance could reasonably be expected to be present in the diet or in the drinking water.
ENVIRONMENTAL CONDITIONS
- Temperature: 15.5 - 23.5°C
- Humidity: 25 - 54 %
- Photoperiod: 12 / 12 hrs dark / hrs light - Route of administration:
- inhalation: dust
- Type of inhalation exposure:
- whole body
- Vehicle:
- air
- Mass median aerodynamic diameter (MMAD):
- > 1.55 - < 3.5 µm
- Remarks on MMAD:
- Examination of the data indicates that the test aerosol did not contain a normal distribution of particle sizes. The test aerosols consisted of at least 2 populations of particles, one with a median aerodynamic diameter of between approximately 1.55 and 3.5 µm and one with median aerodynamic diameter less than 0.5 µm. Size distributions of this type cannot be described by a single mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD).
67.5 to 83.1 % of the population of particles is considered to be less than 6 µm in aerodynamic diameter in each dose group, respectively. These particles are considered to be respirable to the rat. - Details on inhalation exposure:
- GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: exposure chambers were of stainless steel and glass construction, approx. 0.75 m^3 internal volume
- System of generating particulates/aerosols: Wright dust feed mechanism (WDFM)
TEST ATMOSPHERE
- Brief description of analytical method used: Samples of each test atmosphere were withdrawn through 37 mm diameter glass microfibre filters (Type GF/A, Whatman International Ltd, Maidstone, Kent, England) supported in an open-face holder, at a rate of 10 litres per minute. The total volume sampled was measured using an in-line wet-type gas meter (Model DM3D Alexander Wright and Co (Westminster) Ltd). The test substance deposited on the filters was extracted into acetonitrile and the resultant solutions were analysed by an HPLC method.
VEHICLE: air - Analytical verification of doses or concentrations:
- yes
- Duration of treatment / exposure:
- 6 h
- Frequency of treatment:
- 5 days a week
- Dose / conc.:
- 0 mg/m³ air
- Remarks:
- control
- Dose / conc.:
- 0.25 mg/m³ air
- Remarks:
- analytical: 0.30 mg/m^3
- Dose / conc.:
- 1.25 mg/m³ air
- Remarks:
- analytical: 1.16 mg/m^3
- Dose / conc.:
- 6.25 mg/m³ air
- Remarks:
- analytical: 6.70 mg/m^3
- Dose / conc.:
- 0.25 mg/m³ air
- Remarks:
- analytical: 0.23 mg/m^3 (repeat low dose)
- No. of animals per sex per dose:
- Main groups: 10
Satellite groups: 5 - Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Rationale for animal assignment: random
- Post-exposure recovery period in satellite groups: no - Positive control:
- not applicable
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: during each exposure period and at least twice daily
DETAILED CLINICAL OBSERVATIONS: No
BODY WEIGHT: Yes
- Time schedule for examinations: for allocation to groups and weekly, starting one week before exposures commenced until the end of the study
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
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No
WATER CONSUMPTION AND COMPOUND INTAKE: No
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to treatment and during week 13 of exposure
- Dose groups that were examined: all
HAEMATOLOGY: Yes
- Time schedule for collection of blood: during week 13
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: all animals of the main groups
- Parameters examined: packed cell volume (PCV), haemoglobin concentration, erythrocyte count, total and differential white cell count, platelet count, mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV), reticulocyte count, cell morphology, prothrombin time
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during week 13
- Animals fasted: Not specified
- How many animals: all animals of the main groups
- Parameters examined: sodium, potassium, calcium, inorganic phosphorus, chloride, glucose, cholesterol, urea nitrogen, bilirubin, creatinine, total protein, albumin, globulin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AP), gamma glutamyl transpeptidase (gGT)
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes, all animals
ORGAN WEIGHTS: Yes, all animals: liver, kidneys, testes with epididymides main groups: brain
HISTOPATHOLOGY: Yes
- tissues examined:
control and high dosed groups: adrenals, aorta, caecum, colon, duodenum, eyes, gross abnormalities, heart, ileum, jejunum, lymph nodes, mammary gland, nasal passages, oesophagus, pancreas, pituitary, prostate, rectum, salivary gland, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord, spleen, stomach, sternum with marrow, testes with epididymides, thymus, thyroids with parathyroids, trachea, urinary bladder, uterus
all rats: kidneys, liver, lungs (all lobes and bronchi)
control, low, repeat low, and high dosed groups: larynx
main groups only: brain (medullary cerebellar and cortical sections) - Other examinations:
- Plasma and erythrocyte cholinesterase activity was determined in the animals of the satellite groups after 2 and 13 weeks. Brain cholinesterase activity was determined in the satellite groups after terminal sacrifice. For determination of cholinesterase activity the method of Ellman was used.
- Statistics:
- Bartlett’s test: heterogeneity of variance between treatment when the relative frequency of data does not exceed 75%. One-way analysis of variance: for data not showing statistically significant heterogeneity using Bartlett’s test. Kruskal-Wallis analysis for data with significant heterogeneity of variance. Student’s t-test and Williams test for a dose-related response. Analysis of covariance it appropriate. Fischer’s Exact Test for histopathological incidences.
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- During the first three exposures, closed/half closed eyes were seen in the high dosed rats. Red ears were noted following exposures 3, 4, and 5. These effects were considered to be attributable to the irritating potential of the test substance and not of toxicological relevance. There were no other treatment-related signs noted. Clinical signs attributable to cholinesterase inhibition were also not noted.
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- Two male and two female rats of the low dose groups were sacrificed during week 10 on study following the period of overdosing. There were no deaths attributable to treatment with the test substance.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- Mean group body weight and body weight gain were comparable between control and treated dose groups.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- Mean group food consumption was comparable between control and treated dose groups.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Description (incidence and severity):
- Ophthalmoscopy was comparable between controls and treated groups.
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The following statistically significant differences from concurrent control values were observed:
In the high dosed females a greater erythrocyte count, higher PCV, lower MCHC and lower reticulocyte count when compared to controls were observed. In all treated male groups MCV was lower when compared to concurrent controls.
All the above differences were either of low magnitude, not dose-related or inconsistent between sexes. Thus, these changes are not considered to be of toxicological relevance.
Values for haematological parameters of the repeat low dose group were also similar to the controls. - Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The following statistically significant changes in cholinesterase activity determination were noted:
During week 2 a statistically significant lower plasma cholinesterase activity (-21.7%) in high dosed males when compared to concurrent controls and a statistically significant lower erythrocyte cholinesterase activity (-38.9%) in high dosed females when compared to concurrent controls was observed. During week 13 plasma cholinesterase activity in high dosed males was statistically significant lower (-18.2%) when compared to concurrent controls. Brain cholinesterase activity in the mid dosed females (-24.6%) and in males and females of the high dose group was statistically significantly reduced (-16.8% and –35.3%, respectively) when compared to concurrent controls.
Plasma cholinesterase activity is considered to be only a marker of exposure and should not be considered to be of toxicological relevance.
Erythrocyte cholinesterase activity was statistically significant reduced in the high dosed females after two weeks. After 13 weeks of treatment, however, there was no statistically significant difference to concurrent control in erythrocyte cholinesterase activity noted. In the absence of time consistency, reduced erythrocyte cholinesterase activity in the high dose females was not considered to be of toxicological relevance.
At termination, brain cholinesterase activity was statistically significantly reduced in the mid dosed females (-25% lower than concurrent control) as well as in the high dosed males and females (-17% and 36% lower than concurrent control, respectively). However, the trigger of 20% reduction is not reached when females brain cholinesterase activities are compared to the historical control mean. At 1.16 mg/m3, the measured mean brain cholinesterase activity is 10.5% lower than historical control mean, at 6.7 mg/m3 12.3%. A clear dose-response relationship is further not observable.
However, when control brain cholinesterase activities are compared to the historical control data given in the report, one notes that activities show a great variability. Males concurrent control brain cholinesterase activity is 9% lower when compared to historical control mean. In females the concurrent mean control values exceeds the historical mean (+19%).
By inspection of individual data, one can see that the lowest concurrent control value (5.03 µmol/g/min for males and 4.74 µmol/g/min in females) is always comparable to the lowest figure of the treated animals (4.35 µmol/g/min for males and 4.2 µmol/g/min for females).
It is therefore concluded, that brain cholinesterase activity was not adversely affected during the course of the study up to and including the high dose animals and is within the normal biological variation.
The following statistically significant changes in further clinical chemistry parameters were noted: During week 13, higher albumin levels in the high dosed females when compared to controls were observed. ALT activity in the low dosed females and in males and females of the mid and high dosed groups was elevated when compared to concurrent controls.
Changes in albumin and glucose parameters were not considered to be related to treatment because they were inconsistent between sexes, of low magnitude, and/or without a dose response relationship. The increases in ALT activity were within the expected range for rats of this age and strain. In the absence of any histopathological findings in liver, the increase in ALT was not considered to be of toxicological importance.
Clinical chemistry parameters of the repeat low dose group were well within the expected range for animals of this age and strain. - Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Description (incidence and severity):
- Absolute and adjusted organ weights were comparable to control values.
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- There were no changes in macroscopic pathology noted in which could be attributed to test item administration.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Treatment-related histopathological changes were noted larynx and lungs:
In lungs, macrophage aggregates were observed in a slightly higher incidence in the high dosed males when compared to control males. In all treated females and in the low and mid dosed males, the incidence in macrophage aggregates in lungs was comparable to the concurrent control.
Histopathological findings in the larynx were necrosis in the ventral cartilage, epithelial hyperplasia in ventral region, and squamous metaplasia in ventrolateral region in all animals of the high dose group.
In addition, epithelial hyperplasia over the arytenoid projections in all high dosed males and a proportion of the high dosed females was noted. Epithelial ulceration in the ventral region was observed in low incidence in the high dosed males. In some of the high dosed males and females, additionally, atrophy of submucosal glands was noted.
Similar findings (epithelial hyperplasia and/or necrosis of the ventral cartilage) were observed in low incidence in the initial low dosed animals at terminal sacrifice. This was not observed in the animals of this dose group sacrificed during the course of the study. Furthermore, no histopathological lesions were observed in the repeat low dose group.
In conclusion, histopathological lesions in the larynx observed in the initial low dosed animals are not considered to be related to test item treatment due to the lack of a dose-response relationship and thus are considered to be incidental.
The larynx is often damaged during inhalation studies especially in the high dosed animals due to its morphology. Especially the ventral cartilage – a small U-shaped cartilage - is affected. The ventral cartilage lies immediately beneath the thin surface squamous epithelium. The damage of the epithelium normally results in necrosis of the underlying cartilage. Subsequently the epithelium of the cartilage undergoes reparative hyperplasia but the necrotic appearance of the cartilage persists after the epithelium has repaired.
The test item is known to be an irritating substance. The histopathological changes of the larynx in the high dosed animals may thus be associated with the intrinsic irritating properties of the test item.
It should further be considered during evaluation that rats respire obligatory through their nose whereas human tend to respire also through the mouth. The anatomical differences in the nasal passages as well as in the larynx should also be taken into account. For these reasons, histopathological changes seen in larynx are not considered to be of relevance for human exposed to the test item via air.
There were no histopathological changes noted in the other organs. - Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- no effects observed
- Key result
- Dose descriptor:
- LOAEC
- Effect level:
- 6.7 mg/m³ air (analytical)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- histopathology: non-neoplastic
- Key result
- Dose descriptor:
- NOAEC
- Effect level:
- 1.16 mg/m³ air (analytical)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- histopathology: non-neoplastic
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 0.007 mg/L air (analytical)
- System:
- respiratory system: upper respiratory tract
- Organ:
- larynx
- Treatment related:
- yes
- Dose response relationship:
- yes
- Conclusions:
- Based on the results and under the conditions of this study a NOAEC is considered to be 1.16 mg/m^3 based on larynx effects.
- Executive summary:
Treatment of Crl:CD BR rats over 90 days with the test item via the inhalation route at doses of 0, 0.23, 0.3, 1.16, and 6.7 mg/m3 did not influence the survival, the body weight development, or the food consumption of all treated animals. Ophthalmoscopical examination did not show any treatment-related effects on the eyes of the treated rats. There were no treatment-related effects on haematological and clinical chemistry parameters noted.
There were no changes in organ weights noted. Histopathological examination of the organs showed no changes in treated animals when compared to controls except for larynx and lungs of the high dosed animals. In lungs an increased incidence in the aggregation of macrophages was noted in the high dosed males (7 of 15 animals compared to 4 of 15 animals in the concurrent control).
RBC and brain cholinesterase activities were within the normal biological variation.
Histopathological findings in the larynx of the high dose group were necrosis in the ventral cartilage, epithelial hyperplasia in ventral region, and squamous metaplasia in ventrolateral region in all animals. In addition, epithelial hyperplasia over the arytenoid projections in all high dosed males and in 5 of the 15 high dosed females was noted. Epithelial ulceration in the ventral region was observed in low incidence in the high dosed males (4 of 15 animals). In some of the high dosed males and females, additionally, atrophy of submucosal glands was noted (3 and 6 animals of 15, respectively).
Reference
Table 1 Cholinesterase activity determination
Parameter |
unit |
time [week] |
Dose level [mg/m3] |
|||||
0 |
0.3 |
0.23 |
1.16 |
6.7 |
||||
controls |
historical |
|||||||
Males |
|
|
|
|
|
|
|
|
plasma ChE |
µmol/mL/min |
2 |
0.46 |
0.47 |
0.46 |
0.40 |
0.40 |
0.36** |
RBC ChE |
µmol/mL/min |
|
1.41 |
2.03 |
1.37 |
2.83 |
1.52 |
1.59 |
plasma ChE |
µmol/mL/min |
13 |
0.44 |
0.47 |
0.42 |
0.42 |
0.44 |
0.36** |
RBC ChE |
µmol/mL/min |
|
1.68 |
2.03 |
1.74 |
1.37 |
1.54 |
1.07 |
brain ChE |
µmol/g/min |
14 |
5.77 |
6.31 |
5.02 |
7.17 |
5.05 |
4.80* |
Females |
|
|
|
|
|
|
|
|
plasma ChE |
µmol/mL/min |
2 |
0.83 |
1.30 |
0.79 |
1.01 |
0.88 |
0.73 |
RBC ChE |
µmol/mL/min |
|
1.62 |
1.64 |
1.46 |
1.67 |
1.46 |
0.99* |
plasma ChE |
µmol/mL/min |
13 |
1.27 |
1.30 |
1.46 |
1.36 |
1.28 |
1.29 |
RBC ChE |
µmol/mL/min |
|
1.90 |
1.64 |
1.59 |
1.22 |
2.03 |
2.00 |
brain ChE |
µmol/g/min |
14 |
6.59 |
5.55 |
6.38 |
7.21 |
4.97* |
4.87* |
* p < 0.05 compared with control values (William’s test)
** p < 0.01 compared with control values (William’s test)
1 Values in parenthesis indicate the percentage change from concurrent control
2 Values in parenthesis indicate the percentage change from historical control
Table 2 Results of 90-day inhalation toxicity study in rats
Parameter |
Dose level [mg/m3] |
dose-response +/- |
||||||||||
0 |
0.3 |
0.23 |
1.16 |
6.7 |
||||||||
m |
f |
m |
f |
m |
f |
m |
f |
m |
f |
m |
f |
|
number of animals examined |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
Mortality |
0 |
0 |
21) |
21) |
0 |
0 |
0 |
0 |
0 |
0 |
- |
- |
clinical signs |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
- |
- |
body weight |
|
|
ne |
ne |
ne |
ne |
ne |
ne |
ne |
ne |
- |
- |
food consumption |
|
|
ne |
ne |
ne |
ne |
ne |
ne |
ne |
ne |
- |
- |
clinical chemistry |
|
|
ne |
ne |
ne |
ne |
ne |
ne |
ne |
ne |
- |
- |
haematology |
|
|
ne |
ne |
ne |
ne |
ne |
ne |
ne |
ne |
- |
- |
cholinesterase activities |
|
|
ne |
ne |
ne |
ne |
ne |
ne |
ne |
ne |
- |
- |
Larynx |
|
|
|
|
|
|
|
|
|
|
|
|
weight |
nd |
nd |
nd |
nd |
nd |
nd |
nd |
nd |
nd |
nd |
|
|
gross pathology |
ne |
ne |
ne |
ne |
ne |
ne |
ne |
ne |
ne |
ne |
- |
- |
microscopic pathology |
152) |
15 |
13 |
13 |
15 |
15 |
15 |
15 |
15 |
15 |
|
|
necrosis of the ventral cartilage |
0 |
0 |
2 |
0 |
0 |
0 |
nd |
nd |
15 |
15 |
+ |
+ |
epithelial hyperplasia in ventral region |
0 |
0 |
4 |
7 |
0 |
0 |
nd |
nd |
15 |
15 |
+ |
+ |
epithelial hyperplasia over arytenoid |
0 |
0 |
0 |
1 |
0 |
0 |
nd |
nd |
15 |
5 |
+ |
+ |
squamous metaplasia in ventrolateral region |
0 |
0 |
0 |
0 |
0 |
0 |
nd |
nd |
15 |
15 |
+ |
+ |
epithelial ulceration in ventral region |
0 |
0 |
0 |
0 |
0 |
0 |
nd |
nd |
4 |
1 |
+ |
- |
atrophy of submucosal glands |
0 |
0 |
0 |
0 |
0 |
0 |
nd |
nd |
3 |
6 |
+ |
+ |
Lungs |
|
|
|
|
|
|
|
|
|
|
|
|
weight |
|
|
ne |
ne |
ne |
ne |
ne |
ne |
ne |
ne |
- |
- |
gross pathology |
|
|
ne |
ne |
ne |
ne |
ne |
ne |
ne |
ne |
- |
- |
microscopic pathology |
152) |
15 |
13 |
13 |
15 |
15 |
15 |
15 |
15 |
15 |
|
|
aggregates of macrophages |
4 |
1 |
3 |
1 |
1 |
0 |
3 |
3 |
7 |
2 |
+ |
- |
1) sacrificed due to overdosing
2) number of animals examined at terminal sacrifice
3) Please note: the low dosing regime was repeated due to overdosing of the original low dose. However, no concurrent control was used
Abbreviations:
ne no effects
nd not determined
na not applicable (no concurrent control)
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEC
- 1.16 mg/m³
- Study duration:
- subchronic
- Species:
- rat
- Quality of whole database:
- GLP and guideline study
- System:
- respiratory system: upper respiratory tract
- Organ:
- larynx
Repeated dose toxicity: inhalation - local effects
Link to relevant study records
- Endpoint:
- sub-chronic toxicity: inhalation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1994-11-11
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
- Version / remarks:
- 1981
- Deviations:
- yes
- Remarks:
- adrenals were not weighed
- GLP compliance:
- yes
- Limit test:
- no
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 2DR-293-TSI - Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River (UK) Ltd
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation: Group mean weight first delivery: males: 268 - 273 g; females: 188 - 193 g; second delivery: males: 312 g; females: 219 g
- Housing: individually
- Diet: ad libitum
- Water:ad libitum
DETAILS OF FOOD AND WATER QUALITY: There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test substance could reasonably be expected to be present in the diet or in the drinking water.
ENVIRONMENTAL CONDITIONS
- Temperature: 15.5 - 23.5°C
- Humidity: 25 - 54 %
- Photoperiod: 12 / 12 hrs dark / hrs light - Route of administration:
- inhalation: dust
- Type of inhalation exposure:
- whole body
- Vehicle:
- air
- Mass median aerodynamic diameter (MMAD):
- > 1.55 - < 3.5 µm
- Remarks on MMAD:
- Examination of the data indicates that the test aerosol did not contain a normal distribution of particle sizes. The test aerosols consisted of at least 2 populations of particles, one with a median aerodynamic diameter of between approximately 1.55 and 3.5 µm and one with median aerodynamic diameter less than 0.5 µm. Size distributions of this type cannot be described by a single mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD).
67.5 to 83.1 % of the population of particles is considered to be less than 6 µm in aerodynamic diameter in each dose group, respectively. These particles are considered to be respirable to the rat. - Details on inhalation exposure:
- GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: exposure chambers were of stainless steel and glass construction, approx. 0.75 m^3 internal volume
- System of generating particulates/aerosols: Wright dust feed mechanism (WDFM)
TEST ATMOSPHERE
- Brief description of analytical method used: Samples of each test atmosphere were withdrawn through 37 mm diameter glass microfibre filters (Type GF/A, Whatman International Ltd, Maidstone, Kent, England) supported in an open-face holder, at a rate of 10 litres per minute. The total volume sampled was measured using an in-line wet-type gas meter (Model DM3D Alexander Wright and Co (Westminster) Ltd). The test substance deposited on the filters was extracted into acetonitrile and the resultant solutions were analysed by an HPLC method.
VEHICLE: air - Analytical verification of doses or concentrations:
- yes
- Duration of treatment / exposure:
- 6 h
- Frequency of treatment:
- 5 days a week
- Dose / conc.:
- 0 mg/m³ air
- Remarks:
- control
- Dose / conc.:
- 0.25 mg/m³ air
- Remarks:
- analytical: 0.30 mg/m^3
- Dose / conc.:
- 1.25 mg/m³ air
- Remarks:
- analytical: 1.16 mg/m^3
- Dose / conc.:
- 6.25 mg/m³ air
- Remarks:
- analytical: 6.70 mg/m^3
- Dose / conc.:
- 0.25 mg/m³ air
- Remarks:
- analytical: 0.23 mg/m^3 (repeat low dose)
- No. of animals per sex per dose:
- Main groups: 10
Satellite groups: 5 - Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Rationale for animal assignment: random
- Post-exposure recovery period in satellite groups: no - Positive control:
- not applicable
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: during each exposure period and at least twice daily
DETAILED CLINICAL OBSERVATIONS: No
BODY WEIGHT: Yes
- Time schedule for examinations: for allocation to groups and weekly, starting one week before exposures commenced until the end of the study
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
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No
WATER CONSUMPTION AND COMPOUND INTAKE: No
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to treatment and during week 13 of exposure
- Dose groups that were examined: all
HAEMATOLOGY: Yes
- Time schedule for collection of blood: during week 13
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: all animals of the main groups
- Parameters examined: packed cell volume (PCV), haemoglobin concentration, erythrocyte count, total and differential white cell count, platelet count, mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV), reticulocyte count, cell morphology, prothrombin time
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during week 13
- Animals fasted: Not specified
- How many animals: all animals of the main groups
- Parameters examined: sodium, potassium, calcium, inorganic phosphorus, chloride, glucose, cholesterol, urea nitrogen, bilirubin, creatinine, total protein, albumin, globulin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AP), gamma glutamyl transpeptidase (gGT)
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes, all animals
ORGAN WEIGHTS: Yes, all animals: liver, kidneys, testes with epididymides main groups: brain
HISTOPATHOLOGY: Yes
- tissues examined:
control and high dosed groups: adrenals, aorta, caecum, colon, duodenum, eyes, gross abnormalities, heart, ileum, jejunum, lymph nodes, mammary gland, nasal passages, oesophagus, pancreas, pituitary, prostate, rectum, salivary gland, sciatic nerve, seminal vesicles, skeletal muscle, skin, spinal cord, spleen, stomach, sternum with marrow, testes with epididymides, thymus, thyroids with parathyroids, trachea, urinary bladder, uterus
all rats: kidneys, liver, lungs (all lobes and bronchi)
control, low, repeat low, and high dosed groups: larynx
main groups only: brain (medullary cerebellar and cortical sections) - Other examinations:
- Plasma and erythrocyte cholinesterase activity was determined in the animals of the satellite groups after 2 and 13 weeks. Brain cholinesterase activity was determined in the satellite groups after terminal sacrifice. For determination of cholinesterase activity the method of Ellman was used.
- Statistics:
- Bartlett’s test: heterogeneity of variance between treatment when the relative frequency of data does not exceed 75%. One-way analysis of variance: for data not showing statistically significant heterogeneity using Bartlett’s test. Kruskal-Wallis analysis for data with significant heterogeneity of variance. Student’s t-test and Williams test for a dose-related response. Analysis of covariance it appropriate. Fischer’s Exact Test for histopathological incidences.
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- During the first three exposures, closed/half closed eyes were seen in the high dosed rats. Red ears were noted following exposures 3, 4, and 5. These effects were considered to be attributable to the irritating potential of the test substance and not of toxicological relevance. There were no other treatment-related signs noted. Clinical signs attributable to cholinesterase inhibition were also not noted.
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- Two male and two female rats of the low dose groups were sacrificed during week 10 on study following the period of overdosing. There were no deaths attributable to treatment with the test substance.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- Mean group body weight and body weight gain were comparable between control and treated dose groups.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- Mean group food consumption was comparable between control and treated dose groups.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Description (incidence and severity):
- Ophthalmoscopy was comparable between controls and treated groups.
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The following statistically significant differences from concurrent control values were observed:
In the high dosed females a greater erythrocyte count, higher PCV, lower MCHC and lower reticulocyte count when compared to controls were observed. In all treated male groups MCV was lower when compared to concurrent controls.
All the above differences were either of low magnitude, not dose-related or inconsistent between sexes. Thus, these changes are not considered to be of toxicological relevance.
Values for haematological parameters of the repeat low dose group were also similar to the controls. - Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The following statistically significant changes in cholinesterase activity determination were noted:
During week 2 a statistically significant lower plasma cholinesterase activity (-21.7%) in high dosed males when compared to concurrent controls and a statistically significant lower erythrocyte cholinesterase activity (-38.9%) in high dosed females when compared to concurrent controls was observed. During week 13 plasma cholinesterase activity in high dosed males was statistically significant lower (-18.2%) when compared to concurrent controls. Brain cholinesterase activity in the mid dosed females (-24.6%) and in males and females of the high dose group was statistically significantly reduced (-16.8% and –35.3%, respectively) when compared to concurrent controls.
Plasma cholinesterase activity is considered to be only a marker of exposure and should not be considered to be of toxicological relevance.
Erythrocyte cholinesterase activity was statistically significant reduced in the high dosed females after two weeks. After 13 weeks of treatment, however, there was no statistically significant difference to concurrent control in erythrocyte cholinesterase activity noted. In the absence of time consistency, reduced erythrocyte cholinesterase activity in the high dose females was not considered to be of toxicological relevance.
At termination, brain cholinesterase activity was statistically significantly reduced in the mid dosed females (-25% lower than concurrent control) as well as in the high dosed males and females (-17% and 36% lower than concurrent control, respectively). However, the trigger of 20% reduction is not reached when females brain cholinesterase activities are compared to the historical control mean. At 1.16 mg/m3, the measured mean brain cholinesterase activity is 10.5% lower than historical control mean, at 6.7 mg/m3 12.3%. A clear dose-response relationship is further not observable.
However, when control brain cholinesterase activities are compared to the historical control data given in the report, one notes that activities show a great variability. Males concurrent control brain cholinesterase activity is 9% lower when compared to historical control mean. In females the concurrent mean control values exceeds the historical mean (+19%).
By inspection of individual data, one can see that the lowest concurrent control value (5.03 µmol/g/min for males and 4.74 µmol/g/min in females) is always comparable to the lowest figure of the treated animals (4.35 µmol/g/min for males and 4.2 µmol/g/min for females).
It is therefore concluded, that brain cholinesterase activity was not adversely affected during the course of the study up to and including the high dose animals and is within the normal biological variation.
The following statistically significant changes in further clinical chemistry parameters were noted: During week 13, higher albumin levels in the high dosed females when compared to controls were observed. ALT activity in the low dosed females and in males and females of the mid and high dosed groups was elevated when compared to concurrent controls.
Changes in albumin and glucose parameters were not considered to be related to treatment because they were inconsistent between sexes, of low magnitude, and/or without a dose response relationship. The increases in ALT activity were within the expected range for rats of this age and strain. In the absence of any histopathological findings in liver, the increase in ALT was not considered to be of toxicological importance.
Clinical chemistry parameters of the repeat low dose group were well within the expected range for animals of this age and strain. - Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Description (incidence and severity):
- Absolute and adjusted organ weights were comparable to control values.
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- There were no changes in macroscopic pathology noted in which could be attributed to test item administration.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Treatment-related histopathological changes were noted larynx and lungs:
In lungs, macrophage aggregates were observed in a slightly higher incidence in the high dosed males when compared to control males. In all treated females and in the low and mid dosed males, the incidence in macrophage aggregates in lungs was comparable to the concurrent control.
Histopathological findings in the larynx were necrosis in the ventral cartilage, epithelial hyperplasia in ventral region, and squamous metaplasia in ventrolateral region in all animals of the high dose group.
In addition, epithelial hyperplasia over the arytenoid projections in all high dosed males and a proportion of the high dosed females was noted. Epithelial ulceration in the ventral region was observed in low incidence in the high dosed males. In some of the high dosed males and females, additionally, atrophy of submucosal glands was noted.
Similar findings (epithelial hyperplasia and/or necrosis of the ventral cartilage) were observed in low incidence in the initial low dosed animals at terminal sacrifice. This was not observed in the animals of this dose group sacrificed during the course of the study. Furthermore, no histopathological lesions were observed in the repeat low dose group.
In conclusion, histopathological lesions in the larynx observed in the initial low dosed animals are not considered to be related to test item treatment due to the lack of a dose-response relationship and thus are considered to be incidental.
The larynx is often damaged during inhalation studies especially in the high dosed animals due to its morphology. Especially the ventral cartilage – a small U-shaped cartilage - is affected. The ventral cartilage lies immediately beneath the thin surface squamous epithelium. The damage of the epithelium normally results in necrosis of the underlying cartilage. Subsequently the epithelium of the cartilage undergoes reparative hyperplasia but the necrotic appearance of the cartilage persists after the epithelium has repaired.
The test item is known to be an irritating substance. The histopathological changes of the larynx in the high dosed animals may thus be associated with the intrinsic irritating properties of the test item.
It should further be considered during evaluation that rats respire obligatory through their nose whereas human tend to respire also through the mouth. The anatomical differences in the nasal passages as well as in the larynx should also be taken into account. For these reasons, histopathological changes seen in larynx are not considered to be of relevance for human exposed to the test item via air.
There were no histopathological changes noted in the other organs. - Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- no effects observed
- Key result
- Dose descriptor:
- LOAEC
- Effect level:
- 6.7 mg/m³ air (analytical)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- histopathology: non-neoplastic
- Key result
- Dose descriptor:
- NOAEC
- Effect level:
- 1.16 mg/m³ air (analytical)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- histopathology: non-neoplastic
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 0.007 mg/L air (analytical)
- System:
- respiratory system: upper respiratory tract
- Organ:
- larynx
- Treatment related:
- yes
- Dose response relationship:
- yes
- Conclusions:
- Based on the results and under the conditions of this study a NOAEC is considered to be 1.16 mg/m^3 based on larynx effects.
- Executive summary:
Treatment of Crl:CD BR rats over 90 days with the test item via the inhalation route at doses of 0, 0.23, 0.3, 1.16, and 6.7 mg/m3 did not influence the survival, the body weight development, or the food consumption of all treated animals. Ophthalmoscopical examination did not show any treatment-related effects on the eyes of the treated rats. There were no treatment-related effects on haematological and clinical chemistry parameters noted.
There were no changes in organ weights noted. Histopathological examination of the organs showed no changes in treated animals when compared to controls except for larynx and lungs of the high dosed animals. In lungs an increased incidence in the aggregation of macrophages was noted in the high dosed males (7 of 15 animals compared to 4 of 15 animals in the concurrent control).
RBC and brain cholinesterase activities were within the normal biological variation.
Histopathological findings in the larynx of the high dose group were necrosis in the ventral cartilage, epithelial hyperplasia in ventral region, and squamous metaplasia in ventrolateral region in all animals. In addition, epithelial hyperplasia over the arytenoid projections in all high dosed males and in 5 of the 15 high dosed females was noted. Epithelial ulceration in the ventral region was observed in low incidence in the high dosed males (4 of 15 animals). In some of the high dosed males and females, additionally, atrophy of submucosal glands was noted (3 and 6 animals of 15, respectively).
Reference
Table 1 Cholinesterase activity determination
Parameter |
unit |
time [week] |
Dose level [mg/m3] |
|||||
0 |
0.3 |
0.23 |
1.16 |
6.7 |
||||
controls |
historical |
|||||||
Males |
|
|
|
|
|
|
|
|
plasma ChE |
µmol/mL/min |
2 |
0.46 |
0.47 |
0.46 |
0.40 |
0.40 |
0.36** |
RBC ChE |
µmol/mL/min |
|
1.41 |
2.03 |
1.37 |
2.83 |
1.52 |
1.59 |
plasma ChE |
µmol/mL/min |
13 |
0.44 |
0.47 |
0.42 |
0.42 |
0.44 |
0.36** |
RBC ChE |
µmol/mL/min |
|
1.68 |
2.03 |
1.74 |
1.37 |
1.54 |
1.07 |
brain ChE |
µmol/g/min |
14 |
5.77 |
6.31 |
5.02 |
7.17 |
5.05 |
4.80* |
Females |
|
|
|
|
|
|
|
|
plasma ChE |
µmol/mL/min |
2 |
0.83 |
1.30 |
0.79 |
1.01 |
0.88 |
0.73 |
RBC ChE |
µmol/mL/min |
|
1.62 |
1.64 |
1.46 |
1.67 |
1.46 |
0.99* |
plasma ChE |
µmol/mL/min |
13 |
1.27 |
1.30 |
1.46 |
1.36 |
1.28 |
1.29 |
RBC ChE |
µmol/mL/min |
|
1.90 |
1.64 |
1.59 |
1.22 |
2.03 |
2.00 |
brain ChE |
µmol/g/min |
14 |
6.59 |
5.55 |
6.38 |
7.21 |
4.97* |
4.87* |
* p < 0.05 compared with control values (William’s test)
** p < 0.01 compared with control values (William’s test)
1 Values in parenthesis indicate the percentage change from concurrent control
2 Values in parenthesis indicate the percentage change from historical control
Table 2 Results of 90-day inhalation toxicity study in rats
Parameter |
Dose level [mg/m3] |
dose-response +/- |
||||||||||
0 |
0.3 |
0.23 |
1.16 |
6.7 |
||||||||
m |
f |
m |
f |
m |
f |
m |
f |
m |
f |
m |
f |
|
number of animals examined |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
15 |
Mortality |
0 |
0 |
21) |
21) |
0 |
0 |
0 |
0 |
0 |
0 |
- |
- |
clinical signs |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
- |
- |
body weight |
|
|
ne |
ne |
ne |
ne |
ne |
ne |
ne |
ne |
- |
- |
food consumption |
|
|
ne |
ne |
ne |
ne |
ne |
ne |
ne |
ne |
- |
- |
clinical chemistry |
|
|
ne |
ne |
ne |
ne |
ne |
ne |
ne |
ne |
- |
- |
haematology |
|
|
ne |
ne |
ne |
ne |
ne |
ne |
ne |
ne |
- |
- |
cholinesterase activities |
|
|
ne |
ne |
ne |
ne |
ne |
ne |
ne |
ne |
- |
- |
Larynx |
|
|
|
|
|
|
|
|
|
|
|
|
weight |
nd |
nd |
nd |
nd |
nd |
nd |
nd |
nd |
nd |
nd |
|
|
gross pathology |
ne |
ne |
ne |
ne |
ne |
ne |
ne |
ne |
ne |
ne |
- |
- |
microscopic pathology |
152) |
15 |
13 |
13 |
15 |
15 |
15 |
15 |
15 |
15 |
|
|
necrosis of the ventral cartilage |
0 |
0 |
2 |
0 |
0 |
0 |
nd |
nd |
15 |
15 |
+ |
+ |
epithelial hyperplasia in ventral region |
0 |
0 |
4 |
7 |
0 |
0 |
nd |
nd |
15 |
15 |
+ |
+ |
epithelial hyperplasia over arytenoid |
0 |
0 |
0 |
1 |
0 |
0 |
nd |
nd |
15 |
5 |
+ |
+ |
squamous metaplasia in ventrolateral region |
0 |
0 |
0 |
0 |
0 |
0 |
nd |
nd |
15 |
15 |
+ |
+ |
epithelial ulceration in ventral region |
0 |
0 |
0 |
0 |
0 |
0 |
nd |
nd |
4 |
1 |
+ |
- |
atrophy of submucosal glands |
0 |
0 |
0 |
0 |
0 |
0 |
nd |
nd |
3 |
6 |
+ |
+ |
Lungs |
|
|
|
|
|
|
|
|
|
|
|
|
weight |
|
|
ne |
ne |
ne |
ne |
ne |
ne |
ne |
ne |
- |
- |
gross pathology |
|
|
ne |
ne |
ne |
ne |
ne |
ne |
ne |
ne |
- |
- |
microscopic pathology |
152) |
15 |
13 |
13 |
15 |
15 |
15 |
15 |
15 |
15 |
|
|
aggregates of macrophages |
4 |
1 |
3 |
1 |
1 |
0 |
3 |
3 |
7 |
2 |
+ |
- |
1) sacrificed due to overdosing
2) number of animals examined at terminal sacrifice
3) Please note: the low dosing regime was repeated due to overdosing of the original low dose. However, no concurrent control was used
Abbreviations:
ne no effects
nd not determined
na not applicable (no concurrent control)
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEC
- 1.16 mg/m³
- Study duration:
- subchronic
- Species:
- rat
- Quality of whole database:
- GLP and guideline study.
Repeated dose toxicity: dermal - systemic effects
Link to relevant study records
- Endpoint:
- sub-chronic toxicity: dermal
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1990-12-13 to 1991-12-06
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- EPA OPP 82-3 (Subchronic Dermal Toxicity 90 Days)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
- Deviations:
- yes
- Remarks:
- please refer to "principles of method if other than guideline"
- Principles of method if other than guideline:
- Deviations: Salivary glands were not preserved as required by OECD 411 Guideline.
- GLP compliance:
- yes
- Limit test:
- no
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 9011-6010 - Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, Inc., Portage, MI, USA
- Age at study initiation: approx. 54 d
- Weight at study initiation: 222 - 266 g (males); 156 - 192 g (females)
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 11
ENVIRONMENTAL CONDITIONS
- Temperature: 65 - 78° F (13.3 - 25.5 °C)
- Humidity: 40 - 70%
- Photoperiod: 12 /12 hrs dark / hrs light - Type of coverage:
- semiocclusive
- Vehicle:
- polyethylene glycol
- Details on exposure:
- TEST SITE
- Area of exposure: dorsal trunk
- % coverage: approx. 10 % of body surface
- Type of wrap if used: Coban self adherent wrap
- Time intervals for shavings or clipplings: minimum once a week
REMOVAL OF TEST SUBSTANCE
- Washing: skin was wiped with clean gauze moistened with distilled water
- Time after start of exposure: 6 h
TEST MATERIAL
- Amount applied: 6 mL/kg, respectively
- Concentration: 8.33; 33.33; 83.33 mg/mL vehicle
- Constant volume used: 6 mL/kg bw
- For solids, paste formed: yes
VEHICLE
- Justification for use and choice of vehicle: PEG is an accepted vehicle by guidelines
USE OF RESTRAINERS FOR PREVENTING INGESTION: no. Harness and/or plastic collars were used to prevent excessive chewing on bindinw material - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Prior to initiation of the 91-day study, the analytical method was validated. Prestudy homogeneity and stability analyses were performed on concentrations of the test article which encompassed those used in this study. Each fresh dosing preparation was analyzed for test article concentration once per week during the first month and monthly thereafter.
- Duration of treatment / exposure:
- 13 weeks (91 days)
- Frequency of treatment:
- 6 h á 5 days per week
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Dose / conc.:
- 50 mg/kg bw/day (nominal)
- Dose / conc.:
- 200 mg/kg bw/day (nominal)
- Dose / conc.:
- 500 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: preliminary dose-range finder study
- Rationale for animal assignment: random - Positive control:
- not applicable
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily
DERMAL IRRITATION: Yes
- Time schedule for examinations: daily prior to dosing
BODY WEIGHT: Yes
- Time schedule for examinations: weekly and at study termination
FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; weekly
FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No
WATER CONSUMPTION: No
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to testing (day -4) and near conclusion of study (day 87)
- Dose groups that were examined: all
HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 89 for hemolysate analysis and plasma cholinesterase and day 91 or 93 for haematology
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: all
- Parameters examined: Erythrocyte count; Hematocrit (Hct); Hemoglobin concentration (Hgb); Mean corpuscular hemoglobin (MCH); Mean corpuscular hemoglobin concentration (MCHC); Mean corpuscular volume (MCV); Platelet count; Reticulocyte count; Total and differential leukocyte counts.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 91 or 93
- Animals fasted: No
- How many animals: all
- Parameters examined: Alanine aminotransferase (ALT) Albumin; Albumin/globulin (A/G) ratio; Alkaline phosphatase (ALP); Aspartate aminotransferase (AST); Calcium Chloride; Creatinine; Fasting glucose; Gamma glutamyl transpeptidase (GGT); Globulin (calculated); Phosphorus; Potassium; Sodium; Total bilirubin Total serum protein Urea nitrogen (BUN)
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes - Statistics:
- Statistical analyses were performed by a Digital VAX 11/730 computer. All analyses were two-tailed with a minimum significance level of 5%. Continuous data including body weight, weight gain, food consumption, organ weight and clinical pathology were analyzed by One Way Analysis of Variance (ANOVA). When significance was observed with ANOVA, control to treatment group comparisons were performed using Dunnett's Test.
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- No treatment-related clinical signs were observed during the study. Incidental findings, which occurred sporadically throughout the groups, included scab formation at areas other than the exposure site, hairloss at various body regions, swelling of the pinnae, lacrimation, ocular discharge, dark material around the eyes, and nontreatment-related dermal irritation in the binder tape area.
- Dermal irritation:
- effects observed, treatment-related
- Description (incidence and severity):
- No signs of dermal irritation were observed in males or females of the 50 mg/kg/day group.
At the 200 mg/kg/day level, occasional slight erythema was observed in 4 males and 6 females, while slight desquamation was observed occasionally for 8 males and 7 females. For one 200 mg/kg/day female, moderate erythema was observed on one occasion and moderate desquamation was observed on two occasions. Eschar areas covered less than 10% of the exposure site for one 200 mg/kg/day male on three consecutive days which improved to focal and/or pinpoint areas of eschar on the following day. Focal and/or pinpoint areas of eschar were observed for three 200 mg/kg/day females, and on two occasions eschar areas covered less than 10% of the exposure site of one other 200 mg/kg/day female. The dermal irritation at the 200 mg/kg/day level was generally observed within the first few days of treatment and persisted less than two weeks.
Dermal Irritation was more severe at the 500 mg/kg/day level and was observed in all males and females at this level. The most common findings consisted of slight erythema and slight desquamation. Low incidences of slight edema, moderate erythema and moderate desquamation were also observed in both males and females at the 500 mg/kg/day level. Occasional focal and/or pinpoint areas of eschar were observed for 3 males and 6 females at the 500 mg/kg/day level. For one 500 mg/kg/day female, eschar covered less than 25% of the exposure site and showed signs of exfoliation. The dermal irritation for males and females at the 500 mg/kg/day level was generally observed within the first few days of treatment and persisted throughout the study. - Mortality:
- no mortality observed
- Description (incidence):
- All animals survived to scheduled sacrifice on day 92 or 93, with the exception of one 50 mg/kg/day female that died during blood collection on day 89.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- Mean male body weight and body weight gain were similar between the control and 50 mg/kg/day groups throughout the study.
At the 200 mg/kg/day level, male body weight gain was statistically reduced during study days 8-15 when compared to the control group. The reduction in weight gain for males at the 200 mg/kg/day level resulted in cumulative weight gains that were slightly lower than the control group beginning on days 1-15 and continuing throughout the study. Body weight gain for males at the 500 mg/kg/day level was statistically reduced during study days 1-8, while cumulative weight gain for males at this level was statistically reduced during days 1-8, 1-15, 1-22 and 1-29. Cumulative weight gain for males at the 500 mg/kg/day level remained slightly lower than the control group throughout the remainder of the study. In addition, the reductions in male weight gain at the 200 and 500 mg/kg/day levels resulted in group mean body weights that were slightly lower than the control group beginning on day 15 and continuing through the remainder of the study.
Mean body weight and weight gain for females were similar among the groups throughout the study. - Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- Male food consumption calculated as grams/animal/day was similar between the control and 50 mg/kg bw/d groups throughout the study. At the 200 mg/kg bw/d level, food consumption was statistically reduced during study days 8-15, 15-22, 22-29 and 57-64, when compared to the control group. Food consumption for males at the 500 mg/kg bw/d level was statistically lower than the control group during days 15-22 only.
Food consumption for females was similar among the groups throughout the study. - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Description (incidence and severity):
- No treatment-related ocular toxicity was noted in the treated rats.
- Haematological findings:
- no effects observed
- Description (incidence and severity):
- Statistically significant differences in terminal hematology data consisted of increased hemoglobin, hematocrit and eosinophils in the 500 mg/kg/ day females. These differences were not considered to be biologically meaningful since the changes were relatively minor and remained within expected ranges for rats of this age and strain.
- Clinical biochemistry findings:
- no effects observed
- Description (incidence and severity):
- Statistically significant differences in terminal clinical chemistry data consisted of increased total protein in 200 mg/kg bw/d males; decreased glucose in 200 and 500 mg/kg bw/d males; decreased creatinine in 500 mg/kg bw/d males; increased calcium and phosphorus in 500 mg/kg bw/d males; and increased calcium and phosphorus in 500 mg/kg bw/d females. These differences were not considered to be biologically meaningful since the changes were relatively minor, remained within expected ranges for rats of this strain and age, and did not correlate with any abnormal histopathology. It should be noted that concentrations of the test article used in this study showed no evidence of inhibition of either RBC hemolysate or plasma cholinesterase. An incidental statistical increase in RBC hemolysate cholinesterase was observed in the 500 mg/kg bw/d males.
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Description (incidence and severity):
- Absolute, but not relative heart weights of males in the 200 and 500 mg/kg bw/d groups were statistically decreased when compared to the control group. These differences were not considered biologically meaningful since they occurred in the absence of any clear dose response and did not correlate with any abnormal histopathology. No other statistically significant or biologically meaningful differences were noted in the absolute or relative organ weights of the treated rats.
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- Gross necropsy findings were generally unremarkable. Individual findings occurred with a similar frequency among the groups.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Test article-related changes in the skin at the application site were observed in most males and females from the 200 and 500 mg/kg bw/d groups. These changes consisted primarily of minimal to mild hyperkeratosis which occurred in 17/20 rats from the 200 mg/kg bw/d group and 16/20 rats from the 500 mg/kg bw/d group. Hyperkeratosis correlated with the slight desquamation of the application site observed in most rats of the 500 mg/kg bw/d group. In addition, minimal to mild acanthotic thickening of the epidermis was seen in 2/20 rats from the 200 mg/kg bw/d group and 11/20 rats from the 500 mg/kg bw/d group. Mild ulceration and accompanying exudate upon the epidermal surface occurred in a single 500 mg/kg bw/d female. Minimal hyperkeratosis at the application site was also observed microscopically in one male and one female from the 50 mg/kg bw/d group, while minimal acanthosis was observed in one control female. The changes in the control and 50 mg/kg bw/d groups were considered to be due to the experimental procedure rather than a specific effect of the test article. No other treatment-related microscopic changes were observed in rats from any of the study groups.
Overall, the test article effects at the 200 and 500 mg/kg bw/d levels, including the 500 mg/kg bw/d female with mild ulceration, were very mild and would be expected to be quickly reversed upon cessation of test article application. - Histopathological findings: neoplastic:
- not examined
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- 50 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- dermal irritation
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 200 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- dermal irritation
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 500 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- dermal irritation
- Key result
- Critical effects observed:
- no
- Conclusions:
- The test item was applied to Sprague Dawley rats for 91 day in doses of 50, 200, and 500 mg/kg bw/d. Based on observations in this study, NOEL was determined to be 50 mg/kg bw/d, whereas NOAEL was considered to be 200 mg/kg bw/d.
- Executive summary:
The potential toxic effects of the test item were evaluated in this 91 -day dermal toxicity study in Sprague-Dawley rats. The study consisted of a control group and three treatment groups with ten animals per sex per group. The test article was dissolved in PEG 400 and administered five days/week, under semi-occlusive conditions, at levels of 50, 200 and 500 mg/kg bw/d. All doses were given at a constant volume of 6.0 mL/kg. Control animals were administered PEG 400 under the same experimental conditions and at an equivalent dose volume.
The rats were observed daily for clinical signs of toxicity and application sites were examined prior to dosing for adverse skin reactions. Individual body weights and food consumption were measured weekly. Ophthalmological examinations were performed on all animals prior to the initiation and near the conclusion of the study. Blood samples were obtained from all animals on study day 89 for determination of cholinesterase activity. This clinical parameter, cholinesterase activity, was selected by the Sponsor since the test substance is a carbamate pesticide and this class of chemicals is known to inhibit cholinesterase activity. All surviving animals were bled at study termination (day 92 or 93) for evaluation of hematology and biochemistry parameters. A complete gross necropsy examination was performed on all animals. Fresh organ weights were obtained from surviving animals and selected tissues and organs were collected from each animal. All tissues from control and high-dose animals and the treated skin, untreated skin, lungs, liver, kidneys and gross lesions from all low and mid-dose animals were examined microscopically.
No treatment-related mortality or clinical signs of toxicity were observed during the study. Treatment-related decreases in body weight gain and/or food consumption were observed in the 200 and 500 mg/kg/day males during the first four weeks of treatment. The reduced mean body weights of these rats remained slightly lower than controls during the remainder of the study. No adverse body weight or food consumption effects were observed in the 50 mg/kg bw/d males or in females from any of the study groups. There were no apparent test article-related changes observed among the groups concerning ophthalmology, clinical pathology, gross necropsy, or organ weight data. Similarly, no treatment-related differences in RBC hemolysate or plasma cholinesterase levels were noted. Dose-dependent dermal irritation was observed in males and females at 200 and 500 mg/kg bw/d levels. The irritation was mild at the 200 mg/kg bw/d level and consisted primarily of slight erythema and slight desquamation. At the 500 mg/kg bw/d level, the irritation was more severe and consisted of slight erythema, slight desquamation, and low incidences of slight edema, moderate erythema and moderate desquamation. Dermal irritation at the 500 mg/kg bw/d level progressed to focal and/or pinpoint areas of eschar in a few animals. Microscopic evaluation of the treated skin revealed hyperkeratosis and acanthotic epidermal thickening in rats of the 200 and 500 mg/kg bw/d groups. The dermal changes were expected to be reversible upon cessation of treatment. No treatment-related signs of dermal irritation were observed in males or females of the 50 mg/kg bw/d group.
Based on these results, a dosage level of 50 mg/kg bw/d was determined to be a no-observed-effect level for 91-day dermal administration of test item in rats.
Reference
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEL
- 200 mg/kg bw/day
- Study duration:
- subchronic
- Species:
- rat
- Quality of whole database:
- GLP and guideline study.
Repeated dose toxicity: dermal - local effects
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed
Additional information
Repeated dose toxicity: oral route
Chronic, rat
Groups of 50 male and 50 female Sprague-Dawley rats were offered diet ad libitum formulated with test item at concentrations calculated to give dose levels of 20, 40 and 80 mg/kg bw/day. A further group of 50 males and 50 females were offered untreated diet to act as Controls. During Weeks 53/54, 79 and 104 a blood smear was prepared via tailsnip from all surviving animals and a differential blood count performed on Control and High dose group animals. During Week 100 blood samples were taken from 10 males and 10 females from each group in order that a haematology and clinical chemistry screen could be carried out in order to put in context results from earlier time points on the concurrent toxicity study.
After 104 weeks continual dosing all
surviving animals were killed by carbon dioxide asphyxiation followed by
exsaguination and a detailed necropsy, with a selection of organs being
placed in fixative. All organs taken were examined histopathologically
from all Control and High dose animals, as well as premature decedents
from the Low and Intermediate dose groups. In addition liver, kidneys,
lungs, stomach and salivary glands were examined from all survivors in
the Low and Intermediate dose groups.
Regarding mortality, there were no notable intergroup differences observed. There were no clinical signs thought to be attributable to dosing with the test item. There was a reduction in body weight gain in all male dose groups which receive test item ranging from slight in the Low dose group to marked in the High dose group. There was also a moderate reduction in body weight gain in the female Intermediate dose group and a marked reduction in the female High dose group. A slight reduction in food consumption in the male dose group receiving 80 mg/kg bw/day was found. There were no visual intergroup differences observed regarding water consumption.
There were no notable intergroup differences at any time in regards to differential blood counts nor in regards to organ weights in either sex. Intermediate dose males and High dose males and females showed increased incidences of depressed and raised foci in the stomach. Effects of treatment were present in the salivary glands and stomach of Intermediate and High dose groups in both sexes. In the submaxillary salivary gland lobular degeneration was evident in both groups, both sexes. In the keratinized stomach of both groups, in both sexes, notable intergroup differences were seen for submucosal oedema, submucosal inflammation, acanthosis, hyperkeratosis, inflammation in the keratinized layer, ulceration, basal cell proliferation and 'lesions associated with ulcer'.
Accroding to a subsequent pathological
review and interpretation of selected thyroid and forestomach lesions,
lesions reported are not thought to pose a potential carcinogenic risk
to humans.
There were no lesions reported in the thyroid which were thought to be
related to administration of the test item. The increased incidences of
colloid basophilia of the thyroid reported in mid and high dose male
rats represented several artifacts which happened to be reported more
commonly in these treatment groups.
Administration of the test item was associated with non-neoplasitc
proliferative and inflammatory lesions of the forestomach at 40 and 80
mg/kg bw/d in both male and female rats. These lesions are not thought
to pose a potential carcinogenic risk to humans.
Chronic, mouse
There were no differences in mortality, clinical sings, food and water consumption noted between control and treated groups. Mean body weight was statistically significantly reduced in males of the 150 mg/kg bw/day during weeks 60 to 72. Reduced mean body weight was occasionally observed in females of the 150 mg/kg bw/day. Mean body weight gain of the 150 mg/kg bw/day animals was 25 % and 20 % lower when compared to controls in the 150 mg/kg bw/day males and females, respectively. Haematology parameters were not changed in treated animals. Plasma, RBC, and brain cholinesterase activity of the treated animals were comparable to controls. Treatment-related changes in gross pathology were enlarged thyroid in the 150 mg/kg bw/day males (9/50 compared to 1/50 in the control). In thyroids of males and females a statistically significantly higher incidence in small vacuolated cells with uncertain pathogenesis, most likely of follicular origin and general follicular enlargement was noted beginning at 20 mg/kg bw/day when compared to controls. The histopathological findings were of unclear toxicological significance and were not considered to be indicative for a break of thyroid regulation. Males of the 150 mg/kg bw/day dose group had a statistically significantly higher incidence in pneumonitis (9/50) when compared to controls (1/50). Females of the 150 mg/kg bw/day dose group had a higher incidence in pelvic epithelial inflammation (26/50) of one kidney compared to 16/50 in the control. However, the incidence of pelvic epithelial inflammation in the second kidney was comparable to controls. In males at 150 mg/kg bw/day a higher incidence in hepatocellular adenoma (11/50) was observed when compared to controls (4/10). This was, however, not considered to be of biologically significance for the following reasons: (1) The incidence in hepatocellular adenoma observed in this study (11/50) is only slightly outside the observed historical control range for this type of neoplasm (1 to 8/50). (2) There was no statistically significantly increase in the incidence of hepatocellular carcinomas or in foci of cellular alteration. (3) Additionally, there was no evidence of progression to malignant hepatocellular tumours and no effect on tumour multiplicity observable. (4) Hepatocytotoxicity or genotoxicity was not observed. (5) In females, the incidence of hepatocellular adenoma and/or carcinoma was comparable to controls. In conclusion, the test item is not considered to have caused an increased incidence in hepatocellular adenoma in male mice of the 150 mg/kg bw/day dose group.
Sub-chronic, rat
Graded doses (0, 20, 50 and 125 mg/kg bw/d of the test material were formulated in corn oil and administered by oral gavage five times per week to Sprague Dawley rats for thirteen weeks. A separate satellite group received 125 mg/kg bw/d thirteen weeks and was held without treatment for 28 days after the remaining groups were terminated. Each group consisted of 10 males and 10 females.
The animals were observed for mortality, clinical signs, body weight, food intake, ophthalmologic changes and changes to hematology and clinical chemistry parameters. Each animal was necropsied and selected organs were weighed. Complete microscopic evaluation of the tissues was conducted for all control animals and those receiving 125 mg/kg bw/d (group III and Satellite). Selected tissues, target organs and gross lesions were examined for the animals treated with 20 and 50 mg/kg bw/d.
The test material did not cause deaths among the test animals. Clinically, most animals remained normal throughout the test period. However, there was behavioral evidence of a dose-related irritancy of the test material. This irritancy was transient and the animals returned to normal behavior shortly after treatment. Body weight gain was generally lowered in male rats while food intake was not consistently affected in the treated animals. The decrease in body weight was not dramatic and showed a marked recovery when the satellite animals were removed from treatment. Food intake values also increased in satellite animals at this time. There were no changes to any of the hematology or clinical chemistry parameters measured. Ophthalmologic changes were not evident. The livers of animals treated with 125 mg/kg bw/d were larger than control livers. The livers of satellite animals showed a marked weight reduction toward normal values after treatment was stopped. Microscopic evaluation showed evidence of mild irritation of the stomachs in animals treated with the test material as well as those treated with the vehicle alone. Slight physiologic hepatocyte enlargement correlated with the increased liver mass noted. Satellite animals showed normal liver and stomach histology after the recovery period of 28 days.
Sub-chronic, rabbit
This study was designed to assess the sub-chronic toxicity of the test item when administered orally, via the diet, to 30 New Zealand White rabbits. The test material was mixed with the granulated rabbit diet and the mixtures pelletized. Diets at targeted concentration levels of 500, 2000 and 4000 ppm were prepared and animals (five/sex/group) fed these diets for a period of 90 days. Control animals (five/sex) received untreated standard rabbit diet that was granulated and re-pelleted.
Physical observations, ophthalmoscopic examinations, body weight, food consumption, hematology and clinical chemistry evaluations were performed on all animals pretest and at selected intervals during the treatment period.
Following treatment, all surviving animals were sacrificed, selected organs were weighed and organ/body weight and organ/brain weight ratios calculated. Complete macroscopic postmortem examinations were conducted on all animals. Selected tissues were taken from all animals and histopathological evaluation performed for tissues from the control and high-dose animals. Due to findings in the high-dose animals, the livers from the low- and mid-dose animals were also evaluated histopathologically.
Analysis of the first batch of diets confirmed that the preparation procedure used for this study produced homogeneous mixtures.Additional analyses performed either during a two-week pilot study (95-2395) or in this study confirmed that the pelleted diets at the concentration levels used in this study were stable when stored frozen for at least 33 days.The mean analytical concentrations for the diets used on study were 271, 1663 and 3436 ppm and the average dose levels received by the animals consuming these diets over the entire study were approximately 13, 75 and 150 mg/kg bw/day, respectively.
No adverse effect of treatment at the 500 or 2000 ppm dietary levels was evident from body weight or food consumption data. At the 4000 ppm level, animals adjusted more gradually to the test diet, ate less during the early weeks of the study and gained less weight. Treatment produced hepatic changes at the two higher concentration levels. These effects consisted of elevated gamma-glutamyl transferase levels in the high-dose group only, increased liver weights absolute and relative to either brain or terminal body weight, in the females at the two higher dietary levels and microscopic changes in the liver of males and females at the mid- and high-dose levels consisting of swelling of the hepatocytes and brown pigment in the cytoplasm of hepatocytes and sinusoidal macrophages. On the basis of these data, the no-observed-effect level (NOEL) for this study was the 500 ppm dietary level. This was equivalent to a dose level of approximately 13 mg/kg bw/d.
Repeated dose toxicity: inhalation
Sub-chronic, rat
Treatment of Crl:CD BR rats over 90 days with the test item via the inhalation route at doses of 0, 0.23, 0.3, 1.16, and 6.7 mg/m3 did not influence the survival, the body weight development, or the food consumption of all treated animals. Ophthalmoscopical examination did not show any treatment-related effects on the eyes of the treated rats. There were no treatment-related effects on haematological and clinical chemistry parameters noted.
There were no changes in organ weights noted. Histopathological examination of the organs showed no changes in treated animals when compared to controls except for larynx and lungs of the high dosed animals. In lungs an increased incidence in the aggregation of macrophages was noted in the high dosed males (7 of 15 animals compared to 4of 15 animals in the concurrent control).
RBC and brain cholinesterase activities were within the normal biological variation.
Histopathological findings in the larynx of the high dose group were necrosis in the ventral cartilage, epithelial hyperplasia in ventral region, and squamous metaplasia in ventrolateral region in all animals. In addition, epithelial hyperplasia over the arytenoid projections in all high dosed males and in 5 of the 15 high dosed females was noted. Epithelial ulceration in the ventral region was observed in low incidence in the high dosed males (4 of 15 animals). In some of the high dosed males and females, additionally, atrophy of submucosal glands was noted (3 and 6 animals of 15, respectively).
Repeated dose toxicity: dermal route
Sub-chronic, rat
The potential toxic effects of the test item were evaluated in this 91 -day dermal toxicity study in Sprague-Dawley rats. The study consisted of a control group and three treatment groups with ten animals per sex per group. The test article was dissolved in PEG 400 and administered five days/week, under semi-occlusive conditions, at levels of 50, 200 and 500 mg/kg bw/d. All doses were given at a constant volume of 6.0 mL/kg. Control animals were administered PEG 400 under the same experimental conditions and at an equivalent dose volume.
The rats were observed daily for clinical signs of toxicity and application sites were examined prior to dosing for adverse skin reactions. Individual body weights and food consumption were measured weekly. Ophthalmological examinations were performed on all animals prior to the initiation and near the conclusion of the study. Blood samples were obtained from all animals on study day 89 for determination of cholinesterase activity. This clinical parameter, cholinesterase activity, was selected by the Sponsor since the test substance is a carbamate pesticide and this class of chemicals is known to inhibit cholinesterase activity. All surviving animals were bled at study termination (day 92 or 93) for evaluation of hematology and biochemistry parameters. A complete gross necropsy examination was performed on all animals. Fresh organ weights were obtained from surviving animals and selected tissues and organs were collected from each animal. All tissues from control and high-dose animals and the treated skin, untreated skin, lungs, liver, kidneys and gross lesions from all low and mid-dose animals were examined microscopically.
No treatment-related mortality or clinical signs of toxicity were observed during the study. Treatment-related decreases in body weight gain and/or food consumption were observed in the 200 and 500 mg/kg/day males during the first four weeks of treatment. The reduced mean body weights of these rats remained slightly lower than controls during the remainder of the study. No adverse body weight or food consumption effects were observed in the 50 mg/kg bw/d males or in females from any of the study groups. There were no apparent test article-related changes observed among the groups concerning ophthalmology, clinical pathology, gross necropsy, or organ weight data. Similarly, no treatment-related differences in RBC hemolysate or plasma cholinesterase levels were noted. Dose-dependent dermal irritation was observed in males and females at 200 and 500 mg/kg bw/d levels. The irritation was mild at the 200 mg/kg bw/d level and consisted primarily of slight erythema and slight desquamation. At the 500 mg/kg bw/d level, the irritation was more severe and consisted of slight erythema, slight desquamation, and low incidences of slight edema, moderate erythema and moderate desquamation. Dermal irritation at the 500 mg/kg bw/d level progressed to focal and/or pinpoint areas of eschar in a few animals. Microscopic evaluation of the treated skin revealed hyperkeratosis and acanthotic epidermal thickening in rats of the 200 and 500 mg/kg bw/d groups. The dermal changes were expected to be reversible upon cessation of treatment. No treatment-related signs of dermal irritation were observed in males or females of the 50 mg/kg bw/d group.
Based on these results, a dosage level of 50 mg/kg bw/d was determined to be a no-observed-effect level for 91-day dermal administration of test item in rats.
Justification for classification or non-classification
Classification,
Labelling, and Packaging Regulation (EC) No 1272/2008
The
available experimental test data are reliable and suitable for
classification purposes under Regulation (EC) No 1272/2008. Based on
available data on repeated dose toxicity via the inhalation route the
test item is classified and labelled as STOT RE 1 (H372: "Causes damage
to larynx through prolonged or repeated exposure via inhalation route") according
to Regulation (EC) No 1272/2008 (CLP), as amended for the eighth time in
Regulation (EU) No 2016/918.
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