Registration Dossier

Administrative data

Description of key information

The exposure of rats to the test substance for 28 days by inhalation caused concentration-related lesions in larynx, trachea and lung. No histopathological effects were seen in any other organ outside the respiratory tract. The NOAEC for systemic toxicity is the highest tested concentration of 150 mg/m3. The NOAEC for local effects was the lowest tested concentration of 10 mg/m3. In the two-generation oral reproductive toxicity study with the test substance (HCl), the NOAEL for general systemic toxicity was set at 300 mg/kg bw/day based on reduced food consumption and/or body weight gain, as well as organ weight changes unaccompanied by histopathological findings.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
repeated dose toxicity: oral, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 Aug 2006 - 15 Jan 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 416 (Two-generation reproduction toxicity study)
Deviations:
yes
Remarks:
Food consumption was not determined between days 14 and 21 after parturition
Qualifier:
according to
Guideline:
other: Corrigendum to EC Commission Directive 2004/73/EC, Part B: Methods for the determination of toxicity: Two-Generation Reproduction Toxicity Study; Official Journal of the European Communities; No. L216, pp. 236–246
Version / remarks:
29 Apr 2004
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Version / remarks:
Aug 1998
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- No.of test material: 1) 05/0372-2; 2) 05/03723; 3) 05/0372-4
- Lot/batch No.: ad 1) JB116/2+3 (from 09 Aug – 04 Oct 2006); ad 2) JB116/4 (from 04 Oct – 29 Nov 2006); ad 3) JB116/9-17 (from 29 Nov 2006 until the scheduled termination of the in life part of the study)
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, under N2


Species:
rat
Strain:
Wistar
Remarks:
Crl:WI (Han)
Details on species / strain selection:
The rat is the preferred animal species for reproduction studies according to test guidelines. This strain was selected since extensive historical control data were available for Wistar rats.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 44 (+/- 1) days
- Weight at study initiation: (P) Males: 162.1 (142.5 – 186.5) g; Females: 126.2 (110.6 – 145.1) g;
- Fasting period before study: none
- Housing: rats were housed individually in type DK III stainless steel wire mesh cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²), with the following exceptions:
• overnight mating: male and female mating partners were housed together in type DK III cages
• gestation day 18 – lactation day 21: pregnant animals and their litters were housed in Makrolon type M III cages (floor area of about 800 cm²). The M III cages were also supplied by Becker & Co. Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.
- For enrichment wooden gnawing blocks (Typ NGM E-022, supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria) were added. The cages with the test animals were arranged in racks in such a way that uniform experimental conditions (ventilation and light) were ensured.
- Diet: ad libitum, ground Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland
- Water: ad libitum
- Acclimation period: 16 days
- Other: According to a written statement from the breeder, male and female animals were derived from different litters. This was necessary to rule out the possibility of sibling mating.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
The test substance was weighed and thoroughly mixed with a small amount of food. Then corresponding amounts of food, depending on the dose group, were added to this premix in order to obtain the desired concentrations. Mixing was carried out for about 10 minutes in a laboratory mixer. Test diets were prepared at intervals, which guaranteed that the test substance in the diet remained stable throughout the feeding period.

During the first week of the premating period, F0 parental animals received dietary Ethanolamine hydrochloride (EAH) concentrations based on the body weight of randomization and historical food consumption data given below:
Food consumption males: 19 g
Food consumption females: 15 g
The dietary concentration of EAH was calculated using the following formula: BWx . D / FCx = ppm where
BWx = mean body weight on day x [g]
D = desired dose [mg/kg body weight/day]
FCx = mean daily food consumption on day x [g]
ppm = dietary EAH concentration for the week/period following day x

- During the remaining premating period, the dietary concentrations of EAH were adjusted weekly for each group and sex based on body weight and food consumption measurements from the preceding week.
- During the mating period of the F0 parental animals, each group and sex received the concentrations of EAH used during the last week of the premating period. This concentration was maintained throughout the mating period with the following exception: During cohabitation, both sexes received the test substance preparation for females as soon as the male was placed in the cage of its female partner. Both sexes returned to their normal test diet when they were separated the following morning. This test diet cycle remained in effect until there was evidence of successful mating. At that time, the mated animals received the test substance preparations described below at the first opportunity in the specific week.
- During the gestation period, dietary concentrations of EAH for the F0 males were again adjusted weekly on the basis of body weight and food consumption data from the preceding week. The EAH concentrations in the diet of the F0 females were the same as those used during the last week of the premating period.
- During the lactation period, dietary concentrations of EAH for the F0 males continued to be adjusted weekly on the basis of body weight and food consumption data from the preceding week. The EAH concentrations in the diet of the F0 females were 50% of those used during the last week of the premating period. This dietary adjustment, derived from historical body weight and food consumption data, maintained the dams at the desired doses of EAH during this period of increased food intake.
- Post weaning, dietary EAH levels for parental male animals awaiting necropsy were adjusted weekly based on body weight and food consumption data from the preceding week. The EAH concentration of parental female diets was the same as those used during the last week of the preceding premating period.
- Until all litters were weaned (when the last selected F1 pup reached age of day 21 p.p.), the food for the weaned F1 pups selected as F1 parental animals was prepared with EAH concentrations on the basis of historical body weight and food consumption data for rats of similar age.
- During the first week of the premating period of F1 parental animals, dietary EAH concentrations were formulated on the basis of actual body weight on day 0 and historical food consumption data. Subsequently, dietary EAH levels for each F1 dose group and sex were adjusted as described for F0 parental animals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of EAH in the diet over 32 days at room temperature was investigated analytically before the beginning of the study. Homogeneity and concentration control analyses were carried out at the beginning and toward the end of the premating periods. At least one analysis of test substance preparations for female animals was carried out during the gestation and lactation periods.

The analyses were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany.
Duration of treatment / exposure:
semichronic duration (> 75 days)
Frequency of treatment:
continuously
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
in diet
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
in diet
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
in diet
No. of animals per sex per dose:
25
Control animals:
yes, plain diet
Observations and examinations performed and frequency:
MORTALITY:
A check for moribund or dead animals was made twice daily on working days or once daily (Saturday, Sunday or on public holidays). If animals were in a moribund state, they were sacrificed and necropsied. The examinations of these animals were carried out according to the methods established at the pathology laboratory.

CLINICAL OBSERVATIONS:
All parental animals were checked daily for clinically evident signs of toxicity. For technical reasons, however, the clinical observations recorded during the premating periods were printed out on a weekly basis (the daily observations can be found in the raw data). The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only special findings, e.g. disability to deliver, were documented on an individual dam basis. In addition to the evaluations in the mornings, parturition behavior of the dams was also inspected on weekdays (except public holidays) in the afternoons. The day of parturition was considered the 24-hour period from about 3.00 p.m. of one day until about 3.00 p.m. of the following day. Deviations from this procedure were possible on Saturday, Sunday and on public holidays.

FOOD CONSUMPTION:
In general, food consumption was determined once a week (each time for a period of at least 6 days) for the male and female F0 and F1 parental animals. For the females during pregnancy (animals with evidence of sperm), food consumption was determined weekly for days 0-7, 7-14 and 14-20 p.c. During the lactation period (animals with litter), food consumption was determined for days 1-4, 4-7 and 7-14 p.p. Food consumption was not determined between days 14 and 21 after parturition as required in the test guidelines, since during this time pups will begin to consume considerable amounts of solid food offered, and therefore, there was no point in such measurement. Furthermore, food consumption was not determined for females without positive evidence of sperm and for females without litter.

COMPOUND INTAKE:
The intake of test substance was calculated from the amount of food consumed and is expressed as mg/kg body weight/day (mg/kg bw/d). The calculation of the group values/day was carried out according to the following formula: intake of test substance on day x in mg/kg bw/d = (daily food consumption on day x in grams) x (concentration in ppm) / (body weight on day y in grams (last weighing before day x))

BODY WEIGHT DATA:
In general, the body weight of parental animals was determined on the first day of the premating period and then once a week at the same time of day (in the morning). Based on these results, the body weight change of the animals was calculated. The following exceptions are notable for the female parental animals:
a) During each gestation period the F0 and the F1 generation parental females were weighed on the day of positive evidence of sperm (day 0 p.c.) and on days 7, 14 and 20 p.c.
b) Females showing no positive evidence of sperm in vaginal smears were not weighed during the mating interval.
c) Females with litter were weighed on the day after parturition (day 1 p.p.) and on days 4, 7, 14 and 21 p.p.
d) Females without litter were not weighed during the lactation phase.

BLOOD SAMPLINGS:
Blood samples were taken from all F0 and F1 parental animals of each sex and test group during week 10 of premating treatment and the plasma was analyzed for the concentration of the test substance.
Sacrifice and pathology:
All F0 and F1 parental animals were sacrificed by decapitation under Isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention was given to the reproductive organs. As soon as possible after termination, one portion of the liver (lobus medialis) of each 10 dams per group was sampled to be analyzed for choline concentration.

ORGAN WEIGHTS:
Weight assessment was carried out on all animals sacrificed at scheduled dates. The following weights were determined:
1. Anesthetized animals, 2. Liver, 3. Kidneys, 4. Adrenal glands, 5. Testes, 6. Epididymides, 7. Cauda epididymis, 8. Prostate, 9. Seminal vesicles including coagulation glands, 10. Ovaries, 11. Uterus, 12. Spleen, 3. Brain, 14. Pituitary gland, 15. Thyroid glands (with parathyroid glands).

ORGAN/TISSUE FIXATION:
The following organs or tissues of the F0 and F1 generation parental animals were fixed in 4% neutral buffered formaldehyde solution or in BOUIN’s solution, respectively:
1. Vagina, 2. Cervix uteri, 3. Uterus, 4. Ovaries (fixed in BOUIN´s solution), 5. Oviducts, 6. Left testis (fixed in BOUIN´s solution), 7. Left epididymis (fixed in BOUIN´s solution), 8. Seminal vesicles, 9. Coagulation glands, 10. Prostate, 11. Pituitary gland, 12. Adrenal glands, , 3. Liver, 14. Kidneys, 15. Spleen, 16. Brain, 17. Thyroids (with parathyroids), 18. All gross lesions. After fixation, the organs fixed in BOUIN´s solution were embedded in Paraplast. Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings. All gross lesions were examined. Of the fixated organs the organs of all animals in the control group and the high dose group were evaluated. Additionally, the organs for mating pair suspected of reduced fertility were evaluated.
Statistics:
Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
F0: No clinical signs or changes of the general behavior, which may be attributed to the test substance, were detected in F0 male or F0 female parental animals of the test groups 01 and 02 (100 and 300 mg/kg bw/d). Intensively yellow discolored urine was recorded in all F0 males and females of test group 03 (1000 mg/kg bw/d) from study week 3 onwards until the end of the treatment period. This urine discoloration mirrored the systemic availability of the test substance rather than being an adverse effect and was most likely caused by the excreted test compound and/or its metabolites.

Clinical observations for females during gestation of F1 litters:
All F0 females of test group 03 showed intensively yellow discolored urine during the entire gestation period for F1 litter. No other clinical findings were observed in the test groups 00-03 (0, 100, 300 and 1000 mg/kg bw/d). One sperm positive female of test group 02 (300 mg/kg bw/d) and one of test group 03 (1000 mg/kg bw/d) did not deliver F1 pups. This observation was not considered to be associated to the test compound.

Clinical observations for females during lactation of F1 litters:
All F0 females of test group 03 showed intensively yellow discolored urine during the entire lactation period for F1 litter. One high-dose female (1000 mg/kg bw/d) had just one pup (female), which was cannibalized by its mother on lactation day 8. No other clinical findings were observed in the test groups 00-03 (0, 100, 300 and 1000 mg/kg bw/d).

F1: No clinical signs or changes in general behavior, which may be attributed to the test substance, were detected in F1 male or F1 female parental animals of the test groups 11 and 12 (100 and 300 mg/kg bw/d).
Intensively yellow discolored urine was recorded in all F1 males and F1 females of test group 13 (1000 mg/kg bw/d) from study week 0 onwards until the end of the treatment period. This urine discoloration mirrored the systemic availability of the test substance rather than being an adverse effect and was most likely caused by excreted test compound and/or its metabolites. Furthermore, one F1 male animal of test group 11 (100 mg/kg bw/d) had a skin lesion at its throat during study weeks 3-6.

Clinical observations for females during gestation of F2 litters:
All F1 females of test group 13 showed intensively yellow discolored urine during the entire gestation period (F2 litter). No other clinical findings were observed in the test groups 10-13 (0, 100, 300 and 1000 mg/kg bw/d).

One sperm-positive female of test group 10 (control), one of test group 11 (100 mg/kg bw/d) and two of test group 13 (1000 mg/kg bw/d) did not deliver F2 pups. These observations were not considered to be associated to the test compound due to a missing dose-response relationship.

Clinical observations for females during lactation of F2 litters:
All F1 females of test group 13 showed intensive yellow discolored urine during the entire lactation period for F2 litters. No other clinical findings were observed in the test groups 10-13 (0, 100, 300 and 1000 mg/kg bw/d).
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled mortalities of male and female parental animals in any test group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
F0: Mean body weights and average body weight gain of the F0 parental males of all test substance-treated groups (100, 300 and 1000 mg/kg bw/d) were comparable to the controls throughout the entire study period. Observed differences between controls and test groups were regarded as spontaneous in nature. During premating, gestation and lactation periods, the mean body weights and body weight gains of the F0 parental females in the low- and mid-dose groups were generally comparable to the concurrent control group. During premating, gestation and lactation periods, the mean body weights and body weight gains of the F0 parental females in the low- and mid-dose groups were generally comparable to the concurrent control group. Mean body weights and body weight gains of the F0 females in test group 03 (1000 mg/kg bw/d) were similar to the controls throughout the entire premating period. During gestation, these animals gained less weight from gestation day 7 onwards (up to 38%). As a consequence, body weights on gestation day 20 were 8% lower than the control. This effect may have been caused by the statistically significantly increased postimplantation loss and the statistically significantly decreased mean number of delivered pups in test group 03. This is also indicated by the unaffected body weight of the high dose dams on post-delivery day 1. Mean body weights of the high-dose females remained comparable to the controls during entire lactation, whereas the weight gain wavered up and down in the individual lactation sections.

F1: Mean body weights and body weight gain of the F1 parental males in test groups 11-13 (100, 300 and 1000 mg/kg bw/d) were comparable to the control throughout the entire treatment period. The statistically significantly decreased values of body weight gain in the high-dose males during study weeks 6-7 and 9-10 were in the normal range of fluctuation of this group and the control during the course of the study and, therefore, regarded as incidental. Mean body weights and body weight gain of the F1 parental females in test groups 11-12 (100 and 300 1000 mg/kg bw/d) were comparable to the control throughout premating, gestation and lactation periods. Mean body weights and body weight gains of the F1 females in test group 03 (1000 mg/kg bw/d) were similar to the controls throughout the entire premating period, the statistically significantly increased body weight gain of the high-dose F1 females (1000 mg/kg bw/d) during premating week 1-2 was regarded as incidental variance. The average weight gain of these animals was significantly below control (26%) during gestation days 14-20, which led to an averaged decrease of weight gain for the entire gestation of 17%. This effect may have been caused by the statistically significantly increased postimplantation loss and the statistically significantly decreased mean number of delivered pups in test group 03. Mean body weights of the high-dose females remained comparable to the controls during entire lactation.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
F0: Food consumption of the F0 parental males of all test substance-treated groups was generally comparable to the controls throughout the entire study. The statistically significant increase of food consumption in test group 02 (300 mg/kg bw/d) during study week 12-13 was regarded as spontaneous in nature. Food consumption of the F0 parental females of the low- and mid-dose groups (100 and 300 mg/kg bw/d) was comparable to the control animals during the periods of premating, gestation and lactation. Food consumption of the high-dose F0 females (1000 mg/kg bw/d) was also comparable during premating and gestation periods. However, in these animals, food consumption was statistically significantly below controls (up to 18%) in the individual lactation sections: days 1-4, 4-7, and 7-14 p.p.

F1: Food consumption of F1 male and female animals in test groups 11-12 (100, and 300 mg/kg bw/d) was generally comparable to the control group throughout the entire treatment period, covering premating, gestation and lactation periods. Food consumption of the high-dose F0 females (1000 mg/kg bw/d) was also comparable during premating and gestation periods. However, in these animals, food consumption was statistically significantly below controls (-11%) on lactation days 1-4, and remained below control on lactation days 4-7 and 7-14 (-7%), although not statistically significant.

For all test groups the intake of Ethanolamine hydrochloride correlated well with the desired target doses. For the actual test substance intake see 'Any other information on results incl. tables'.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
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):
In the top-dose F0 and F1 males the test substance administration led to a decrease of absolute and relative organ weights of cauda epididymidis and epididymides. Furthermore, prostate weight and the number of homogenization resistant caudal epididymal sperm was slightly, but significantly decreased in the F0 males. These findings were considered to be treatment-related effects, whereas histomorphological correlates were missing.
A statistically significant increase of absolute and relative kidney weights was noted in male and female F1 animals of the mid (300 mg/kg bw/day) and top-dose (1000 mg/kg bw/day) groups. Because no histomorphological correlate was detected, the treatment-related weight increase was considered to be of no toxicological concern. As compared to control animals, the kidneys of low-, mid-, and top-dose male and female animals revealed a low incidence of basophilic tubules in a slightly higher number of animals. The severity (minimal to slight) was comparable between controls and treated animals and a clear dose-response relationship was missing. Thus this finding was considered to have no toxicological relevance.
Gross pathological findings:
no effects observed
Description (incidence and severity):
F0: All gross lesions observed in test animals occurred singularly. They are considered to be spontaneous lesions in origin and are not related to treatment. The female animal which was not pregnant as well as the male mating partner did not show relevant gross lesions.
F1: All gross lesions observed in test animals occurred singularly. They are considered to be spontaneous lesions in origin and are not related to treatment.
One non-pregnant female animal did show a bilaterally severe reduced size of the ovaries as well as a moderate thickening of the uterus wall. The other three non-pregnant females did not show any gross lesions. The four male mating partners did not show any gross lesions either.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
F0:
- Extramedullar hematopoiesis was seen in the spleen of 1000 mg/kg male animals in a slightly higher number of animals compared to control animals, but only in a minimal (grade 1) to slight (grade 2) severity. A treatment-related increase seems unlikely, because no weight deviations were observed, the severity was very low and comparable to the control animals. All other findings noted were single observations either, or were similarly in distribution pattern and severity in control rats compared to treatment groups. All of them are consideredto be incidental and/or spontaneous in origin and without any relation to treatment.
- Fertility: The non-pregnant female and the male mating partner did not show histopathological findings explaining the infertility. Only a minimal focal atrophy of the prostate was present, which is considered an incidental finding.

F1:
- As compared to control animals, the kidneys of low, mid, and top dose male and female animals revealed a low incidence of basophilic tubules in a slightly higher number of animals. The severity (minimal to slight) was comparable between controls and treated animals and a clear dose-response relationship was missing. The cauda epididymis and epididymides of top dose males showed no histomorphological correlates to the decreased organ weights. All other findings noted were single observations either, or were similarly in distribution pattern and severity in control rats compared to treatment groups. All of them are considered to be incidental and/or spontaneous in origin and without any relation to treatment.
- Fertility: One non-pregnant female showed a bilateral moderate diffuse stromal hyperplasia and a unilateral severe focally extensive chronic inflammation of the ovaries as well as an ovarian cyst. There were still corpora lutea present and the histopathological findings did not correlate with the gross lesion. The gross lesion “thickening of uterine wall” had no corresponding histological finding. The findings on the ovaries might explain the infertility. The male mating partner did not reveal lesions affecting the fertility. One female Animal showed within the uterus a severe multifocal endometrial and glandular degeneration and within the oviducts a severe diffuse epithelial degeneration, which explains the infertility of this animal. The male mating partner did not reveal lesions affecting the fertility. The other two mating pairs did not show any lesions affecting the fertility.
Histopathological findings: neoplastic:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
Key result
Critical effects observed:
no

Test substance stability:

The stability of test substance in rat diet was demonstrated for a period of 32 days at room temperature in a different batch of comparable quality, which was not used for the study. The homogeneity of the mixtures was verified. The concentration control analyses of the samples taken revealed that the values were within a range of 90-110% of the nominal concentration in all analyses at all time points, with the exception of one concentration in the feed of the high-dose group (88%).

Plasma concentrations of 2-aminoethanol were below 3 mg/kg for all control animals, <3 - 4 mg/kg for the low dose animals, 8 - 11 mg/kg for the mid dose animals and 60 – 81 mg/kg for the high dose animals.

Toxicokinetic data of 2-aminoethanol (calculated as 2-aminoethanol hydrochloride) from this two-generation reproduction toxicity study show a dose dependency of the plasma levels of 2 -aminoethanol in the experimental animals and there with prove the bioavailability of 2 -aminoethanol hydrochloride in principle.

 

Tables

Mean test substance intake (mg/kg bw/d; minimum value / maximum value)

 

Test group 01
(100 mg/kg bw/d)

Test group 02
(300 mg/kg bw/d)

Test group 03
(1000 mg/kg bw/d)

F0 males

94.3 (72.4 / 102.5)

283.2 (218.4 / 309.4)

943.3 (716.7 / 1032.6)

F0 females (premating)

96.7 (80.5 / 100.7)

289.6 (241.2 / 304.9)

964.4 (792.4 / 1017.8)

F0 females
(F1 litter)
- gestation period
- lactation period*



103.5 (92.6 / 111.6)
99.2 (81.6 / 120.2)



315.2 (284.8 / 337.9)
306.7 (249.7 / 370.3)



1043.2 (989.4 / 1084.7)
866.0 (668.6 / 1053.9)

* = Days 1–14 p.p. only

Absolute organ weights (P-generation)

Compared to the controls (= 100%), the following values (in %) were significantly changed (printed in bold):

 

Male animals

Female animals

Group

01

100 mg/kg bw/d

02

300 mg/kg bw/d

03

1000 mg/kg bw/d

01

100 mg/kg bw/d

02

300 mg/kg bw/d

03

1000 mg/kg bw/d

Brain

99%

100%

97%*

 

 

 

Cauda epididymis

99%

102%

88%**

 

 

 

Epididymides

100%

101%

92%**

 

 

 

Prostate

92%

99%

86%**

 

 

 

Spleen

 

 

 

105%*

107%

97%

 

*: p≤0.05; **: p≤0.01

 

All other mean absolute weight parameters did not show significant differences compared to the control groups.

The decrease of absolute weights of cauda epididymis, epididymides, and prostate in male top-dose animals (1000 mg/kg bw/d) were considered as treatment-related effects.

The decrease of brain weights in top-dose males (1000 mg/kg bw/d) as well as the increase of spleen weights in low-dose females (100 mg/kg bw/d) was considered as incidental and not treatment-related due to a missing dose-response relationship.

Absolute organ weights (F1 generation)

Compared to the controls (= 100%), the following values (in %) were significantly changed (printed in bold):

 

Male animals

Female animals

Group

11

100 mg/kg bw/d

12

300 mg/kg bw/d

13

1000 mg/kg bw day

11

100 mg/kg bw/d

12

300 mg/kg bw/d

13

1000 mg/kg bw/d

Cauda epididymis

96%

99%

88%**

 

 

 

Epididymides

100%

101%

91%**

 

 

 

Kidneys

99%

106%*

111%**

103%

106%**

115%**

Spleen

99%

103%

92%*

 

 

 

Thyroid glands

106%

99%

109%*

110%

118%**

111%*

 

*: p≤0.05; **: p≤0.01

All other mean absolute weight parameters did not show significant differences compared to the control groups.

The decrease of absolute weights of cauda epididymis and epididymides in male top-dose animals (1000 mg/kg bw/d) were considered to be treatment-related.

 

The increase of absolute kidney weights of male and female animals in mid- (300 mg/kg bw/d) and top-dose (1000 mg/kg bw/d) groups, respectively, was statistically significant. Because no histomorphological correlate was detected, a treatment-related weight increase was less likely.

 

The decrease of spleen weights in top-dose males as well as the increase of thyroid glands in top-dose males and mid- and top-dose females, respectively, is considered incidental and not treatment-related due to a missing dose-response relationship.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
Read Across OECD TG 416

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Qualifier:
according to
Guideline:
EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: MEOA K 540
- Test substance No.: 08/0924-1

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The stability under storage conditions over the exposure period was guaranteed by the manufacturer, and the manufacturer holds this responsibility.
- Stability under test conditions: Ambient

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation: about 7 weeks
- Weight at study initiation: male ± 228 g; female ± 165 g
- Housing: The rats were housed together (up to 5 animals per cage) in Polysulfon cages (H-Temp [PSU]) (floor area about 2065 cm2). Bedding in the Polycarbonate cages were Type Lignocel fibres, dust free bedding. For enrichment wooden gnawing blocks (Typ NGM E-022), were added.
- Diet (e.g. ad libitum): mouse/rat laboratory diet “GLP”, 10 mm pellets (Provimi Kliba SA, Kaiseraugst, Basel Switzerland), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: mixture of vapour and aerosol / mist
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 1.1 - <= 1.2 µm
Remarks on MMAD:
MMAD / GSD: The measurements of particle size in test group 3 resulted in MMADs of 1.1 and 1.2 µm with a GSD of 5.3 and 6.4.
The calculated mass fractions of particles below 3 µm aerodynamic size were 70.0 and 70.3 %.
Thus the aerosols were highly respirable for rats and a very high proportion of the aerosol particles reached the lungs.
Details on inhalation exposure:
Generation of the inhalation atmospheres
Generator systems:
• Continuous infusion pumps PERFUSOR (B. Braun)
• Two-component atomizers (stainless steel, Schlick mod. 970)
Generation procedure:
The test substance was used unchanged.
For each concentration the test substance was supplied to a two-component atomizer at a constant rate by means of a metering pump. The aerosol was generated with compressed air mixed with conditioned dilution air into the inhalation system.
The control group was exposed to conditioned air.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations of the inhalation atmospheres were analysed by a gas chromatography analysis (GC) in all test groups including control. The vapor and liquid aerosol concentration were determined separately.
Daily means were calculated based on two measured samples per concentration and exposure. From the daily mean values of each concentration, mean concentrations and standard deviations for the entire study were derived.
In these groups, the constancy of concentrations in the inhalation systems in the chambers were continuously monitored using scattered light photometers.
In the control group (test group 0) one sample was analysed over the study period.

The particle size analysis was carried out with a cascade impactor. In test group 3, particle size distribution was determined two times during the exposure period. In this test group significant amount liquid aerosol was found and the concentration was high enough for this measurement. In test group 2, due to the low aerosol concentration, long sampling time was necessary. During ongoing sampling, deposited aerosol would get evaporated again and the measured particle size distribution would not reflect the real size distribution. Therefore, no cascade impactor measurement was performed in this test group. In test group 1, no significant aerosol fraction was determined.
Duration of treatment / exposure:
28 days
Frequency of treatment:
6 hours/day, 5 days/week
Dose / conc.:
10 mg/m³ air
Remarks:
Target concentration
Dose / conc.:
50 mg/m³ air
Remarks:
Target concentration
Dose / conc.:
150 mg/m³ air
Remarks:
Target concentration
Dose / conc.:
10.2 mg/m³ air (analytical)
Remarks:
± 2.7
Dose / conc.:
49.1 mg/m³ air (analytical)
Remarks:
± 8.3
Dose / conc.:
155.9 mg/m³ air (analytical)
Remarks:
± 23.4
No. of animals per sex per dose:
5
Control animals:
yes
Details on study design:
The concentrations to be tested in this study were selected based on the results of the 5-day range finding study.
Summarizing the results, inhalation exposure to MEA for 6 hours per day on 5 consecutive days caused histological changes all over the respiratory tract. Most pronounced effects were observed in the upper respiratory tract.
At 500 mg/m3, minimal to mild inflammatory cell infiltrates in the submucosa of the ventral meatus in level I of the nasal cavity. In addition, four of the five animals revealed (multi)focal perivascular hemorrhage in this region. One animal showed necrosis of the squamous epithelium. In the area of the transition from squamous to the respiratory epithelium, four of the five animals revealed minimal to mild squamous metaplasia of the respiratory epithelium. In level II of the nasal cavity three of the five animals of the same concentration group showed minimal to moderate inflammatory cell infiltrates. In the larynx, minimal to severe epithelial necrosis, mild to severe inflammatory cell infiltrates, and minimal to moderate squamous metaplasia was observed. In level I of the larynx, inflammation was accompanied by necrosis of the submucosal glands. Moreover, cellular atypia within the metaplastic epithelium was observed in level I and II of the larynx. These findings were less severe in level III. Inflammatory cell infiltrates, focal epithelial necrosis and minimal diffuse epithelial hyperplasia could still be observed.
In the carina (trachea) respiratory epithelium hyperplasia and degeneration intermingled with inflammatory cell infiltrates in almost all animals of the high concentration group. In the lung minimal to mild hyperplasia of the bronchiolar epithelium in the areas of bifurcation of large bronchi was observed.
At 200 mg/m3 similar findings were noted in the above mentioned organs and tissues with less incidence and severity. At 20 mg/m3, no adverse effects were observed.
The observed effects seem to be associated with aerosol exposure. Considering the histological findings in the respiratory tract, 150 mg/m3 was selected as the high concentration for the main study to cause toxic effect. The mid concentration for the main study should be 50 mg/m3 because this concentration was around the saturated vapor concentration in the inhalation system. The low concentration should be 10 mg/m³, as the expected No Observed Adverse Effect Concentration (NOAEC).
150 mg/m³(61 ppm) as high concentration causing toxic effects
50 mg/m³ (20 ppm): as mid concentration
10 mg/m³ (4 ppm): as low concentration and expected NOAEC
Observations and examinations performed and frequency:
Mortality: The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) on working days and once a day (in the morning) on Saturdays, Sundays and public holidays.

Clinical observations: The clinical condition of the test animals was recorded once during the pre-exposure period and on the post-exposure observation day and at least 3 times (before, during and after exposure) on exposure days. During exposure only a group wise examination was possible.

Body weight data: The body weight of the animals was determined at the start of the pre-exposure, at the start of the exposure period and then, as a rule, once a week as well as prior to gross necropsy. As a rule, the animals were weighed at the same time of the day. Body weight change was calculated as the difference between body weight on the respective exposure day and body weight on the day of the first exposure. Group means were derived
from the individual differences.

Food consumption: Food consumption was determined weekly and calculated as mean food consumption in grams per animal and day. The animals were maintained in social-housing cages, with 5 animals per cage, during the whole study period. Therefore, the food consumption was determined cage-wise. The food consumption per animal and day was calculated by dividing food consumption of the day of a respective cage by the 5 animals per cage. As the animals of each test group were housed in only one cage per sex, no statistical evaluation of food consumption is possible.

Ophthalmology: Before the start of the exposure period (day -3) the eyes of all animals, and at the end of the study (day 26) the eyes of all animals were examined for any changes in the refracting media with an ophthalmoscope (HEINE Optotechnik, Herrsching, FRG) after administration of a mydriatic (Mydrum, Chauvin ankerpharm GmbH, Rudolstadt, Germany).

CLINICAL PATHOLOGY
In the morning, blood was taken from the retro-orbital venous plexus from fasted animals. The animals were anaesthetized using isoflurane (Isoba®, Essex GmbH Munich, Germany). The blood sampling procedure and the subsequent analysis of the blood and serum samples were carried out in a randomized sequence. The assays of blood and serum parameters were performed under internal laboratory quality control conditions with commercial reference controls to assure reliable test results. The results of the clinical pathology examinations are expressed in units of the International System (SI). The following examinations were carried out in 5 animals per test group and sex.

Hematology: Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes, Prothrombin time.

Clinical chemistry: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL), Magnesium (MG).
Sacrifice and pathology:
Necropsy: All animals were sacrificed under Narcoren anesthesia by exsanguination from the abdominal aorta and vena cava. The exsanguinated animals were necropsied and assessed by gross pathology.

Organ weights: The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals
2. Adrenal glands
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Lungs
9. Spleen
10. Testes
11. Thymus
12. Thyroid glands
3.10.3. Organ / Tissue fixation
The following organs or tissues were fixed in 4% buffered formaldehyde:
1. All gross lesions
2. Adrenal glands
3. Brain with olfactory bulb
4. Bone marrow (femur)
5. Eyes with optic nerve
6. Heart
7. Kidneys
8. Larynx/Pharynx
9. Liver
10. Lungs
11. Lymph nodes (tracheobronchial and mediastinal lymph nodes)
12. Nose (nasal cavity)
13. Esophagus
14. Ovaries
15. Seminal vesicle
16. Spinal cord (cervical, thoracic and lumbar cords)
17. Stomach (forestomach and glandular stomach)
18. Spleen
19. Testes
20. Thyroid glands
21. Thymus
22. Trachea
23. Urinary bladder
24. Uterus
From the liver, each one slices of the Lobus dexter medialis and the Lobus sinster lateralis were fixed in Carnoy’s solution and embedded in paraplast.

Histotechnical processing / Examination by light microscopy and assessment of findings: Fixation was followed by histotechnical processing and examination by light microscopy and assessment of findings according to the list below: Organs and tissues of main group animals designated for histological processing and light microscopic examination
1. All gross lesions
2. Nasal cavity (4 levels)
3. Larynx (3 levels)
4. Trachea
5. Lungs (5 lobes)
6. Lymph nodes (tracheobronchial and mediastinal lymph nodes)
7. Adrenal glands
8. Bone marrow (femur)
9. Brain
10. Heart
11. Kidneys
12. Liver
13. Esophagus
14. Ovaries
15. Seminal vesicles
16. Spinal cord (cervical, thoracic and lumbar cords)
17. Spleen
18. Stomach (forestomach and glandular stomach)
19. Testes
20. Thyroid glands
21. Thymus
22. Uterus
A correlation between gross lesions and histopathological findings was performed.
Statistics:
Body weight, body weight change: A comparison of each group with the control group was performed using DUNNETT's test (twosided) for the hypothesis of equal means.

Clinical pathology parameters, urine volume, urine specific gravity: Non-parametric one-way analysis using KRUSKALWALLIS test (two-sided).If the
resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using Wilcoxon-test (two-sided) for the equal medians.

Weight parameters: Non-parametric one-way analysis using KRUSKALWALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pair wise comparison of each concentration group with the control group was performed using the WILCOXON test for the hypothesis of equal medians.
Clinical signs:
no effects observed
Description (incidence and severity):
During the pre-exposure period and the post-exposure observation day the animals showed no clinical signs and findings different from normal. During the exposure period the animals of the control group showed no clinical signs and findings different from normal. During the exposure period a few animals crossbench all test groups showed salivation after exposure
Mortality:
no mortality observed
Description (incidence):
No deaths were recorded throughout the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weights of the test substance exposed groups were not statistically significantly different from the control group 0.
The mean body weight changes of the test substance exposed groups were not statistically significantly different from the control group 0.
Absolute weights: All mean absolute weight parameters did not show significant differences when compared with the control group 0.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No substance-related changes of food consumption were observed during the whole study period.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Description (incidence and severity):
The ophthalmologic examinations did not show any impairment of the refracting media. Spontaneous findings such as remainders of the pupillary membrane or corneal stippling, striation of lens and opacity were observed in several animals of all test groups and the control group without any concentration-response relationship.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
No treatment-related, adverse changes among hematological parameters were measured. In male rats of dose group 2 and 3 (50 mg/m3 and 150 mg/m3) the mean corpuscular hemoglobin concentration (MCHC) was higher compared to controls. The increase of this calculated parameter was not accompanied by an alteration of any other red blood cell parameter value. Therefore, the MCHC increase is regarded as possibly treatment-related, but not adverse.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among clinical chemistry parameters were measured. At the end of the study, in male rats of all dose groups the creatinine values were higher compared to controls, whereas in females of dose group 1 (10 mg/m3) the urea levels were lower compared to controls. The values were not changed dose-dependently. Therefore, they are regarded as incidental and not treatment-related. In male rats of dose group 3 (150 mg/m3) the triglyceride values were decreased. This was the only altered clinical chemistry parameter and it was especially not accompanied by any change of protein, glucose or cholesterol levels. Therefore, this decreased triglyceride concentrations were regarded as not adverse (ECETOC Technical Report No. 85, 2002).
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
When compared with control group 0 (=100%), the mean relative weights of liver in male treatment groups were significantly decreased. All other mean relative weight parameters did not show significant differences when compared with the control group 0. The decrease of mean liver weights in treated males was not concentration dependent and there were no histopathological correlates. Therefore, the reduced liver weights in males of all treatment groups were regarded to be incidental and not related to treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross lesions in treated male and female animals.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathology Larynx: At the base of epiglottis (level I), a submucosal inflammation that was characterized by infiltrates of granulocytes and lymphoid cells occurred in all males and females of test groups 2 (50 mg/m3) and 3 (150 mg/m3). In animals of test group 3 (150 mg/m3), the inflammation was accompanied by degeneration of the submucosal glands. In addition, 4 males and 3 females of test group 3 (150 mg/m3) showed a focal epithelial necrosis at the base of epiglottis. In the same region, a focal squamous cell metaplasia was observed in 3 males and 2 females of test group 2 (50 mg/m3) as well as in all males and females of test group 3 (150 mg/m3). All these findings were related to treatment. The occurrence of a minimal inflammation at the base of epiglottis in one female of test group 1 (10 mg/m3) was considered incidental. A minimal or slight epithelial alteration was observed in 2 males and 3 females of the control group, in 4 males and one female of test group 1 (10 mg/m3), as well as in 2 males and 3 females of test group 2 (50 mg/m3). The epithelial alteration was located at the base of epiglottis and was characterized by a slight focal flattening of epithelial cells. The epithelial alteration was regarded as a spontaneous lesion. At the entrance to the ventral pouch (larynx, level II), a minimal (grade 1) focal squamous metaplasia was seen in one female of test group 2 (50 mg/m3) as well as in one male and two females of test group 3 (150 mg/m3). A minimal focal epithelial hyperplasia occurred in all males and in 4 females. A mostly minimal inflammation was observed in 2 males and 3 females of test group 2 (50 mg/m3) as well as in all males and 4 females of test group 3 (150 mg/m3). All findings were considered treatment-related.

Histopathology Trachea: In males, a minimal or slight focal squamous metaplasia that was located in the area of the carina occurred in 3 animals of test group 3 (150 mg/m3). A minimal or slight inflammation was observed in one male of test group 1 (10 mg/m3) and in 4 males of test group 3 (150 mg/m3). The occurrence of squamous metaplasia and of inflammation in males of test group 3 (150 mg/m3) was related to treatment. In females, a minimal focal inflammation was only seen in one control animal.

Histopathology Lungs: A minimal or slight focal or multifocal mucous cell hyperplasia was seen in single or few large bronchi in all males and 2 females of test group 3 (150 mg/m3). In affected bronchi, the number of goblet cells was minimally or slightly increased. The occurrence of mucous cell hyperplasia was regarded as treatment-related.

Histopathology rest: All other findings occurred either individually or were biologically equally distributed over the control group and the treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOAEC
Remarks:
local effects
Effect level:
10 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOEC
Remarks:
systemic effects
Effect level:
150 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse systemic effects were reported
Key result
Critical effects observed:
no
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
150 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
OECD TG 412

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Qualifier:
according to
Guideline:
EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: MEOA K 540
- Test substance No.: 08/0924-1

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The stability under storage conditions over the exposure period was guaranteed by the manufacturer, and the manufacturer holds this responsibility.
- Stability under test conditions: Ambient

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation: about 7 weeks
- Weight at study initiation: male ± 228 g; female ± 165 g
- Housing: The rats were housed together (up to 5 animals per cage) in Polysulfon cages (H-Temp [PSU]) (floor area about 2065 cm2). Bedding in the Polycarbonate cages were Type Lignocel fibres, dust free bedding. For enrichment wooden gnawing blocks (Typ NGM E-022), were added.
- Diet (e.g. ad libitum): mouse/rat laboratory diet “GLP”, 10 mm pellets (Provimi Kliba SA, Kaiseraugst, Basel Switzerland), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: mixture of vapour and aerosol / mist
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 1.1 - <= 1.2 µm
Remarks on MMAD:
MMAD / GSD: The measurements of particle size in test group 3 resulted in MMADs of 1.1 and 1.2 µm with a GSD of 5.3 and 6.4.
The calculated mass fractions of particles below 3 µm aerodynamic size were 70.0 and 70.3 %.
Thus the aerosols were highly respirable for rats and a very high proportion of the aerosol particles reached the lungs.
Details on inhalation exposure:
Generation of the inhalation atmospheres
Generator systems:
• Continuous infusion pumps PERFUSOR (B. Braun)
• Two-component atomizers (stainless steel, Schlick mod. 970)
Generation procedure:
The test substance was used unchanged.
For each concentration the test substance was supplied to a two-component atomizer at a constant rate by means of a metering pump. The aerosol was generated with compressed air mixed with conditioned dilution air into the inhalation system.
The control group was exposed to conditioned air.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations of the inhalation atmospheres were analysed by a gas chromatography analysis (GC) in all test groups including control. The vapor and liquid aerosol concentration were determined separately.
Daily means were calculated based on two measured samples per concentration and exposure. From the daily mean values of each concentration, mean concentrations and standard deviations for the entire study were derived.
In these groups, the constancy of concentrations in the inhalation systems in the chambers were continuously monitored using scattered light photometers.
In the control group (test group 0) one sample was analysed over the study period.

The particle size analysis was carried out with a cascade impactor. In test group 3, particle size distribution was determined two times during the exposure period. In this test group significant amount liquid aerosol was found and the concentration was high enough for this measurement. In test group 2, due to the low aerosol concentration, long sampling time was necessary. During ongoing sampling, deposited aerosol would get evaporated again and the measured particle size distribution would not reflect the real size distribution. Therefore, no cascade impactor measurement was performed in this test group. In test group 1, no significant aerosol fraction was determined.
Duration of treatment / exposure:
28 days
Frequency of treatment:
6 hours/day, 5 days/week
Dose / conc.:
10 mg/m³ air
Remarks:
Target concentration
Dose / conc.:
50 mg/m³ air
Remarks:
Target concentration
Dose / conc.:
150 mg/m³ air
Remarks:
Target concentration
Dose / conc.:
10.2 mg/m³ air (analytical)
Remarks:
± 2.7
Dose / conc.:
49.1 mg/m³ air (analytical)
Remarks:
± 8.3
Dose / conc.:
155.9 mg/m³ air (analytical)
Remarks:
± 23.4
No. of animals per sex per dose:
5
Control animals:
yes
Details on study design:
The concentrations to be tested in this study were selected based on the results of the 5-day range finding study.
Summarizing the results, inhalation exposure to MEA for 6 hours per day on 5 consecutive days caused histological changes all over the respiratory tract. Most pronounced effects were observed in the upper respiratory tract.
At 500 mg/m3, minimal to mild inflammatory cell infiltrates in the submucosa of the ventral meatus in level I of the nasal cavity. In addition, four of the five animals revealed (multi)focal perivascular hemorrhage in this region. One animal showed necrosis of the squamous epithelium. In the area of the transition from squamous to the respiratory epithelium, four of the five animals revealed minimal to mild squamous metaplasia of the respiratory epithelium. In level II of the nasal cavity three of the five animals of the same concentration group showed minimal to moderate inflammatory cell infiltrates. In the larynx, minimal to severe epithelial necrosis, mild to severe inflammatory cell infiltrates, and minimal to moderate squamous metaplasia was observed. In level I of the larynx, inflammation was accompanied by necrosis of the submucosal glands. Moreover, cellular atypia within the metaplastic epithelium was observed in level I and II of the larynx. These findings were less severe in level III. Inflammatory cell infiltrates, focal epithelial necrosis and minimal diffuse epithelial hyperplasia could still be observed.
In the carina (trachea) respiratory epithelium hyperplasia and degeneration intermingled with inflammatory cell infiltrates in almost all animals of the high concentration group. In the lung minimal to mild hyperplasia of the bronchiolar epithelium in the areas of bifurcation of large bronchi was observed.
At 200 mg/m3 similar findings were noted in the above mentioned organs and tissues with less incidence and severity. At 20 mg/m3, no adverse effects were observed.
The observed effects seem to be associated with aerosol exposure. Considering the histological findings in the respiratory tract, 150 mg/m3 was selected as the high concentration for the main study to cause toxic effect. The mid concentration for the main study should be 50 mg/m3 because this concentration was around the saturated vapor concentration in the inhalation system. The low concentration should be 10 mg/m³, as the expected No Observed Adverse Effect Concentration (NOAEC).
150 mg/m³(61 ppm) as high concentration causing toxic effects
50 mg/m³ (20 ppm): as mid concentration
10 mg/m³ (4 ppm): as low concentration and expected NOAEC
Observations and examinations performed and frequency:
Mortality: The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) on working days and once a day (in the morning) on Saturdays, Sundays and public holidays.

Clinical observations: The clinical condition of the test animals was recorded once during the pre-exposure period and on the post-exposure observation day and at least 3 times (before, during and after exposure) on exposure days. During exposure only a group wise examination was possible.

Body weight data: The body weight of the animals was determined at the start of the pre-exposure, at the start of the exposure period and then, as a rule, once a week as well as prior to gross necropsy. As a rule, the animals were weighed at the same time of the day. Body weight change was calculated as the difference between body weight on the respective exposure day and body weight on the day of the first exposure. Group means were derived
from the individual differences.

Food consumption: Food consumption was determined weekly and calculated as mean food consumption in grams per animal and day. The animals were maintained in social-housing cages, with 5 animals per cage, during the whole study period. Therefore, the food consumption was determined cage-wise. The food consumption per animal and day was calculated by dividing food consumption of the day of a respective cage by the 5 animals per cage. As the animals of each test group were housed in only one cage per sex, no statistical evaluation of food consumption is possible.

Ophthalmology: Before the start of the exposure period (day -3) the eyes of all animals, and at the end of the study (day 26) the eyes of all animals were examined for any changes in the refracting media with an ophthalmoscope (HEINE Optotechnik, Herrsching, FRG) after administration of a mydriatic (Mydrum, Chauvin ankerpharm GmbH, Rudolstadt, Germany).

CLINICAL PATHOLOGY
In the morning, blood was taken from the retro-orbital venous plexus from fasted animals. The animals were anaesthetized using isoflurane (Isoba®, Essex GmbH Munich, Germany). The blood sampling procedure and the subsequent analysis of the blood and serum samples were carried out in a randomized sequence. The assays of blood and serum parameters were performed under internal laboratory quality control conditions with commercial reference controls to assure reliable test results. The results of the clinical pathology examinations are expressed in units of the International System (SI). The following examinations were carried out in 5 animals per test group and sex.

Hematology: Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes, Prothrombin time.

Clinical chemistry: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL), Magnesium (MG).
Sacrifice and pathology:
Necropsy: All animals were sacrificed under Narcoren anesthesia by exsanguination from the abdominal aorta and vena cava. The exsanguinated animals were necropsied and assessed by gross pathology.

Organ weights: The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals
2. Adrenal glands
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Lungs
9. Spleen
10. Testes
11. Thymus
12. Thyroid glands
3.10.3. Organ / Tissue fixation
The following organs or tissues were fixed in 4% buffered formaldehyde:
1. All gross lesions
2. Adrenal glands
3. Brain with olfactory bulb
4. Bone marrow (femur)
5. Eyes with optic nerve
6. Heart
7. Kidneys
8. Larynx/Pharynx
9. Liver
10. Lungs
11. Lymph nodes (tracheobronchial and mediastinal lymph nodes)
12. Nose (nasal cavity)
13. Esophagus
14. Ovaries
15. Seminal vesicle
16. Spinal cord (cervical, thoracic and lumbar cords)
17. Stomach (forestomach and glandular stomach)
18. Spleen
19. Testes
20. Thyroid glands
21. Thymus
22. Trachea
23. Urinary bladder
24. Uterus
From the liver, each one slices of the Lobus dexter medialis and the Lobus sinster lateralis were fixed in Carnoy’s solution and embedded in paraplast.

Histotechnical processing / Examination by light microscopy and assessment of findings: Fixation was followed by histotechnical processing and examination by light microscopy and assessment of findings according to the list below: Organs and tissues of main group animals designated for histological processing and light microscopic examination
1. All gross lesions
2. Nasal cavity (4 levels)
3. Larynx (3 levels)
4. Trachea
5. Lungs (5 lobes)
6. Lymph nodes (tracheobronchial and mediastinal lymph nodes)
7. Adrenal glands
8. Bone marrow (femur)
9. Brain
10. Heart
11. Kidneys
12. Liver
13. Esophagus
14. Ovaries
15. Seminal vesicles
16. Spinal cord (cervical, thoracic and lumbar cords)
17. Spleen
18. Stomach (forestomach and glandular stomach)
19. Testes
20. Thyroid glands
21. Thymus
22. Uterus
A correlation between gross lesions and histopathological findings was performed.
Statistics:
Body weight, body weight change: A comparison of each group with the control group was performed using DUNNETT's test (twosided) for the hypothesis of equal means.

Clinical pathology parameters, urine volume, urine specific gravity: Non-parametric one-way analysis using KRUSKALWALLIS test (two-sided).If the
resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using Wilcoxon-test (two-sided) for the equal medians.

Weight parameters: Non-parametric one-way analysis using KRUSKALWALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pair wise comparison of each concentration group with the control group was performed using the WILCOXON test for the hypothesis of equal medians.
Clinical signs:
no effects observed
Description (incidence and severity):
During the pre-exposure period and the post-exposure observation day the animals showed no clinical signs and findings different from normal. During the exposure period the animals of the control group showed no clinical signs and findings different from normal. During the exposure period a few animals crossbench all test groups showed salivation after exposure
Mortality:
no mortality observed
Description (incidence):
No deaths were recorded throughout the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The mean body weights of the test substance exposed groups were not statistically significantly different from the control group 0.
The mean body weight changes of the test substance exposed groups were not statistically significantly different from the control group 0.
Absolute weights: All mean absolute weight parameters did not show significant differences when compared with the control group 0.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No substance-related changes of food consumption were observed during the whole study period.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Description (incidence and severity):
The ophthalmologic examinations did not show any impairment of the refracting media. Spontaneous findings such as remainders of the pupillary membrane or corneal stippling, striation of lens and opacity were observed in several animals of all test groups and the control group without any concentration-response relationship.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
No treatment-related, adverse changes among hematological parameters were measured. In male rats of dose group 2 and 3 (50 mg/m3 and 150 mg/m3) the mean corpuscular hemoglobin concentration (MCHC) was higher compared to controls. The increase of this calculated parameter was not accompanied by an alteration of any other red blood cell parameter value. Therefore, the MCHC increase is regarded as possibly treatment-related, but not adverse.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among clinical chemistry parameters were measured. At the end of the study, in male rats of all dose groups the creatinine values were higher compared to controls, whereas in females of dose group 1 (10 mg/m3) the urea levels were lower compared to controls. The values were not changed dose-dependently. Therefore, they are regarded as incidental and not treatment-related. In male rats of dose group 3 (150 mg/m3) the triglyceride values were decreased. This was the only altered clinical chemistry parameter and it was especially not accompanied by any change of protein, glucose or cholesterol levels. Therefore, this decreased triglyceride concentrations were regarded as not adverse (ECETOC Technical Report No. 85, 2002).
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
When compared with control group 0 (=100%), the mean relative weights of liver in male treatment groups were significantly decreased. All other mean relative weight parameters did not show significant differences when compared with the control group 0. The decrease of mean liver weights in treated males was not concentration dependent and there were no histopathological correlates. Therefore, the reduced liver weights in males of all treatment groups were regarded to be incidental and not related to treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no gross lesions in treated male and female animals.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathology Larynx: At the base of epiglottis (level I), a submucosal inflammation that was characterized by infiltrates of granulocytes and lymphoid cells occurred in all males and females of test groups 2 (50 mg/m3) and 3 (150 mg/m3). In animals of test group 3 (150 mg/m3), the inflammation was accompanied by degeneration of the submucosal glands. In addition, 4 males and 3 females of test group 3 (150 mg/m3) showed a focal epithelial necrosis at the base of epiglottis. In the same region, a focal squamous cell metaplasia was observed in 3 males and 2 females of test group 2 (50 mg/m3) as well as in all males and females of test group 3 (150 mg/m3). All these findings were related to treatment. The occurrence of a minimal inflammation at the base of epiglottis in one female of test group 1 (10 mg/m3) was considered incidental. A minimal or slight epithelial alteration was observed in 2 males and 3 females of the control group, in 4 males and one female of test group 1 (10 mg/m3), as well as in 2 males and 3 females of test group 2 (50 mg/m3). The epithelial alteration was located at the base of epiglottis and was characterized by a slight focal flattening of epithelial cells. The epithelial alteration was regarded as a spontaneous lesion. At the entrance to the ventral pouch (larynx, level II), a minimal (grade 1) focal squamous metaplasia was seen in one female of test group 2 (50 mg/m3) as well as in one male and two females of test group 3 (150 mg/m3). A minimal focal epithelial hyperplasia occurred in all males and in 4 females. A mostly minimal inflammation was observed in 2 males and 3 females of test group 2 (50 mg/m3) as well as in all males and 4 females of test group 3 (150 mg/m3). All findings were considered treatment-related.

Histopathology Trachea: In males, a minimal or slight focal squamous metaplasia that was located in the area of the carina occurred in 3 animals of test group 3 (150 mg/m3). A minimal or slight inflammation was observed in one male of test group 1 (10 mg/m3) and in 4 males of test group 3 (150 mg/m3). The occurrence of squamous metaplasia and of inflammation in males of test group 3 (150 mg/m3) was related to treatment. In females, a minimal focal inflammation was only seen in one control animal.

Histopathology Lungs: A minimal or slight focal or multifocal mucous cell hyperplasia was seen in single or few large bronchi in all males and 2 females of test group 3 (150 mg/m3). In affected bronchi, the number of goblet cells was minimally or slightly increased. The occurrence of mucous cell hyperplasia was regarded as treatment-related.

Histopathology rest: All other findings occurred either individually or were biologically equally distributed over the control group and the treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOAEC
Remarks:
local effects
Effect level:
10 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOEC
Remarks:
systemic effects
Effect level:
150 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse systemic effects were reported
Key result
Critical effects observed:
no
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
10 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
OECD TG 412

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Oral

No reliable conventional repeated dose toxicity studies with MEA are available. However, MEA HCl was tested in an oral two generation reproduction toxicity study according to OECD TG 416 and GLP-conditions (BASF SE, 2009). The test substance was administered to groups of 25 male and 25 female healthy young Wistar rats (F0 parental generation) as a homogeneous addition to the food in different concentrations, which were adjusted regularly to obtain target dose levels of 0, 100, 300 and 1000 mg/kg bw/day. At least 75 days after the beginning of treatment, F0 animals were mated to produce a litter (F1 generation).

The detailed description of the study results regarding reproduction toxicity is provided in the section on reproductive toxicity.

Regarding general repeated dose toxicity, the dose level of 1000 mg/kg bw/day caused systemic toxicity in parental females, as was indicated by reduced food consumption and/or body weight gain during gestation and lactation. In the mid and high dose F1 animals the absolute and relative kidney weights were statistical significantly increased without histopathological correlate findings. In the top-dose F0 and F1 males the test substance administration led to a decrease of absolute and relative organ weights of cauda epididymidis and epididymides. Furthermore, prostate weight and the number of homogenization resistant caudal epididymal sperm was slightly, but significantly, decreased in the F0 males. These findings were considered to be treatment-related effects, whereas histomorphological correlates were missing.

Based on this study, the NOAEL for general toxicity was set at 300 mg/kg bw/day.

Inhalation

The toxicity of MEA was studied in a 28 days inhalation toxicity study performed according to OECD guideline 412 and GLP compliant (REACH Ethanolamines consortium, 2010). Groups of five male and five female Wistar rats per test group were exposed nose-only to dynamic atmosphere of MEA for 6 hours per day on 5 consecutive days per week for 4 weeks (28-day study). The target concentrations were 10 mg/m3, 50 mg/m3and 150 mg/m3. A concurrent control group was exposed to conditioned air. Clinical observations, body weight determinations and food consumption determinations were performed for all animals. Ophthalmological examinations were performed prior to exposure and towards the end of the exposure. After the last exposure, blood was sampled from the animals and hematology and clinical chemistry parameters were determined. The animals were then subjected to gross necropsy (including macroscopic examination of the major internal organs and collection of organ weight data). Selected tissues were processed histopathologically and were evaluated by light microscopy. Histological examinations were performed according to standardised methods with particular emphasis on the nasal cavity (4 levels) and larynx (3 levels).

The exposure of rats to MEA caused concentration-related lesions in larynx, trachea and lung. At the high concentration (150 mg/m3), submucosal inflammation (levels I, II) in males and females, degeneration of submucosal glands (level I) in males and females, focal epithelial necrosis (level I) in males and females, focal squamous metaplasia, (level I) in males and females; (level II) in one male and 2 females and focal epithelial hyperplasia (level II) in males and females were observed in the larynx. In the trachea, focal squamous metaplasia (carina) accompanied by inflammation in males was observed.

At the mid concentration (50 mg/m3), submucosal inflammation (level I and II) in males and females and squamous metaplasia (level I and II) in few males and females in the larynx was reported.

No treatment-related weight changes, gross lesions or microscopic findings at the low concentration (10 mg/m3).

No histopathological effects were seen in any other organ outside the respiratory tract. The NOAEC for systemic toxicity is the highest concentration of 150 mg/m3. The NOAEC for local effect was the lowest tested concentration of 10 mg/m3 under the current test conditions.



Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. The substance is not considered to be classified for repeated dose toxicity according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.