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Description of key information

A NOAEC of 250 ppm (1060 mg/m3) is selected for inhalation exposure based on organ (liver, adrenals and kidneys) weight increases seen in the 90 day study with male and female F-344 rats.
For the oral route, a NOAEL of 125 mg/kg bw/day is selected based on increased adrenal (absolute and relative) and kidney (relative) weights at higher doses in male rats in the 28 day study.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP compliant, comparable to guideline study with acceptable restrictions.
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
not specified
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Age at start of study: 6-8 weeks
- Weight at study initiation: 200-250 gram (males); 150-200 gram (females)
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
dosage by gavage at a dose volume of 2 ml/kg
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
29 days
Frequency of treatment:
once per day, 7 days per week
Remarks:
Doses / Concentrations:
0.125, 0.5, 1.0 g/kg bw/day
Basis:

No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
Post-exposure period: none
Observations and examinations performed and frequency:
Rats were observed daily for clinical sings and body weight prior to the first dosing and weekly thereafter. Food consumption was measured weekly. Haematology and clinical chemistry parameters were determined from the blood sample collected at necropsy.
Sacrifice and pathology:
Organ weights were measured for the kidneys, adrenals, liver, testes and ovaries. These organs together with the heart and spleen from the control and high-dose group were microscopically examined and any tissues appearing abnormal (as well as these organs from animals that died during the study).
Statistics:
Data from the treated groups were compared to those of the control group using the following tests. Comparisons were limited to within sex analysis. Bartlett's test of homogeneity of variance was used to determine if the groups had equivalent variance at the 1% level. If the variances were not statistically different, the groups were computed using a standard one-way analysis of variance. If significant differences among the means were indicated, Dunnett's test was used to determine which treatment groups differed from controls. Where groups did not have equivalent variance, the non-parametric Kruskall-Wallis test was used to assess differences in group means. If the means were different, Dunn's rank test was used to determine which treatment group differed from control.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Details on results:
Four animals died in the high dose group. The deaths occurred between the days 6 and 9 and two of them were presumed test material related, although the precise cause of death was not identified.
The majority of animals did not exhibit unusual clinical signs during the experiment. Lung rales and anogenital staining was observed at low frequency in 1000 mg/kg animals.
Mean body weights were significantly lower in the 1000 mg/kg males on 7 (-14%), 21 (-18%) and 28 (-19%). Females had mean body weights comparable to controls. Food consumption was significantly lower in the 1000 mg/kg males and females during week 1 and also during week 2 in the 1000 mg/kg males.
A dose-related increase of relative (body) and absolute adrenal weight was noted and it was statistically significant in the 500 (mid) and the 1000 (high) mg/kg males. The following mean adrenal weights were measured (grams): ctrl: 0.057 ± 0.010, 125 mg/kg: 0.074 ± 0.015, 500 mg/kg: 0.082 ± 0.008*, 1000 mg/kg: 0.100 ± 0.010** (*p<0.05, **p < 0.01). The mid and high dose males also had increased relative kidney weights. The organ weight increases in the kidney and adrenals were not accompanied by any histopathological changes. The liver weights were not statistically significantly affected. Females displayed no statistically significant differences in any organ weights.
The high dose males had increased activated partial thromboblastin time and increased blood glucose but the biological significance of these findings was unknown.
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
other: adrenal (absolute and relative) and kidney (relative) weight increase
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
125 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant guideline study, no restrictions, fully adequate for assessment.
Reference:
Composition 0
Qualifier:
according to
Guideline:
EPA OTS 798.2450 (90-Day Inhalation Toxicity)
Qualifier:
according to
Guideline:
other: US EPA Neurotoxicity testing guidelines: 40 CFR Part 798 subpart G (7-1-93 Edition)
GLP compliance:
yes
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 7 weeks
- Weight at study initiation: 102-159 gram
- Housing: doubly housed during the acclimatisation period , individually during exposure
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-27
- Humidity (%): 18-95
- Photoperiod (hrs dark / hrs light): 12/12


Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure chamber: Harford 6000 litre glass and stainless steel chamber
- Source and rate of air: houseline air
- System of generating particulates/aerosols: test material was evaporated under nitrogen. A laboratory pump equipped with a piston pumped the test material directly from a S-gallon gas can via 1/8" Teflon tubing. The test material was delivered onto the glass helix of a counter-current volatilization chamber.
- Temperature, humidity, pressure in air chamber: temperature and humidity were monitored and recorded every half hour during exposure and maintained within the following ranges: 20-24 °C and 40-60%
- Air flow rate: 1252 -1336 Lpm
- Air change rate: 4.5-4.8 min; the exposure chambers were operated dynamically under slight negative pressure at a calibrated airflow rate calculated to provide one complete air change every 5 minutes (12 changes per hour) and a 99% equilibrium time (T99) of 23 minutes or less. The chamber size and airflow rate was considered adequate to maintain the animal loading factor below 5%o and oxygen at or above 19%.
- Method of particle size determination: TSI Aerodynamic Particle Sizer
-Treatment of exhaust air: the chamber was exhausted through a system of a coarse filter, a HEPA filter and activated charcoal bed.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
samples from the exposure chamber were analysed four times (once for the control groups) each day (MIRAN ambient air analyser).
Duration of treatment / exposure:
13 weeks / at least 65 exposures
Frequency of treatment:
6 hours/day, 5 days/week
Remarks:
Doses / Concentrations:
250, 1500, 3500 ppm
Basis:
other: target concentrations
Remarks:
Doses / Concentrations:
251±4, 1500±45, 3519±61 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
There were 51 rats/sex/treatment in the control and high dose groups, and 41 rats/sex/treatment in the low- and mid-dose groups.
Control animals:
yes, concurrent vehicle
Details on study design:
Exposure levels were selected based on available toxicity data and limiting the highest exposure to less than 75% of the vapour explosion limit for the test substance.
Post-exposure period: 4 weeks
Observations and examinations performed and frequency:
All animals were observed twice daily (cage side) for mortality and obvious signs of toxicity and given a detailed examination weekly. In addition, all visible animals were observed as a group at least once during each exposure.

Body weights were monitored twice pre-test, weekly and terminally. Food consumption was recorded one week before treatment and after that weekly.

Ophthalmology evaluations were performed pretest, at termination and at the end of the recovery period.

Samples for haematology evaluations were collected on weeks 5, 14 and 18 (recovery).

Clinical chemistry samples were taken on weeks 5, 6, 14, and 18.

A satellite group of 10 rats/sex/group was evaluated based on a Functional Observation Battery after a single 6-hour exposure 1, 6 and 24 hours after the exposure. FOB was also performed to another group of 10 rats/sex/group of repeated exposure in weeks 2, 3, 5, 9 and 14. A modified version of Schulze’s procedure was used to monitor motor activity in weeks 5, 9 and 14 with a group of 10 rats/sex/group. The FOB was performed to all animals before evaluation of motor activity. A neuropathology examination was performed to 6/10 animals.
Sacrifice and pathology:
A complete macroscopic examination was performed on all animals at termination and recovery sacrifice. Selected organs were weighed; brain length and width measurements were taken for neuropathology animals. Selected tissues and organs were taken and preserved for all animals. Microscopic evaluation was conducted on 39 organs for the high dose and control groups; the lungs were examined in all groups. Adrenals, brain, heart, kidneys, liver, lungs, ovaries and testes were weighed.
The following organs were collected from the animals subjected to a neuropathological examination: brain, spinal cord, sciatic nerve, sural nerve, tibial nerve, gasserian ganglia dorsal root ganglia, dorsal and ventral root fibres.
Other examinations:
Using separate animals treated similarly 5 male and 5 female rats per group were subjected to renal nephropathy and proliferation studies on weeks 1, 4 and 13. Proliferation was examined of 5-bromo-2'-deoxyuridine (BrdU) incorporation into tissues after 1 ,4, and 13 weeks of exposure. Nephropathy in the rat was also evaluated by the presence of hyaline droplets and specific staining for α-2u-globulin in the proximal convoluted tubules.
Statistics:
STATISTICAL ANALYSIS
The following parameters were analyzed statistically:
- body weight
- cumulative body weigh change (from Week 0)
- food consumption
- motor activity counts
- forelimb and hindlimb grip strength measurements
- landing foot splay measurements
- hematology
- clinical chemistry
- terminal organ weights, organ:body weights ratios, organ/brain weight ratios,
and brain length and width
- proliferation assessment in rat kidney and mouse liver

METHOD OF ANALYSIS
Mean values of all dose groups were compared to the mean value for the control group at each time interval.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
effects observed, treatment-related
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Mortality and clinical observations
Two high dose animals, one of both sexes, were found dead on days 36 and 33. Although the deaths occurred soon after blood sampling (day 32) they were believed to have been treatment-related. The cause of death could not, however, be determined in the post-mortem macroscopic and microscopic evaluations. In the high dose group, most animals were prostrate during the exposure of TAME. Also the animals in the mid dose group were prostrate or lethargic during the first month of the exposure. The latter half of the study few animals of this group had laboured breathing and lethargy. The low dose group (250 ppm) showed no abnormal signs during the study. The observations in the high dose group during the recovery period were comparable to the control group.

Ophthalmology
At termination of the study, there seemed to be a dose related increase of corneal scarring in the males and females of the 1500 and 3500 ppm dose groups. The incidences were 20% and 28% in the high dose males and females, respectively and 12.5% in the mid exposure males and females. Corneal scarring was not observed in the 250 ppm group. The control incidence was 3.8% in each group. After the 4-week recovery scarring was seen in 30% of the high exposure males and 20% high exposure females, but not in the control animals. However, corneal scarring occurs increasingly with age in F-344 rat. In this study TAME has increased the onset in the 1500 ppm and 3500 ppm exposure groups.

Body weights and food consumption
The mean body weight and body weight increase were significantly decreased in the 3500 ppm dose group. The decrease was slightly more pronounced in the males than in the females: the mean body weight was 6.9% lower in females and 10% lower in males. The mean body weights were still decreased after the recovery period on week 18, with males 9.4% and females 3%. The weight gain was comparable to control. The mean body weights and mean body weight increases in the 250 and 1500 ppm dose groups were significantly increased with about the same magnitude as they were decreased in the high dose groups, but this change is not considered to be toxicologically significant. The food consumption was statistically significantly decreased in the males and females of the high dose group during the initial 2 weeks of exposure. The low and mid dose group animals had sporadic decreases and increases in food consumption.

Neurobehavioral studies
After the single 6-hour exposure (3500 ppm) to the satellite group of 10 rats/dose/sex dose related effects on the central nervous system and neuromuscular junction after one hour interval were described. The effects included depression of the central nervous system and neuromuscular junction impairment. The effects were no longer evident after 6 or 24 hours acute exposure and they were not seen after repeated exposure to TAME. In the 1500 ppm dose group, these effects were only seen in male rats. There were no neuropathological changes at any exposure level. The NOAEL for acute neurobehavioral effects of TAME was 250 ppm in males and 1500 ppm in females.

Haematology
Platelet counts were statistically significantly increased in weeks 5 and 14 in the males and females of the 3500 ppm and 1500 ppm dose groups. The values returned to normal after the 4-week recovery period. There other slight but statistically significant decreases were seen in haemoglobin, haematocrit and/or red blood cell counts compared to controls in all treated males. However, the authors did not consider these changes toxicologically significant since all values were within normal physiological range and changes were not related to the exposure range. In white blood cells, there were no decreases in the total count but an increase in the absolute neutrophils count in high dose males and females and a concomitant decrease in absolute lymphocyte count in high exposure males and females in week 5 and high exposure males in week 14. The authors regarded the changes as usually indicative of stress if they are not accompanied by a change in total white blood cell count. The neutrophils and lymphocyte values had returned to control level, except in the high dose females, which had slight, but statistically significant increases in total white blood cell count and absolute lymphocyte counts.

Clinical chemistry
Total protein was significantly increased in the 1500 and 3500 ppm male and female rats. The changes were accompanied by an increase of albumin and globulin with a decrease in albumin/globulin ratio. These changes reversed after the 4-week recovery period. Alkaline phosphatase activity was significantly decreased in all treated males at week 5 and in 250 ppm and 3500 ppm groups also at week 14. In addition, there was a significant decrease in female alkaline phosphatase activity at 1500 and 3500 ppm. Apart from the high dose male group the activity levels were normal after the recovery period. The changes were within normal variability and were not considered toxicologically relevant. There was also a slight increase in blood urea nitrogen (BUN) in low- and mid-dose males at week 5 and in high exposure males at week 19. In addition, there was a series of other sporadically and non-consistently occurring changes in clinical chemistry parameters, which were not of toxicological significance.

Organ weights and microscopic findings
A statistically significant 4-5% decrease was noted in the mean absolute brain weight of the 3500 ppm males and females at termination of exposure. This was accompanied by a significant increase in the brain-to-body weight ratio in the 3500 ppm males. This change remained after the recovery period in males, although the mean absolute weight increase was now at 3%. The female brain weights, absolute or relative to body weight, were normal at the end of the recovery period. The findings in the male brain weight decrease were not supported by the dimension measurements in the neuropathology animals, which had brain weights comparable to control group. However, the results were obtained by whole-body perfusion, which together with the fixation process may have caused small changes go unnoticed. Liver weights (absolute, relative to body weight and relative to brain weight) were increased in all treated males (250, 1500 and 3500 ppm) and females of 1500 ppm and 3500 ppm groups. The percent changes in absolute weight compared to control rats were 19% (250 ppm), 18% (1500 ppm) and 22% (3500 ppm). When expressed as relative to brain weight, the differences between control and the treated groups were 15% (250 ppm), 17% (1500 ppm) and 26% (3500 ppm). The liver weights returned to normal after the recovery period. No histopathologic findings were seen in the livers, which would have correlated with the weight changes. Kidney to body weight and kidney to brain weigh ratios were increased in the 3500 ppm males and females. In females, the kidney to body weight changes to control was 15% and relative to brain weight the change was 13%. The respective male values were 21% and 13%. Absolute and relative kidney weights were increased in 1500 ppm females. Absolute kidney and kidney to brain weight ratios were statistically significantly increased relative to control males in the 1500 ppm (18% abs., 15% org/brn) and 250 ppm (12% abs., 8% org/brn) group males. The increase in the latter groups was consistent with the increase in terminal body weights (250 ppm: ~9% and 1500 ppm 15% higher than the control). In microscopy, evidence of cell proliferation and male rat specific α2u-globulin syndrome were seen. These findings contributed to the weight increase in males but were not present in the females. There was also statistically significant increase in the heart to body weight in the 3500 ppm males (15%) and females (7%), which remained increased (10%) only in males after the recovery period. Absolute heart weight was increased in the 250 ppm group (11%) and in the 1500 ppm males, absolute (17%) and heart weight relative to brain weight was increased (14%). However, these changes were attributed to increase in body weight since the heart to body weight ratios were comparable to controls. Adrenal (absolute, relative to brain and body) weights were statistically significantly increased in the 3500 ppm dose males (55%) and females (45%). In the 1500 ppm group, the absolute adrenal weight increase in males was 20% and in females 16%. Also the adrenal to brain weight ratios were increased in this dose group, 17% in males and 15% in females. In the 250 ppm group, only males had an increased absolute adrenal weight (14% abs, 10% relative to brain weight). Following the four-week recovery period, the increases in adrenal weights had dropped in the high dose females to 11% increase in the absolute weights. In the high exposure recovery-group males the mean absolute adrenal weight was comparable to the control group adrenal weight. Only the adrenal to body and adrenal to brain weights were statistically significantly increased in the high dose males. This was attributed to lower terminal body and brain weight in these animals. There were no microscopic lesions found and the weight changes were attributed to stress due to exposure. There were no adverse treatment-related histopathological findings seen in either the whole body exposure or the neuropathology portion of the study.
Dose descriptor:
NOAEC
Effect level:
250 ppm
Sex:
male/female
Basis for effect level:
other: organ weight increases in male and female liver, adrenals and kidneys
Dose descriptor:
NOAEC
Remarks:
acute neurobehavioral effects
Effect level:
250 ppm
Sex:
male/female
Basis for effect level:
other: depression of the central nervous system and neuromuscular junction impairment
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
1 060 mg/m³
Study duration:
subchronic
Species:
rat

Mode of Action Analysis / Human Relevance Framework

Additional information

Regarding the oral route, a subacute toxicity with rats is available. Five male and five female Sprague-Dawley rats were administered 0, 125, 500 or 1000 mg/kg bw TAME in corn oil by gavage (Daughtrey et al., 1995). The dose was given once daily, 7 days a week for a period of 29 days. The critical effects observed were an increased adrenal (absolute and relative) and kidney (relative) weight at 500 and 1000 mg/kg bw in males. The organ weight increases in the kidney and adrenals were not accompanied by any histopathological changes. Liver weights were not affected significantly. The NOAEL was set at 125 mg/kg bw/day.

For inhalation repeated dose toxicity, two 90 days studies are available, one with rats and one with mice (Huntingdon Life Sciences, 1997a/b). In addition, a subacute toxicity study with rats is also available (IIT Research Institute, 1992). The 90 days studies are considered key (longer exposure duration, lower NOAEC). In these studies the exposure concentrations were 250, 1500 and 2500 / 3500 ppm (1060, 6360, 10600 / 14840 mg/m3).

 

In mice, the critical effect was an increase in the labeling index of hepatocytes in high- and mid-exposure males and females and low-exposure females. There was microscopic evidence of centrilobular, hepatocellular hypertrophy in high- and mid-exposure males and females. Based on these findings, the NOAEC for male mice exposed to TAME was 250 ppm (1060 mg/m3); a NOAEC for female mice was not established by the study authors. However, given the absence of dose-response in the female liver proliferation findings and their transient nature, the EU concluded in their risk assessment report that the NOAEC can be set at 250 ppm (1060 mg/m3) for both males and females.

In rats, effects critical for setting the NOAEC were (relative) organ weights increases in male and female liver, adrenals and kidney. Increased absolute and relative adrenal weights in the 3500 and 1500 ppm males and females and 250 ppm males were explained by the investigators as a response to stress due to exposure to the test material. They did not consider this a direct effect of the test material because there were no microscopic lesions in the adrenals. Only male rats showed α-2µ-microglobulin-related cell proliferation of the kidney tubules, which could have contributed to the weight change in male kidneys at 3500 ppm. The increase in the female kidney weights at 3500 and 1500 ppm could not be explained by any toxicological phenomenon. After the 4-week recovery period, the high dose males and females had increased kidney to body weights, although the absolute and kidney to brain weights were comparable to control values.

The investigators set the NOAEC to 250 ppm (1060 mg/m3) for females based on the increased liver and kidney weights at 1500 ppm. Based on the liver weight increase in all dose groups, no NOAEC can be set for males. While liver weight increase is a typical form of adaptation and could be seen as non-adverse, it is more difficult to account for the adrenal or especially the kidney weight increases simply as adaptive. It is debatable whether, e.g., the adrenal organ weight increases with no adverse changes in microscopy or blood values can be discounted as “indirect” or “stress-related”. However, although there was an 8% increase in kidney/brain weight and a 10% increase in adrenal/brain weight in males at 250 ppm, this level was not considered as the LOAEC by the EU (conclusion from the EU risk assessment report). The weight increases were statistically significant only when presented as absolute weight or organ/brain weight values. Since the body weight of the low dose males also increased (+9%), a factor that may have played a contributing or confounding role, and since there was no histopathological damage present in these animals, the exposure level of 250 ppm (1060 mg/m3) was considered as a NOAEC.


Repeated dose toxicity: via oral route - systemic effects (target organ) glandular: adrenal gland; urogenital: kidneys

Repeated dose toxicity: inhalation - systemic effects (target organ) digestive: liver; glandular: adrenal gland; neurologic: central nervous system; urogenital: kidneys

Justification for classification or non-classification

In accordance with the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, classification is not necessary for repeated dose toxicity.