2,2'-butyliminodiethanol

Endpoint summary

Description of key information

Oral

DBEA (CAS 102-81-8)

- Oral, 90 d (OECD 408), rat: NOAEL = 50 mg/kg bw

- Oral, 28 d (OECD 407), rat: NOEL = 50 mg/kg bw/d; NOAEL = 100 mg/kg bw/d; LOAEL = 400 mg/kg bw/d 

- Oral, 5 weeks (OECD 407), rat: NOAEL (male) = 430 mg/kg bw/d; NOAEL (female) = 330mg/kg bw/d

BEA (CAS 111-75-1)

- Oral, 90 d (OECD 408), rat: NOAEL = 240 mg/kg bw/d (highest dose tested)
- Oral, screening (OECD 422), rat: NOAEL (reproductive and developmental parameters; males and females) = 240 mg/kg bw/d (highest dose tested), NOAEL (systemic; males) = 120 mg/kg bw/d, NOAEL (systemic; females) = 240 mg/kg bw/d

 

Inhalation

DBEA (CAS 102-81-8)

- Inhalative, screening (OECD 422), rat: NOAEC (reproductive and developmental parameters; males and females) = 236.3 mg/m³, NOAEC (systemic; males and females) = 236.3 mg/m³ (highest dose tested), NOAEC (local, males and females) = 20.6 mg/m³

- inhalative, 6 month, rat: NOAEC = 156 mg/m³ (males)

- inhalative, 5 days, rat: NOAEC = 234 mg/m³  (= 33 ppm); LOAEC = 495 mg/m³  (=70 ppm)

 

Dermal

no study available

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
In this justification, the read-across (bridging) concept is applied, based on the chemical structure of the potential analogues, their toxicokinetic behaviour and other available (eco-)toxicological data.
Please refer to a full version of Read-across statement attached in the section 13 "Assessment reports".

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The underlying hypothesis for the read-across is that the target and the source substance have similar toxicological properties (including the same target organs) due to their structural similarity, resemblance to their chemical reactivity, and therefore a similar mode of action (impairment of choline homeostasis). The substances share the same ethanolamine moiety and can be considered as derivatives of mono-ethanolamine (CAS 141-43-5). Ethanolamines have structural similarity with choline, an ubiquitous physiological molecule (e.g. involved in phospholipid synthesis like phosphatidylcholine and acetylcholine).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

source substance: 2-Dibutylethanolamine (or 2-Dibutylaminoethanol)
structural formula: C10H23NO
Smiles: CCCCN(CCCC)CCO
Molecular weight: 173.30
CAS 102-81-8
EC No 203-057-1
purity: not specified

target substance: Butyldiethanolamine
structural formula: C8H19NO2
Smiles: CCCCN(CCO)CCO
Molecular weight: 161.24
CAS 102-79-4
EC No 203-055-0
purity: not specified
No additional information is available on purity of the source and the target substances. Both substances are normally of high purity, containing only minor amounts of impurities that do not influence the read-across validity.

3. ANALOGUE APPROACH JUSTIFICATION
The systemic effects in the repeated dose toxicity studies with ethanolamines by oral route of exposure are generally manifested by adverse effects in liver and kidneys as a consequence of perturbation in choline metabolism.
Since DBEA and BDEA are both tertiary amines, their structural difference that could impact prediction is due to the additional butyl- alkyl group in the source substance DBEA. Both substances share one ethanol group while the second ethanol group in BDEA does not influence systemic toxicity to a such extent as the additional butyl group does. As it was observed in studies with different ethanolamines, including those with shorter aliphatic groups (methyl, ethyl, propyl), the number of ethanol groups in an amine seems not to impair choline homeostasis significantly: the more ethanol groups are, the weaker the strength of systemic toxicity effects is (please refer to data sets of monoethanolamine (MEA; CAS 141-43-5), diethanolamine (DEA, CAS 111-42-2) and triethanolamine (TEA, CAS 102-71-6). TEA was the least toxic chemical compared to DEA and MEA). Thus, the potency of ethanolamines to perturb choline homeostasis seems to decline with the number of ethanol groups. Thus, one could speculate that BDEA would be a chemical with the weakest systemic toxicity effects among butyl ethanolamines (BEA and DBEA). Regarding the additional butyl- alkyl chain in DBEA, if it was associated with other mode(s) of action than choline impairment, then these mode(s) of action would be identified in this study. However, the effects observed in this 90-day study including the same target organ: kidney support the already established mode-of-action for ethanolamines and therewith allow extending the read-across approach to butyl ethanolamine representatives. Therefore, the target substance BDEA, containing only one butyl-alkyl chain, would not possess other mode(s) of action than impairment of choline homeostasis, already confirmed in studies with one alkyl chain (MDEA, CAS 105-59-9; MMEA, CAS 109-83-1). Moreover, it was shown in an in vitro study that DBEA was more potent than BEA to inhibit choline esterase activity (Hartung and Cornish, 1968). Thus, BDEA is expected to be a less potent butyl amine with regard to impairment of choline homeostasis than the source substance DBEA.
Thus, based on above arguments, the same or even weaker than DBEA systemic toxicity by oral route of exposure is predicted for BDEA. Thus, the level of systemic toxicity caused by DBEA in the oral 90-day study would represent worst-case for BDEA and would not lead to an underestimation of systemic effects if the same dose levels were used in an oral repeated dose toxicity study with BDEA.

4. DATA MATRIX
Please refer to the full version of the read-across statement.

Reason / purpose for cross-reference:

read-across source

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

see attached document: summary clinical observation
All male and all female animals of test group 3 (150 mg/kg bw/d) showed slight salivation directly after treatment on several days of the application period. Salivation was also observed in 5 male and 3 female animals of test group 2 (50 mg/kg bw/d) and in 6 male animals in test group 1 (15 mg/kg bw/d).
From the temporary, short appearance immediately after dosing it was concluded the findings were induced by a bad taste of the test substance or local affection of the upper digestive tract. The effect was related to the test substance but assessed as being non-adverse as no lesions in the upper digestive tract were observed in male and female animals during pathological examinations.
A protruding right eyeball was observed one in female animal of test group 1 (15 mg/kg bw/d) between study days 91-93. This finding was considered to be incidental and not related to treatment.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

see attached document; summary bw_bwc
No test substance-related, adverse effects on body weight development were obtained in any test group (15, 50 and 150 mg/kg bw/d).
Mean body weights and body weight change values of male and female animals did not show any significant deviations to the control.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

see attached document: summary ophthalmological findings
No treatment-related findings were observed.
All other apparent findings were assessed as being incidental in nature since they occurred in control as well as in treated animals and did not show a dose-response relationship
see attached document: ophthalmological findings

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

see attached document: summary haematological and chlinical chemistry
At the end of the administration period, in females of test groups 1 and 2 (15 and 50 mg/kg bw/d) absolute and relative lymphocyte counts were significantly decreased whereas relative neutrophil counts were significantly increased. Additionally, in females of test group 2 (50 mg/kg bw/d) relative, large unstained cell (LUC) counts were significantly decreased.
However, all mentioned alterations were not dose-dependent and, therefore, they were regarded as incidental and not treatment-related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

see attached document: summary haematological and chlinical chemistry
At the end of the administration period, in males of test group 3 (150 mg/kg bw/d) potassium levels were significantly increased whereas chloride levels were significantly decreased.
Chloride values were within, potassium values slightly above the historical control range (males, chloride 96.4-106.1 mmol/L, potassium 4.52-5.04 mmol/L). The unique potassium increase among these individuals was regarded to be potentially treatment-related but non-adverse (ECETOC Technical Report No. 85, 2002), whereas the chloride decrease was regarded as incidental and not treatment-related.

Thyroid hormones
No treatment-related alterations of T3, T4 and TSH levels were observed.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the study end, in males of test group 2 (50 mg/kg bw/d) higher incidences of triple phosphate crystals were observed in the urine sediment. This change was not dose-dependent and, therefore, it was regarded as incidental and not treatment-related.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

see attached document: FOB
Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, were without a dose-response relationship or occurred in single rats only, these observations were considered to have been incidental.
Open field observations: No test substance-related effects were observed.
Sensorimotor tests/reflexes: No test substance-related effects were observed.
Quantitative parameters No test substance-related effects were observed.
In male animals of test group 2 (50 mg/kg bw/d), grip strength of hindlimbs was significantly increased (+17 %), but a dose-response relationship did not occur. The deviation was assessed as being spontaneous in nature.

Immunological findings:

not examined

Description (incidence and severity):

see attached document: summary organ and body weight
All mean absolute and relative weight parameters did not show significant differences when compared to the control group 0.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

see attached document: summary gross lesions and microscopic findings
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Vacuolation of epithelial cells of the collecting ducts was characterized by macrovesicular clear vacuoles in the collecting duct epithelium of the inner medulla. The outer part of the inner medulla was most prominently affected with a gradual decrease towards the tip of papilla. The vacuoles did not stain with HE, Sudan Black or Oil-Red-O. Vacuolation affected 8 out of 10 males and 1 out of 10 females of test group 3 (150 mg/kg bw/d), with a severity ranging from minimal to severe in males, the one female animal was only minimally affected.
No signs of necrosis, cellular degeneration, inflammation or edema were present.
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Description (incidence and severity):

see attached document: summary motor activity
Regarding the overall motor activity as well as single intervals, no test substance-related and relevant deviations to the control animals were noted for male and female animals of test groups 1-3 (15, 50, and 150 mg/kg bw/d).
In female animals of test group 2 (50 mg/kg bw/d) and test group 3 (150 mg/kg bw/d), the overall motor activity was significantly higher when compared to the controls. However, a clear dose-response relationship did not occur, and the changes were assessed to be incidental and not related to treatment.
Comparing the single intervals with the control groups, significantly higher values were measured for male animals of test group 1 (15 mg/kg bw/d) at interval No. 8 and for male animals of test group 3 (150 mg/kg bw/d) at interval Nos. 11 and 12.
Significantly higher values were also obtained for female animals of test groups 2 (50 mg/kg bw/d) and 3 (150 mg/kg bw/d) at interval No. 5 as well as for female animals of test group 2 at interval No. 6.
All changes were regarded to be incidental and not related to treatment as single intervals were not changed in a dose-dependent manner.

see attached document: summary estrous cycle
Estrous cycle
No test substance-related, adverse effects on estrous cycles were obtained in any test group (15, 50 and 150 mg/kg bw/d).

see attached document: summary spermanalysis
Sperm analysis
Concerning motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as sperm head counts in the testis and in the cauda epididymidis no treatment-related effects were observed

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day (actual dose received)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

   

Kidneys	Male animals				Female animals
Test group (mg/kg bw/d)	0 (0)	1 (15)	2 (50)	3 (150)	0 (0)	1 (15)	2 (50)	3 (150)
No. of animals	10	10	10	10	10	10	10	10
Vacuolation,collecting ducts	0	0	0	8	0	0	0	1
·      Grade1				4				1
·      Grade2				1
·      Grade3				2
·      Grade4				1

 

Conclusions:

The administration of Dibutylethanolamine by gavage for 3 months to male and female Wistar rats caused test substance-related findings at a dose level of 150 mg/kg bw/d taking the vacuolation of the collecting duct epithelium in kidneys into account. Therefore, under the conditions of the present study the NOAEL was 50 mg/kg bw/d for male and female Wistar rats.

Executive summary:

Dibutylethanolamine was administered orally by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0 mg/kg b/day (mg/kg bw/d; test group 0), 15 mg/kg bw/d (test group 1), 50 mg/kg bw/d (test group 2) and 150 mg/kg bw/d (test group 3) over a period of 3 months. Corn oil served as vehicle, control animals were dosed daily with the vehicle only.

With regard to clinical examinations, signs of general systemic toxicity were not observed even at a dose level of 150 mg/kg bw/d of Dibutylethanolamine. In addition, no test substance-related effects on estrous cycle length and the number of cycles were obtained.

Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose of the compound of 150 mg/kg bw/d. The unique potassium increase observed for male animals of test group 3 (150 mg/kg bw/d) was considered to be potentially treatment-related but regarded to be non-adverse (ECETOC Technical Report No. 85, 2002). A relation to the kidney-related findings observed in these male animals of the test group 3 was excluded.

Regarding pathology, neither treatment-related weight changes nor gross lesions were observed. The target organ were the kidneys. In 8 out of 10 males and 1 out of 10 females of test group 3, macrovesicular vacuolation of the epithelial cells of the collecting ducts was observed. No signs of necrosis, degeneration or inflammation were present. 

Therefore, under the conditions of the present study the NOAEL was 50 mg/kg bw/d for male and female Wistar rats.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
In this justification, the read-across (bridging) concept is applied, based on the chemical structure of the potential analogues, their toxicokinetic behaviour and other available (eco-)toxicological data.
Please refer to a full version of Read-across statement attached in the section 13 "Assessment reports".

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The underlying hypothesis for the read-across is that the target and the source substance have similar toxicological properties (including the same target organs) due to their structural similarity, resemblance to their chemical reactivity, and therefore a similar mode of action (impairment of choline homeostasis). The substances share the same ethanolamine moiety and can be considered as derivatives of mono-ethanolamine (CAS 141-43-5). Ethanolamines have structural similarity with choline, an ubiquitous physiological molecule (e.g. involved in phospholipid synthesis like phosphatidylcholine and acetylcholine).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

source substance: Butylethanolamine (or Butylaminoethanol)
structural formula: C6H15NO
Smiles: CCCCNCCO
Molecular weight: 117.19
CAS 111-75-1
EC No 203-904-5
purity: not specified

target substance: Butyldiethanolamine
structural formula: C8H19NO2
Smiles: CCCCN(CCO)CCO
Molecular weight: 161.24
CAS 102-79-4
EC No 203-055-0
purity: not specified

No additional information is available on purity of the source and the target substances. Both substances are normally of high purity, containing only minor amounts of impurities that do not influence the read-across validity.

3. ANALOGUE APPROACH JUSTIFICATION
The systemic effects in the repeated dose toxicity studies with ethanolamines by oral route of exposure are generally manifested by adverse effects in liver and kidneys as a consequence of perturbation in choline metabolism. In this study BEA no significant adverse effects were noted in the animals treated during 90 days by the test substance orally (gavage) up to the highest dose of 240 mg/kg bw/day.

Since BDEA is a tertiary amine, while BEA is a secondary amine their structural difference that could impact prediction is due to the additional ethanol-group in the target substance BDEA. Both substances share one butyl group. As it was observed in studies with different ethanolamines, including those with shorter aliphatic groups (methyl, ethyl, propyl), the number of ethanol groups in an amine seems not to impair choline homeostasis significantly: the more ethanol groups are, the weaker the strength of systemic toxicity effects is (please refer to data sets of monoethanolamine (MEA; CAS 141-43-5), diethanolamine (DEA, CAS 111-42-2) and triethanolamine (TEA, CAS 102-71-6). TEA was the least toxic chemical compared to DEA and MEA). Thus, the potency of ethanolamines to perturb choline homeostasis seems to decline with the number of ethanol groups. Thus, one could speculate that BDEA would be a chemical with the weakest systemic toxicity effects among butyl ethanolamines (BEA and DBEA).

Thus, based on above arguments, the same or even weaker than BEA systemic toxicity by oral route of exposure is predicted for BDEA. Thus, the level of systemic toxicity caused by BEA in the oral 90-day study would represent worst-case for BDEA and would not lead to an underestimation of systemic effects if the same dose levels were used in an oral repeated dose toxicity study with BDEA.

4. DATA MATRIX
Please refer to the full version of the read-across statement.

Reason / purpose for cross-reference:

read-across source

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

All rats were normal, throughout the dosing period, in control, low, and mid dose groups. Mild salivation in male and female rats and gasping in male rats of high dose treated group were observed.
Salivation started approximately 20 to 25 minutes post-dosing and lasted until approximately at 45 to 60 minutes post-dosing. Salivation observed in high dose group was considered a response to the dose solution and considered as non-adverse in nature. Gasping was observed only in male rats of high dose treated group. Gasping was seen in 3 out of 10 male rats in high dose group and there were no microscopic changes observed in respiratory organs. Hence, it was considered as possibly related an irritancy response to the test material and non-adverse in nature. Additionally, lethargy and weakness were observed on day 84 in the female rat N° 75 of high dose. Rat N° 75 was found dead after showing lethargy and weakness, 1 day prior.
Please also refer to attached Tables below.

Mortality:

no mortality observed

Description (incidence):

No mortality or morbidity were observed during the study period in the control, low, and mid dose groups. One female rat from the high dose group (rat N° 75) died on day 85 of dosing after showing clinical signs like, lethargy and weakness. However, there were no treatment related changes observed in body weight, food consumption, clinical pathology, gross and microscopic examination, in female rats of high dose group. Hence, this death was considered not to be related to treatment.
Please also refer to attached Tables below.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

According to the study director’s detailed data analysis, no statistically significant difference in mean body weights were noted for the treated male and female groups when compared to the same sexed controls.
The mean body weight change in low dose of male rats and all treatment groups of female rats, was comparable with that of the vehicle control group. The body weight gains for mid dose male rats were statistically significantly lower than control for weeks 7 through 13, however the difference between mid dose males and control animals consistently remained <10% and was thus not considered as toxicologically relevant. Similarly, body weight gains for the high dose male group showed statistically significantly lower body weight gains during week 2, 4, and 6 with numerically lower body weight gains at other intervals. However, differences in body weight gain between high dose males and control animals did not reach statistical significance at any time point after week 6, so this was not considered as a test item-related effect. In sum, in the absence of statistically significant changes in body weights for mid and high dose male rats and no effects on body weight or body weight gain in female rats, the lower body weight gains for the mid and high dose males are not considered an adverse effect of test item administration.
Please also refer to attached Tables below.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The mean food consumption of rats from the treatment groups were comparable with that of the vehicle control group, except statistically significant decrease in the food consumption observed during the week 3 in male rats of high dose group. This reduction in food consumption was marginal and inconsistent. Hence, it was not considered as treatment related.
Please also refer to attached Tables below.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ophthalmological examination did not reveal any abnormalities.
Please also refer to attached Tables below.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related changes in the haematology parameters were observed in male and female rats of the treatment groups.
- Platelet counts for high dose male and female groups were statistically significantly higher than those of the same-sexed vehicle control group. The increases were minimal and considered to be incidental in nature and non-treatment related.
- Statistically significant increases were noted in RBC and HCT in low, mid and high females. Effect lacked dose dependency and consistency between sexes. Moreover, values of these parameters of all treated females were within 95 percentile historical ranges (except one female of low dose), hence, effect was not related to test item treatment.
- Dose independent statistically significant increase was noted in haemoglobin in low and mid dose female, but values of these parameters of all treated females were within 95 percentile historical ranges (except one female of low dose), hence, effect was not related to test item treatment.
- Statistically significant increase was noted in reticulocytes in high dose male, which was not related to treatment as decrease in red cell mass (RBC, haemoglobin and haematocrit) and it also lacked consistency between sexes. Moreover, values of all male rats of high dose were within historical ranges.
- Statistically significant decrease was noted in APTT in high dose male which was not considered as toxicologically significant.
- Statistically significant decrease was noted in reticulocytes in mid dose female, which was considered unrelated to test item treatment due to lack of dose dependency.
Please also refer to attached Tables below.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment related changes in the clinical chemistry parameters was observed in male and female rats of the treatment group.
- Statistically significant decreases were noted in albumin in high dose male and female, and total protein in high female. The lower values were minimal in nature and, in general, were within the historical control range and no other clinical chemistry changes related to liver function were noted for this high dose group. The lower total protein and albumin values were considered incidental in nature and not treatment related.
- Statistically significant increases were noted in LDL and inorganic phosphorus in high dose males, while decrease was noted in chloride in high dose females, which were considered unrelated to test item treatment, due to lack of consistency between sexes. Moreover, though values of 3 males of high dose were above historical ranges, it was considered as associated with false elevation due to increase in platelets (Lutomski and Bower, 1994). Similarly, in high dose females only one rat had values below historical range, while historical range for LDL is not available.
- Statistically significant increases were observed in glucose, urea and BUN in mid dose males, and HDL and LDL in mid dose females, which were considered unrelated to test item treatment, due to lack of dose dependency.
Please also refer to attached Tables below.

Endocrine findings:

no effects observed

Description (incidence and severity):

Hormone Analysis: The study director concluded that thyroid hormone level (TSH, T3 and T4) of male and female rats of the treatment groups was comparable with that of the vehicle control group.
In more detail, serum T4 level was statistically significantly increased in female rats. However, serum T4 level was not statistically significantly altered in males, both serum TSH and T3 levels were unchanged and no changes were observed in thyroid weight or histopathology. In the absence of any correlating effect and considering the unchanged thyroid hormone levels in the OECD 414 and OECD 422 studies, this was not considered as a relevant test item-related effect.
Please also refer to attached Tables below.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Urine analysis parameters did not reveal any alteration related to treatment.
- Statistically significant increase was observed in volume and pH in males of high dose animals, which was considered unrelated to test item treatment, due to lack of consistency between sexes and normal kidneys function tests (serum creatinine, urea/BUN concentration and histopathology of kidneys)
Please also refer to attached Tables below.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Neurobehavioural Observations:
Home Cage Observations:
- In the home cage, all rats from the treatment (G2, G3, and G4) and the vehicle control groups (G1) showed normal posture, asleep (curled up often asleep), sitting A (sitting but with head hung down),sitting B (sitting normally, feet tucked in), sitting C (sitting or standing alert, watching) and rearing. Clonic and tonic movements were absent in home cage during NBO
Please also refer to attached Tables below.

Handling Observations:
NBO performed during handling of rats did not reveal any abnormalities. All rats revealed normal behaviour during removal (very easy - rats sit quietly) and handling (easy - alert, limbs put against the body). No rat showed lacrimation, salivation, or piloerection. Eyelids were wide open in all rats. Eye and skin examination did not reveal any abnormality in any rat.

Open Field Observation:
In the open field, all rats from the treatment and vehicle control groups showed normal gait, mobility, arousal, and respiration during the ‘three-minute observation period’. Clonic and tonic movements, vocalisation, stereotypy, and bizarre behaviour were absent in this assessment. The rearing, urination, and defecation counts of male and female rats from the treatment groups were generally comparable with those of the vehicle control group with a few exceptions as follows:
- Statistically significant increase in rearing count (week 12, male rats, mid dose)
- Statistically significant increase in the urination count (week 3, male rats, low, mid, high dose)
- Statistically significant decrease in the urination count (week 2, female rats, high dose) and increase in the urination count (week 6 female rats, mid dose)
- Statistically significant increase in the defecation count (week 2, male rats, high dose)
These findings were stand-alone findings and were not supported by any other neurobehavioural parameters and motor function findings in the high dose group. Hence, the finding were not considered treatment related.

Functional Observational Battery (FOB):
- Motor Activity: The motor activity data of rats from the treatment groups were comparable with that of the control group except statistically significant decrease in the fine activity during 21-30 minute interval for high dose females. These changes were not supported with rearing count or any other neuromuscular activity parameters. Hence, this finding was considered incidental in nature.
- Sensory Reactivity Observations, Grip Strength, Hindlimb Foot Splay: Treatment groups were comparable with control group.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The test item did not produce any treatment related alteration in the absolute organ weights.
- Statistically significantly higher relative weights of liver for mid and high dose males, and thyroid with parathyroid for high dose males were noted. These changes were considered to be related to the lower terminal body weights noted for these groups and not a treatment effect.
- Statistically significant increase was also noted in relative weights of kidneys in mid dose male animals, which was considered unrelated to test item treatment, due to absence of dose dependency.
Please also refer to attached Tables below.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Gross examination (external and internal) of rats of either sex across various groups (G1 to G4) did not reveal any abnormality except for a single mid dose male (Rat No 45) which showed bilateral reduced size of testes and epididymides considered spontaneous or incidental in nature and not related to the test item treatment.
A gross pathological examination of the found dead rat No 75 revealed a generalised autolysis.
Please also refer to attached Tables below.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

The test item treatment did not lead to any alteration in the microscopic examination.
An examination of the found dead rat No 75 revealed varying degrees of autolytic changes in various organs. In the absence of any significant gross and histopathological observation, cause of death of the rat could not be defined and relation of death with the test item treatment could not be established.
Please also refer to attached Tables below.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Dose descriptor:

NOAEL

Effect level:

240 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

ophthalmological examination

organ weights and organ / body weight ratios

urinalysis

other: Hormone analysis

Critical effects observed:

no

Estrous Cycle Status of Termination

Estrous status of female rats on day of necropsy was as mentioned below in this table

Rat N°	Phase	Rat N°	Phase	Rat N°	Phase	Rat N°	Phase
11	D	31	D	51	E	71	P
12	E	32	E	52	E	72	E
13	P	33	D	53	D	73	D
14	P	34	D	54	P	74	E
15	D	35	E	55	D	75	-
16	P	36	E	56	D	76	M
17	P	37	P	57	E	77	E
18	E	38	D	58	D	78	P
19	E	39	E	59	D	79	D
20	P	40	E	60	E	80	D

  Key: E = Estrous, P = Proestrous, M = Metestrous, D = Diestrous and - = Not applicable

Please also refer to attached additional Tables below.

Conclusions:

- Based on above results, it is concluded that N-Butylaminoethanol did not produce any toxicity or test-item related adverse effect up to 240 mg/kg bw/day when administered orally through gavage for 90 consecutive days in Wistar rats.
- Therefore, the No Observed Adverse Effect Level (NOAEL) of N-Butylaminoethanol is 240 mg/kg bw/day under the conditions and procedure followed in this study.

Executive summary:

This study was conducted to determine the adverse effects occurring as a result of the daily repeated oral (gavage) administration of N-Butylaminoethanol for a period of 90 consecutive days in Wistar rats.

 

Method:

Dose levels	G1 (vehicle control): 0 mg/kg bw/day G2 (low dose): 60 mg/kg bw/day G3 (mid dose):120 mg/kg bw/day G4 (high dose): 240 mg/kg bw/day
Number of rats	10 rats/sex/group
Vehicle	RO Water

 

Based on the results from a stability study, the test item was found to be stable for 7 days in dose formulation, using RO water as the vehicle. Three samples of dose formulation (upper, middle, and lower layers) from each concentration and one sample from the vehicle control (middle layer) were collected on days 1, 29, 57, and 85 of dosing, for homogeneity and active ingredient (a.i.) concentration analysis.

Each rat was observed twice daily for clinical signs, morbidity, and mortality, during the treatment period. Body weights were recorded weekly for all rats. Body weight change and food consumption were calculated weekly. Neurobehavioural observation (NBO) was performed on all rats, prior to the initiation of the treatment and weekly, thereafter. The ophthalmological examination was performed on all rats from each group, prior to the initiation of the treatment and on surviving rats, prior to the terminal sacrifice. A Functional Observational Battery (FOB) was performed on all rats, during the 12th week of treatment. Haematological and clinical chemistry analyses were performed at the end of the treatment. Urine was collected from all surviving rats, at the end of the treatment for urine analysis.

All surviving rats were sacrificed by carbon dioxide asphyxiation at end of the treatment period. All surviving and found dead rats were subjected to a full gross necropsy. On the day of necropsy, vaginal smears were examined in all surviving female rats to determine the stage of estrous cycle. Absolute organ weights were recorded, and relative organ weights were calculated for the adrenals, brain, epididymides, heart, kidneys, liver, pituitary gland, spleen, seminal vesicles with coagulating glands and prostate, thymus, testes, thyroid with parathyroid and uterus with cervix. Detailed histopathological examination was carried out in all rats from the vehicle control and high dose groups.

 

 

Results:

Results of the active ingredient concentration and homogeneity analysis of the dose formulation samples collected on days 1, 29, 57, and 85 were within the acceptable ranges of ± 10 % of the nominal concentration.

Mortality was observed in one high dose female rat on day 85. However, there were no treatment related changes observed in body weight, food consumption, clinical pathology, gross and microscopic examination, in female rats of high dose group. Hence, this death was considered not to be related to treatment.

Mild salivation in male and female rats and gasping in male rats of high dose treated group were observed on multiple days of study. Salivation observed in high dose group was considered as a response to the dose solution and considered as non-adverse in nature. Gasping was not supported with any microscopic change observed in respiratory organs. Hence, it was considered as possibly related an irritancy response to the test material and non-adverse in nature.

 

No treatment related change was observed during the ophthalmological examination of the treated rats.

No treatment related changes were observed in the neurobehavioural observation and functionalobservational battery performed in rats from the reatment groups.

No treatment related changes were observed in the mean body weight in females and food consumption oof all treatment groups as compared to control. In males, body weight gain was slightly (<10 %) decreased in mid dose males from week 7 until week 13. Due to the marginal extend, this was not considered as toxicologically relevant. Occasional, unique statistically significant decreases in body weight gain observed in high dose males at weeks 2, 4 and 6, but not thereafter, were not considered as a consistent test-item-related effect.

No treatment related changes were observed in the heamatology, clinical chemistry and urinalysis parameters of the treatment groups as compared to control.

 

The test item did not produce any treatment related alteration in the terminal body weight and absolute organ weight. A gross examination (external and internal) of rats of either sex across various groups (G1 to G4) did not reveal any abnormality with the exception of a male rat from the mid dose group (Rat No 45) showed bilateral reduced size of testes and epididymides, which was spontaneous or incidental in nature and not related to the test item treatment. A gross pathological examination of the found dead rat revealed a generalised autolysis. The test item treatment did not lead to any alteration in the microscopic examination. An examination of the found dead rat No 75 revealed varying degree of autolytic change in various organs. In the absence of any significant gross and histopathological observation, cause of death of the rat could not be defined and relation of death with the test item treatment could not be established.

 

Conclusion:

During this study, there was no treatment related adverse change observed in the body weight, food consumption, clinical pathology, and histopathology, up to dose level 240 mg/kg bw/day.

Based on above results, it is concluded that N-Butylaminoethanol did not produce any relevant toxicity or test-item related adverse effect up to 240 mg/kg bw/day when administered orally through gavage for 90 consecutive days in Wistar rats. Therefore, the No Observed Adverse Effect Level (NOAEL) of N-Butylaminoethanol is 240 mg/kg bw/day under the conditions and procedure followed in this study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP guideline study (Klimisch 1).

System:

urinary

Organ:

kidney

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 May 2012 (Experimental starting date (Arrival of test animals)) to 18 Apr 2013 (Experimental completion date (check the raw data for its completeness)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: U.S. EPA OPPTS Guidelines 870.3650

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS: Wistar Crl:WI(Han)
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation: about 10 - 11 weeks; male/female (no siblings)
- Weight at study initiation: not reported
- Fasting period before study: no
- Housing: individually in Makrolon type M III cages (floor area of about 800 cm²) except mating period during which one male and one female were housed together. Pregnant animals and their litters were housed together until PND 4 (end of lactation). For motor activity (MA) measurements the animals were housed individually in polycarbonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany with wire covers from Ehret, Emmendingen, Germany (floor area of about 800 cm²) and small amounts of bedding material (the present supplier is documented in the raw data).

Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.

Dust-free wooden bedding was used in this study (the present supplier is documented in the raw data). Wooden gnawing blocks (Type NGM E-022), supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria, were added for environmental enrichment.

The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured.

- Diet (e.g. ad libitum): ad libitum (Kliba maintenance diet mouse-rat “GLP” meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland) except the exposure period. Besides, the animals were sacrificed after a fasting period (withdrawal of food) for at least 16-20 hours.
- Water (e.g. ad libitum): ad libitum (from water bottles) except the exposure period.
- Acclimation period: 8 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:

Route of administration:

inhalation: vapour

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: No details are given

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Inhalation apparatus type INA
The inhalation atmosphere was maintained inside aerodynamic exposure systems (INA 120, volume V ≈ 155 L, BASF SE until study day 7 (control group) or INA 60, volume V ≈ 90 L, BASF SE from study day 8 through to the end of the study (control and test groups)) consisting of a cylindrical inhalation chamber made of stainless steel sheeting and cone shaped outlets and inlets.
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 into the inhalation system. Due to the high vapor pressure of the test substance, the liquid droplets evaporate spontaneously and form vapor inhalation atmospheres. The control group was exposed to conditioned air.

- Method of holding animals in test chamber: The rats were restrained in glass exposure tubes. Their snouts projected into the inhalation chamber and thus they inhaled the aerosol.
- Source and rate of air: conditioned supply air and humidified air (without further details),
- Method of conditioning air: filtered through activated charcoal

- System of generating particulates/aerosols:
• Continuous infusion pumps PERFUSOR (B. Braun Melsungen AG, Melsungen, Germany)
• Two-component atomizers (stainless steel, Model 970; Düsen-Schlick GmbH, Untersiemau/Coburg, Germany)

- Temperature, humidity, pressure in air chamber:
Conditioned supply air is activated charcoal filtered air conditioned to about 50 % ± 20 % relative humidity and 22 °C ± 2 °C. Compressed air is filtered air pressurized to about 6 bar.

- Air flow rate and air change rate: The test pump rates, substance flow and air flows were scheduled (presented in table 2 in "Any othet information on materials and methods").
- Method of particle size determination: The test substance in the concentration to be tested is a vapor. Therefore no cascade impactor measurement had been performed.
- Treatment of exhaust air: A positive pressure was maintained inside the exposure systems by adjusting the air flow of the exhaust air system. This ensured that the aerosol in the breathing zones of the animals was not diluted by laboratory air.

In order to accustom the animals to exposure they were treated with supply air under conditions comparable to exposure on two days before start of exposure (pre-exposure period).

TEST ATMOSPHERE
- Brief description of analytical method used:
The concentrations of the inhalation atmospheres were analyzed online by propane-calibrated total hydrocarbon analyzer (FID). By means of response factor provided by the manufacture, the measured concentration of propane in each test group less the background concentration, were converted to concentration of the test substance. To verify the correctness of the FID measurement, absorption samples were drawn from the atmospheres. The absorption samples were analyzed by gas chromatography in all test groups.

Daily means were calculated based on three 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 total hydrocarbon analyzers.

The control group was analyzed on three days during the exposure period.

- Samples taken from breathing zone: yes

VEHICLE: air

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

MEASUREMENT OF THE EXPOSURE CONDITIONS
The atmospheric concentrations were measured online by propane-calibrated total hydrocarbon analyzer (FID) and confirmed by GC analyses of absorption samples.
Recording of exposure parameters is presented in table 3 in "Any othet information on materials and methods".
No surveillance of the oxygen content in the inhalation system was performed. The air change within the inhalation systems was judged to be sufficient to prevent oxygen depletion by the breathing of the animals and the concentrations of the test substance used could not have a substantial influence on oxygen partial pressure.

Principles of recording with the automated measuring system:

Each parameter was measured at appropriate measuring points using suitable measuring equipment (sensors, orifice plates etc.). The measurements were standardized (0 20 or 4 20 mA) and transferred to instrumentation consoles. There, the measured values were displayed in an analogous way (where this is provided for) and some were used as actual value for regulating the specific parameter.

In addition, the measured values were scanned every 10 seconds, converted from analog to digital, transferred to a personal computer, displayed on its screen, and saved on hard disk. The computer checked the arriving values against preset threshold values, displayed warnings if violations of thresholds occurred and recorded the start and the end of threshold violations for each measured parameter affected. After the end of each exposure all data gathered during this exposure were backed up on optical media.

Daily protocols were prepared from the recorded values using suitable software. The protocols include start and stop times of exposure and possible threshold violations, and daily means of each parameter. The records saved on optical media and the printed daily records are considered as raw data.

Duration of treatment / exposure:

Males (28 exposure days):
a) 14 days premating
b) up to 14 days mating
c) Sacrifice after a minimum of 28 days after the first application (on day 29)

Females (50 exposure days)
a) 14 days premating
b) up to 14 days mating
c) during the pregnancy up to and including GD 19
d) after necropsy of the pups total 4 exposures on 4 consecutive days including the day before scheduled killing (on day 51)

Frequency of treatment:

6h/day; 5 days/week

Dose / conc.:

20.6 mg/m³ air (nominal)

Dose / conc.:

72.1 mg/m³ air (nominal)

Dose / conc.:

236.3 mg/m³ air (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Based on available data, the following concentrations were selected for the present study:

225 mg/m³ as the high concentration causing adverse effect
75 mg/m³ as the mid concentration causing some effect
25 mg/m³ as the low concentration and the expected no adverse effect concentration
- Other: no exposure on the day of functional observation battery/motor activity measurement (FOB/MA). Observations of FOB/BA were performed on study day 12 in females and on study day 26 in males.
- Rationale for animal assignment (if not random): randomized

Positive control:

None.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
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.
- Time schedule: The clinical condition of the test animals was recorded once during the pre-exposure period and on non-exposure days and at least 3 times (before, during and after exposure) on exposure days.
During exposure only a group wise examination was possible.

Abnormalities and changes were documented daily for each animal. Individual data of 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 particular findings (e.g. inability to deliver) were documented on an individual dam basis.

On weekdays (except Saturday, Sunday and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings.

The day of parturition was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first exposure and weekly thereafter.

For observation, the animals were therefore be removed from their cages and placed in a standard arena (50 x 37.5 x 25 cm). The scope of examinations and the scoring of the findings that are observed will be based on the current index of findings in PDS ToxData® and includes but is not limited to the following parameters listed:
1. Abnormal behavior in “handling”
2. Fur
3. Skin
4. Posture
5. Salivation
6. Respiration
7. Activity/arousal level
8. Tremors
9. Convulsions
10. Abnormal movements
11. Gait abnormalities
12. Lacrimation
13. Palpebral closure
14. Exophthalmos
15. Assessment of the feces discharged during the examination (appearance/consistency)
16. Assessment of the urine discharged during the examination
17. Pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: once a week at the same time of the day (in the morning) until sacrifice.

The following exceptions are notable for the female animals:
- During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
- Females with litter were weighed on the day of parturition (PND 1) and on PND 4.
- After the pups were sacrificed the females were exposed for 4 consecutive days. The F0 females were weight once before the exposure period and once on the last exposure day.
Females without positive evidence of sperm, without litter or waiting for necropsy, were weighed weekly. These body weight data were solely used for the calculations of the dose volume

FOOD CONSUMPTION
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
- Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female F0 animals).
- Food consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0 - 7 7 - 14, and 14 - 20.
- Food consumption of F0 females, which gave birth to a litter, was determined on PND 1 - 4.
- Food consumption of the females during the 4 exposure days after necropsy of the pups.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at the time of the detailed clinical observations, the animals were tested for the presence of exophthalmos.
- Dose groups that were examined: all animals.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: towards the end of the exposure period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5 animals per sex and group
- Parameters checked in table [4] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: towards the end of the exposure period
- Animals fasted: Yes
- How many animals: 5 animals per sex and group
- Parameters checked in table [4] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: A functional observational battery was performed in 5 parental male and 5 parental female animals per group and for the males the FOB were carried out at the end of the administration period, for the females at the end of the premating period.
- Dose groups that were examined: all dose groups.
- Battery of functions tested: sensory activity, grip strength, motor activity

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. All parental animals were sacrificed under pentobarbitone anesthesia by exsanguination from the abdominal aorta and vena cava. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.
HISTOPATHOLOGY: Yes (see table 5)

Other examinations:

Organ weights
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals
2. Epididymides
3. Testes

The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations):

1. Adrenal glands
2. Brain
3. Heart
4. Kidneys
5. Liver
6. Lung
7. Spleen
8. Thymus

Statistics:

Statistical methods are summarised in "Overall remarks, attachments".

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

body weights were slightly reduced in male and female animals during premating and mating period. During gestation stage, mean body weights of group 3 females were significantly reduced on gestation day 7 and 14.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

food consumptions were slightly reduced in male and female animals during premating and mating period and significantly decreased during gestation stage.

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Endocrine findings:

not specified

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

increased: epididymides, testes, spleen, liver and thymus

Gross pathological findings:

no effects observed

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment – related findings in the larynx, nasal cavities, testes and epididymides.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no test substance-related or spontaneous mortalities in any of the groups.
During the pre-exposure period, the pre-mating and the mating period the animals showed no clinical signs and findings different from normal.

During the post-mating day the male animals showed no clinical signs and findings different from normal. During the post-mating period one female animal of the control group (No. 104) showed vaginal discharge.

During the gestation period six female animals of the control group, eight female animals of the low concentration (25 mg/m³), eight female animals of the mid concentration (75 mg/m³) and seven female animals of the high concentration (225 mg/m³) showed vaginal discharge. As this finding was distributed evenly in the controls and the groups exposed to the test substance, it was considered to be treatment-related (head-nose exposure), but not substance-related.
During the lactation period the female animals showed no clinical signs and findings different from normal.
During the 4-day exposure period of the females after the pups were sacrificed the animals showed no clinical signs and findings different from normal.
One sperm positive low concentration female (No. 117) did not deliver F1 pups.
The detailed clinical observations did not reveal any abnormalities in male and female animals of all test groups.

BODY WEIGHT AND WEIGHT GAIN

Body weight in premating period:
The mean body weights of the test substance exposed male and female animals were not statistically significantly different from the control group 0.

Body weigh in mating period:
The mean body weights of the test substance exposed male animals were not statistically significantly different from the control group 0, although the mean body weights of the males test group 3 seems to be slightly lower than those of other groups.
Body weight in gestation:
The mean body weights of group 3 females were slightly lower than the control on gestation days (GD) 7 and 14 (p < 0.05).
Body weight during lactation and on PND 4:
The mean body weights of F0 female animals were not statistically significantly different from the control group 0.
Body weight change / during the exposure period:

The body weight change of the F0 male animals of the high concentration group (225 mg/m³) was statistically significantly lower than the controls during premating period (-12.9 g, p < 0.05) at the beginning of the mating period. This effect was diminished in course of the exposure and was not observed any further in the second week of the pre-mating period, as well as during mating period.
During pre-mating period, the mean body weight changes of the female animals were not statistically different when compared with the control.
From gestation day 0 to 7, the mean body weight change of F0 female animals was significantly lower (-37 %, p < 0.01) than the controls. This effect was not observed any further at later time points. The body weight changes were in other test groups not significantly different to the control

FOOD CONSUMPTION
In high concentration (225 mg/m³) food consumption during pre-mating was significantly decreased in male animals between study days 0-7 (-9 %) and 7-14 (-11 %) as well as in female animals between study days 0-7 (-7 %) and 7-14 (-5 %). These findings were considered to be substance-related.
During the gestation the food consumption in the high concentration (225 mg/m³) was significantly decreased in female animals during the whole period (-11 %) and in the mid concentration (75 mg/m³) was significantly decreased in female animals between study days 0 - 7 (-9 %).
No other findings were observed for male and female animals in test group 1 and 2 (25 and 75 mg/m³).

OPHTHALMOSCOPIC EXAMINATION (included in detailed clinical observation)
No effects.

HAEMATOLOGY
Prior to blood sampling, male rats were dosed for four weeks and female rats were dosed for two weeks. No treatment-related changes among hematological parameters were observed.

CLINICAL CHEMISTRY
No treatment-related changes among clinical chemistry parameters were observed.
In male rats of test group 3 (225 mg/m3), globulin values were lower compared to controls. However, this parameter was not dose-dependently changed. Additionally, this was the only changed parameter in clinical pathology. Therefore, this alteration was regarded as incidental and not treatment-related.

NEUROBEHAVIOUR
On the day of the performance of the Functional Observation Battery, the animals were not exposed to the test substances. Observations were performed on study day 12 in females and on study day 26 in males.
- Home cage observations: No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation;
- Open field observations: The open field observations did not reveal any test substance-related findings in male and female animals of all test groups;
- Sensorimotor tests/reflexes: There were no test substance-related findings in male and female animals of all test groups;
- Quantitative parameters: In general no test substance-related impaired parameters were observed in male and female animals of all test groups.
In F0 females of test group 1, slightly though significantly higher grip strength of forelimbs was determined. This parameter was not increased in other groups, it was considered to be incidental due to the lack of concentration-response relationship;
- Motor activity measurement (MA): No statistically significant changes on motor activity data (summation of all intervals) as well as on single intervals were observed in the male and female animals of all dose groups in comparison to the concurrent control group.

ORGAN WEIGHTS
When compared with control group 0 (=100%), the mean absolute weights presented in the tables 2-5 were significantly increased or decreased in one or more test groups (statistically significant changes printed in bold).

GROSS PATHOLOGY
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Fertility:
The mating pair (Nos. 17/117), which did not produce offspring did not show relevant gross lesions.


HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment-related findings were observed in level I of larynx, and level I and II of nasal cavity, in males and females as well as in epididymis and testis in male animals with incidences and grading shown in the tables 6-9 below.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
No neoplastic lesions are reported.

OTHER FINDINGS
- Male and female reproduction and delivery data were similar to controls (please refer to section 7.8.1. and 7.8.2. for detailed information);
- Pup number, status at delivery, viability/mortality, sex ratio, clinical observations, pup body weight and pup necropsy observations were all similar to controls (please refer to section 7.8.1. and 7.8.2. for detailed information).

Dose descriptor:

NOAEC

Remarks:

local

Effect level:

20.6 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on histopathological changes in larynx and nasal cavity

Dose descriptor:

NOAEC

Remarks:

systemic

Effect level:

236.3 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOAEC

Remarks:

reproductive and developmental parameters

Effect level:

236.3 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: the highest dose tested, no adverse effects observed

Critical effects observed:

not specified

The atmospheric concentrations were measured online by FID and confirmed by GC analyses of absorption samples. The concentrations measured by FID are presented in table 1. GC analyses confirmed largely the values of FID measurement. Details see part II of the report.

 

Table 1: Study means and standard deviations of test substance concentrations measured by FID

Test group	Target concentration (mg/m³)	Measured concentration (mg/m³)		Nominal concentration (mg/m³)	Effectiveness of vapor generation (%)
		Mean	SD
1	25	20.6	6.0	21.5	95.8
2	75	72.1	20.0	86.0	83.8
3	225	236.3	39.5	301.0	78.5

The vapor generation effectiveness was as expected for these high concentrations.

 

Measurementsconcerning operation conditions

The air flows were constantly maintained in the desired range. An air change of about 67 times per hour can be calculated by dividing the supply air flow through the volume of each inhalation system.

 

Relative humidities in the inhalation systems ranged between 39.3 and 63.8 %. In the test groups the relative humidity was slightly lower than those in the control chamber, because the test substance was sprayed by means of compressed air, which is dry. Nonetheless, all values were within the range suggested by the respective testing guidelines. 

The temperatures in the inhalation systems ranged between 21.7 and 24.1 °C.

 

Food, drinking water and bedding enrichment analyses

On the basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants the food, drinking water and bedding enrichment were found to be suitable.

 

Absolute and relative organ weights

When compared with control group 0 (= 100 %), the mean absolute weights presented in the tables 2-5 were significantly increased in one or more test groups (statistically significant changes printed in bold):

 

Table 2: Relative increase of absolute weights in males

Absolute weights	Males
Group (mg/m³)	1 (25)	2 (75)	3 (225)
Epididymides	88 %**	93 %	90 %**
Spleen	137 %**	96 %	90 %
Testes	98 %	93 %*	92 %*

* : p <= 0.05, **: p <= 0.01

 

Table 3: Relative increase of absolute weights in females 

Absolute weights	Females
Group (mg/m³)	1 (25)	2 (75)	3 (225)
Liver	102 %	103 %	89 %*

* : p <= 0.05, **: p <= 0.01

 

Table 4: Relative increase of relative weights in males 

Relative weights	Males
Group (mg/m³)	1 (25)	2 (75)	3 (225)
Epididymides	88 %**	94 %	94 %

*: p <= 0.05, **: p <= 0.01

 

Table 5: Relative increase of relative weights in females

Relative weights	Females
Group (mg/m³)	1 (25)	2 (75)	3 (225)
Thymus	129 %*	96 %	107 %

*: p <= 0.05, **: p <= 0.01

 

Weight changes in thymus, spleen and liver were regarded as incidental as no dose response relationship and/or histopathological correlates were detected.

The decrease in epididymis and testis weights was considered to be treatment-related even in the absence of a clear dose response relationship or, in the case of the testis, no changes in relative weights as there was a histopathological correlate.

All other mean absolute or relative weight parameters did not show significant differences when compared to the control group 0.

 

 

Histopathology

Treatment-related findings were observed in level I of larynx, and level I and II of nasal cavity, in males and females as well as in epididymis and testis in male animals with incidences and grading shown in the table below:

 

Larynx

Treatment – related findings in the larynx were epithelial alteration characterized by slight modification of epithelial cells (i.e., three to four cell layers, focally flattened and stratified) indicating beginning metaplastic transformation and minimal to slight squamous metaplasia (characterized by three to four cell layers of flattened, stratified epithelium with no signs of keratinization and only affecting the epiglottis when graded minimal and up to five cell layers of flattened, stratified epithelium, occasionally minimal focal keratinization, with/without focal desquamation of superficial cells when graded slight).

 

Table 6: Incidence and grading of histological findings in larynx

	Male animals				Female animals
Test group (mg/m³)	0 (0)	1 (25)	2 (75)	3 (225)	0 (0)	1 (25)	2 (75)	3 (225)
No. of animals	10	10	10	10	9	10	10	10
Epithelial alteration	1	6	8	5	1	6	8	8
Grade 1	1	6	8	4	1	6	4	8
Grade 2				1			4
Metaplasia, squamous			2	5			1	2
Grade 1			2	4			1	2
Grade 2				1

 

Nasal cavity, level I

The following treatment- related findings were noted in the nasal cavity:

Degeneration / regeneration of transitional and respiratory epithelium was characterized by variable vacuolation, presence of few apoptotic bodies, minimal infiltrates of inflammatory cells, increased size and basophilia of nuclei and minimal disorganization of cells.

Metaplasia, squamous was characterized by flattened epithelial cells with variably present minimal keratinization.

 

Table 7: Incidence and grading of histological findings in nasal cavity

	Male animals				Female animals
Test group (mg/m³)	0 (0)	1 (25)	2 (75)	3 (225)	0 (0)	1 (25)	2 (75)	3 (225)
No. of animals	10	10	10	10	9	10	10	10
Degeneration /regeneration transitional epithelium				2			1	8
Grade 1				2			1	7
Grade 2								1
Metaplasia, squamous, transitional epithelium				1				3
Grade 1				1				3
Degeneration /regeneration respiratory epithelium				2
Grade 1				2

 

Nasal cavity, level II:

One male test group 3 (225 mg/m³) animal (No 134) showed minimal degeneration / regeneration of the olfactory epithelium, in level I it showed degeneration/regeneration of the transitional epithelium)

 

Testes

Tubular degeneration was observed in a higher incidence in treated test groups. This finding was characterized by randomly affected (not stage specific) tubules with sloughed spermatogenic cells, vacuolation of the spermatogenic epithelium or missing germ cell layers.

 

Table 8: Incidence and grading of histological findings in testes

	Male animals
Test group (mg/m³)	0 (0)	1 (25)	2 (75)	3 (225)
No. of animals	10	10	10	10
Degeneration, tubular	4	6	7	8
Grade 1	3	2	2	3
Grade 2		3	3	4
Grade 3	1	1	2	1
Sperm plug				1
present				1

 

Epididymides

Debris in the epididimydes was characterized by sloughed spermatogenic cells and noted with increased incidence in treated animals.

 

Table 9: Incidence and grading of histological findings in epididymides

	Male animals
Test group (mg/m³)	0 (0)	1 (25)	2 (75)	3 (225)
No. of animals	10	10	10	10
Debris	2	4	4	7
present	2	4	4	7

 

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

 

Fertility

The male partner of the mating pair (Nos. 17/117), which did not produce offspring showed testicular degeneration and debris in the epididymis which might have impaired fertility even if animals with similar findings in the same group did produce offspring.

Conclusions:

Inhalation exposure to dibutylethanolamine to maximum of 236.3 mg/m³ during 2 weeks premating, mating, gestation period did not cause any adverse effect with regard to reproductive and developmental parameters, but only transiently reduced food consumption, body weight and body weight gain. No adverse parental or pup findings were evident at any concentration. In histopathology lesions of nasal epithelia was observed in animals exposed to 72.1 and 236.3 mg/m³ test substance. Thus, concerning reproductive and developmental parameters the no observed adverse effect concentration (NOAEC) for dibutylethanolamine was determined to be >236.3 mg/m3. Considering histopathological changes the NOAEC for dibutylethanolamine was 20.6 mg/m³.

Executive summary:

To evaluate the toxicity profile of Dibutylethanolamine (DBEA) after inhalation exposure, groups of ten male and ten female Wistar rats (F0 animals) per test group were exposed nose-only to dynamic atmosphere of DBEA for 6 hours per day on each day. The duration of treatment covered a 2-week pre-mating and 2-week mating period in both sexes, 1 day post-mating in males, and the entire gestation period of the females. After the lactation period and after necropsy of the pups total all parental females were exposed to the test substance on 4 consecutive days.

 

The target concentrations were 25, 75 and 225 mg/m³ (correspond to measured concentrations 20.6, 72.1 and 236.3 mg/m³, respectively). A concurrent control group was exposed to conditioned air. For adaptation to the experimental conditions all animals were kept in glass restraining tubes identical to those used in the study and were exposed nose-only to fresh air on two days before start of the exposure period.

 

After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm was detected in the vaginal smear. F0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of post-implantation loss for all F0 females.

 

A detailed clinical observation (DCO) was performed in all animals before initial test substance exposure and, as a rule, thereafter at weekly intervals. Clinical observation was performed at least three times on exposure days and once a day during the other days.

 

Food consumption of the F0 parents was determined once weekly during premating. In dams food consumption was determined for gestation days 0 - 7, 7 - 14, 14 - 20 and lactation days 1 - 4. During the 4 exposure days after necropsy of the pups the food consumption was determined also.

 

Body weights of F0 parents were determined once a week, in males throughout the study and in females during premating. During gestation and lactation period, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, on the day after parturitionpostnatal day [PND] 1) and on PND 4. After the pups are sacrificed the females that were exposed for 4 consecutive days were weight once before the exposure period and once on the last exposure day.

 

A functional observational battery (FOB) was performed and motor activity was measured in 5 parental males and females per group. The FOB of the female animals was on study day 12. The male animals were performed at the end of the exposure period on study day 26.

 

The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1 and on PND 4. Their viability was recorded. At necropsy on PND 4, all pups were sacrificed with CO₂, under isoflurane anesthesia, and examined macroscopically for external and visceral findings.

 

Clinico-chemical and hematological examinations were performed in 5 animals per sex and group towards the end of the exposure period.

 

A complete necropsy including gross pathological evaluation and weighing of selected organs was performed. Organs and tissues were examined histopathologically as required by the corresponding test guidelines.

 

Results

Test group 3 (225 mg/m³)

 

Significantly decreased food consumption during pre-mating (p < 0.05) in male animals between study days 0-7 (-9 %) and 7-14 (-11 %) and in female animals between study days 0-7 (-7 %) and 7-14 (-5 %);

Significantly decreased food consumption during gestationin female animals during the whole period (-11 %,p < 0.05);

Significantly lowered mean body weights F0 females on gestation days (GD) 7 (-5.7 %, p < 0.05) and 14 (5.2 %, p < 0.05);

Significantly reduced body weight gain of the F0 male animals during premating period (-12.9 g versus -1.8 g in the control, p < 0.05);

Significantly lowered mean body weight gain of F0 female animals from gestation day 0 to 7 (-37 %, p < 0.01).

 

Nasal cavity, level I

 

Degeneration /regeneration transitional epithelium in 2/10 male (graded minimal) and 8/10 female animals (graded minimal in 7/10 and slight in 1/10 animals);

Metaplasia, squamous, transitional epitheliumgraded minimal in 1/10 male and 3/10 female animals.

Degeneration /regeneration respiratory epithelium in 2/10 male animals graded minimal;

 

Nasal cavity, level II

 

Degeneration /regeneration olfactory epithelium graded minimal in one male animal.

 

Test group 2 (75 mg/m³)

Significantly decreased food consumption during gestation in female animals between study days 0 - 7 (-9 %, p < 0.05).

 

Nasal cavity, level I

 

Degeneration /regeneration transitional epithelium in one female animal (graded minimal)

 

Test group 1 (25 mg/m³)

 

No adverse effect observed

 

Conclusion

Inhalation exposure to dibutylethanolamine to maximum of 236.3 mg/m³ during 2 weeks premating, mating, gestation period did not cause any adverse effect with regard to reproductive and developmental parameters, but only transiently reduced food consumption, body weight and body weight gain. No adverse parental or pup findings were evident at any concentration. In histopathology lesions of nasal epithelia was observed in animals exposed to 72.1 and 236.3 mg/m³ test substance. Thus, concerning reproductive and developmental parameters the no observed adverse effect concentration (NOAEC) for dibutylethanolamine was determined to be >236.3 mg/m³. Considering histopathological changes the NOAEC for dibutylethanolamine was 20.6 mg/m³.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

236.3 mg/m³

Study duration:

subacute

Experimental exposure time per week (hours/week):

30

Species:

rat

Quality of whole database:

GLP guideline study on a structurally similar chemical.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
In this justification, the read-across (bridging) concept is applied, based on the chemical structure of the potential analogues, their toxicokinetic behaviour and other available (eco-)toxicological data.
Please refer to a full version of Read-across statement attached in the section 13 "Assessment reports".

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The underlying hypothesis for the read-across is that the target and the source substance have similar toxicological properties (including the same target organs) due to their structural similarity, resemblance to their chemical reactivity, and therefore a similar mode of action (impairment of choline homeostasis). The substances share the same ethanolamine moiety and can be considered as derivatives of mono-ethanolamine (CAS 141-43-5). Ethanolamines have structural similarity with choline, an ubiquitous physiological molecule (e.g. involved in phospholipid synthesis like phosphatidylcholine and acetylcholine).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

source substance: 2-Dibutylethanolamine (or 2-Dibutylaminoethanol)
structural formula: C10H23NO
Smiles: CCCCN(CCCC)CCO
Molecular weight: 173.30
CAS 102-81-8
EC No 203-057-1
purity: not specified

target substance: Butyldiethanolamine
structural formula: C8H19NO2
Smiles: CCCCN(CCO)CCO
Molecular weight: 161.24
CAS 102-79-4
EC No 203-055-0
purity: not specified
No additional information is available on purity of the source and the target substances. Both substances are normally of high purity, containing only minor amounts of impurities that do not influence the read-across validity.

3. ANALOGUE APPROACH JUSTIFICATION
The repeated dose toxicity by inhalation of the amines is generally characterized by local respiratory and ocular effects because ethanolamines are caustic. Indeed, histopathological lesions of nasal epithelia were observed in animals exposed to 72.1 and 236.3 mg/m³ the source substance DBEA. The same or even weaker local effects are expected in a repeated inhalation study if it were conducted with the target substance BDEA. This can be explained by slightly different reactivity to tissues due to additional butyl group in the source substance DBEA. The source substance DBEA and the target substance BDEA are both tertiary amines but basicity of DBEA is the higher than the basicity of BDEA because of two butyl groups. Both substance share one ethanol group while the second ethanol group in BDEA does not influence basicity to such extent as the additional butyl group does. As it was observed in studies with different ethanolamines, including those with shorter aliphatic groups (methyl, ethyl, propyl), the more ethanol groups are, the weaker irritating properties are (please refer to IUCLID file of Triethanolamine (102-71-6), which is not irritating to skin and eyes). Thus, one could speculate that BDEA would be a less reactive chemical to tissues compared to DBEA.
Regarding systemic toxicity, if an additional butyl- alkyl chain in DBEA was associated with other mode(s) of action than choline impairment, then these mode(s) of action would be identified in this study. However, the effects were only decreased body weights and body weight gains as well as decreased food consumption. No other clinical signs that would point to another mode of action were observed. There were no mortalities, gross pathological changes or findings at necropsy. Since the number of ethanol groups in an amine seems not to impair choline homeostasis significantly (please refer to data sets of monoethanolamine (MEA; CAS 141-43-5), diethanolamine (DEA, CAS 111-42-2) and triethanolamine (TEA, CAS 102-71-6), the target substance BDEA, containing only one butyl-alkyl chain, would not possess other mode(s) of action than impairment of choline homeostasis, already confirmed in studies with one alkyl chain (MDEA, CAS 105-59-9; MMEA, CAS 109-83-1). Based on a number of study results with ethanolamines, they all affect liver and kidney. The liver is known to be the primary site of choline uptake and metabolism followed by the kidney. According to the various study results, presented in the read-across statement (attached below), the potency of ethanolamines to perturb choline homeostasis seems, however, to decline with the number of ethanol groups (the least pronounced systemic effects were observed with TEA). It was shown in an in vitro study that DBEA was more potent than BEA to inhibit choline esterase activity (Hartung and Cornish, 1968). Thus, BDEA is expected to be a less potent butyl amine with regard to impairment of choline homeostasis than the source substance DBEA.
Thus, based on above arguments, one could speculate that BDEA would be the least reactive chemical to tissues and, if BDEA was tested in a repeated dose toxicity study by inhalation, higher concentrations would be possible compared with DBEA. Therefore, having used the same concentrations for BDEA as used in the present study with DBEA, the strength of local effects caused by BDEA is expected to be lower than the strength of effects observed for the source substance DBEA. With other words, the differences in the reactivity would not lead to an underestimation of the local effects of BDEA if the same dose levels were used in a repeated dose toxicity study by inhalation. So, DBEA would represent a worst-case for local effects of BDEA. For systemic effects, it can be thought that lower reactivity of BDEA to tissues may allow higher concentrations to assess systemic effects in repeated inhalation studies. The higher concentrations seem however not achievable because vapour pressure of BDEA is lower than vapour pressure of DBEA. Thus, the concentrations that already tested in the study with DBEA would represent also the achievable concentration for BDEA in worst case. In conclusion, the same or even weaker than DBEA systemic toxicity by inhalation is predicted for BDEA. Thus, the level of systemic toxicity caused by DBEA in the inhalation study would represent worst-case for BDEA and would not lead to an underestimation of systemic effects if the same dose levels were used in a repeated inhalation toxicity study with BDEA.

4. DATA MATRIX
Please refer to the full version of the read-across statement.

Reason / purpose for cross-reference:

read-across source

Remarks on MMAD:

MMAD / GSD: No details are given

Dose / conc.:

20.6 mg/m³ air (nominal)

Dose / conc.:

72.1 mg/m³ air (nominal)

Dose / conc.:

236.3 mg/m³ air (nominal)

Positive control:

None.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

body weights were slightly reduced in male and female animals during premating and mating period. During gestation stage, mean body weights of group 3 females were significantly reduced on gestation day 7 and 14.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

food consumptions were slightly reduced in male and female animals during premating and mating period and significantly decreased during gestation stage.

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Endocrine findings:

not specified

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

increased: epididymides, testes, spleen, liver and thymus

Gross pathological findings:

no effects observed

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment – related findings in the larynx, nasal cavities, testes and epididymides.

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no test substance-related or spontaneous mortalities in any of the groups.
During the pre-exposure period, the pre-mating and the mating period the animals showed no clinical signs and findings different from normal.

During the post-mating day the male animals showed no clinical signs and findings different from normal. During the post-mating period one female animal of the control group (No. 104) showed vaginal discharge.

During the gestation period six female animals of the control group, eight female animals of the low concentration (25 mg/m³), eight female animals of the mid concentration (75 mg/m³) and seven female animals of the high concentration (225 mg/m³) showed vaginal discharge. As this finding was distributed evenly in the controls and the groups exposed to the test substance, it was considered to be treatment-related (head-nose exposure), but not substance-related.
During the lactation period the female animals showed no clinical signs and findings different from normal.
During the 4-day exposure period of the females after the pups were sacrificed the animals showed no clinical signs and findings different from normal.
One sperm positive low concentration female (No. 117) did not deliver F1 pups.
The detailed clinical observations did not reveal any abnormalities in male and female animals of all test groups.

BODY WEIGHT AND WEIGHT GAIN
Body weight in premating period:
The mean body weights of the test substance exposed male and female animals were not statistically significantly different from the control group 0.

Body weigh in mating period:
The mean body weights of the test substance exposed male animals were not statistically significantly different from the control group 0, although the mean body weights of the males test group 3 seems to be slightly lower than those of other groups.
Body weight in gestation:
The mean body weights of group 3 females were slightly lower than the control on gestation days (GD) 7 and 14 (p < 0.05).
Body weight during lactation and on PND 4:
The mean body weights of F0 female animals were not statistically significantly different from the control group 0.

Body weight change / during the exposure period:
The body weight change of the F0 male animals of the high concentration group (225 mg/m³) was statistically significantly lower than the controls during premating period (-12.9 g, p < 0.05) at the beginning of the mating period. This effect was diminished in course of the exposure and was not observed any further in the second week of the pre-mating period, as well as during mating period.
During pre-mating period, the mean body weight changes of the female animals were not statistically different when compared with the control.
From gestation day 0 to 7, the mean body weight change of F0 female animals was significantly lower (-37 %, p < 0.01) than the controls. This effect was not observed any further at later time points. The body weight changes were in other test groups not significantly different to the control

FOOD CONSUMPTION
In high concentration (225 mg/m³) food consumption during pre-mating was significantly decreased in male animals between study days 0-7 (-9 %) and 7-14 (-11 %) as well as in female animals between study days 0-7 (-7 %) and 7-14 (-5 %). These findings were considered to be substance-related.
During the gestation the food consumption in the high concentration (225 mg/m³) was significantly decreased in female animals during the whole period (-11 %) and in the mid concentration (75 mg/m³) was significantly decreased in female animals between study days 0 - 7 (-9 %).
No other findings were observed for male and female animals in test group 1 and 2 (25 and 75 mg/m³).

OPHTHALMOSCOPIC EXAMINATION (included in detailed clinical observation)
No effects.

HAEMATOLOGY
Prior to blood sampling, male rats were dosed for four weeks and female rats were dosed for two weeks. No treatment-related changes among hematological parameters were observed.

CLINICAL CHEMISTRY
No treatment-related changes among clinical chemistry parameters were observed.
In male rats of test group 3 (225 mg/m3), globulin values were lower compared to controls. However, this parameter was not dose-dependently changed. Additionally, this was the only changed parameter in clinical pathology. Therefore, this alteration was regarded as incidental and not treatment-related.

NEUROBEHAVIOUR
On the day of the performance of the Functional Observation Battery, the animals were not exposed to the test substances. Observations were performed on study day 12 in females and on study day 26 in males.
- Home cage observations: No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation;
- Open field observations: The open field observations did not reveal any test substance-related findings in male and female animals of all test groups;
- Sensorimotor tests/reflexes: There were no test substance-related findings in male and female animals of all test groups;
- Quantitative parameters: In general no test substance-related impaired parameters were observed in male and female animals of all test groups.
In F0 females of test group 1, slightly though significantly higher grip strength of forelimbs was determined. This parameter was not increased in other groups, it was considered to be incidental due to the lack of concentration-response relationship;
- Motor activity measurement (MA): No statistically significant changes on motor activity data (summation of all intervals) as well as on single intervals were observed in the male and female animals of all dose groups in comparison to the concurrent control group.

ORGAN WEIGHTS
When compared with control group 0 (=100 %), the mean absolute weights presented in the tables 2-5 were significantly increased or decreased in one or more test groups (statistically significant changes printed in bold).

GROSS PATHOLOGY
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Fertility:
The mating pair (Nos. 17/117), which did not produce offspring did not show relevant gross lesions.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment-related findings were observed in level I of larynx, and level I and II of nasal cavity, in males and females as well as in epididymis and testis in male animals with incidences and grading shown in the tables 6-9 below.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
No neoplastic lesions are reported.

OTHER FINDINGS
- Male and female reproduction and delivery data were similar to controls (please refer to section 7.8.1. and 7.8.2. for detailed information);
- Pup number, status at delivery, viability/mortality, sex ratio, clinical observations, pup body weight and pup necropsy observations were all similar to controls (please refer to section 7.8.1. and 7.8.2. for detailed information).

Dose descriptor:

NOAEC

Remarks:

local

Effect level:

20.6 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on histopathological changes in larynx and nasal cavity

Dose descriptor:

NOAEC

Remarks:

systemic

Effect level:

236.3 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOAEC

Remarks:

reproductive and developmental parameters

Effect level:

236.3 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: the highest dose tested, no adverse effects observed

Critical effects observed:

not specified

The atmospheric concentrations were measured online by FID and confirmed by GC analyses of absorption samples. The concentrations measured by FID are presented in table 1. GC analyses confirmed largely the values of FID measurement. Details see part II of the report.

 

Table 1: Study means and standard deviations of test substance concentrations measured by FID

Test group	Target concentration (mg/m³)	Measured concentration (mg/m³)		Nominal concentration (mg/m³)	Effectiveness of vapor generation (%)
		Mean	SD
1	25	20.6	6.0	21.5	95.8
2	75	72.1	20.0	86.0	83.8
3	225	236.3	39.5	301.0	78.5

The vapor generation effectiveness was as expected for these high concentrations.

 

Measurementsconcerning operation conditions

The air flows were constantly maintained in the desired range. An air change of about 67 times per hour can be calculated by dividing the supply air flow through the volume of each inhalation system.

 

Relative humidities in the inhalation systems ranged between 39.3 and 63.8 %. In the test groups the relative humidity was slightly lower than those in the control chamber, because the test substance was sprayed by means of compressed air, which is dry. Nonetheless, all values were within the range suggested by the respective testing guidelines. 

The temperatures in the inhalation systems ranged between 21.7 and 24.1 °C.

 

Food, drinking water and bedding enrichment analyses

On the basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants the food, drinking water and bedding enrichment were found to be suitable.

 

Absolute and relative organ weights

When compared with control group 0 (=100%), the mean absolute weights presented in the tables 2-5 were significantly increased in one or more test groups (statistically significant changes printed in bold):

 

Table 2: Relative increase of absolute weights in males

Absolute weights	Males
Group (mg/m³)	1 (25)	2 (75)	3 (225)
Epididymides	88 %**	93 %	90 %**
Spleen	137 %**	96 %	90 %
Testes	98 %	93 %*	92 %*

* : p <= 0.05, **: p <= 0.01

 

Table 3: Relative increase of absolute weights in females 

Absolute weights	Females
Group (mg/m³)	1 (25)	2 (75)	3 (225)
Liver	102 %	103 %	89 %*

* : p <= 0.05, **: p <= 0.01

 

Table 4: Relative increase of relative weights in males 

Relative weights	Males
Group (mg/m³)	1 (25)	2 (75)	3 (225)
Epididymides	88 %**	94 %	94 %

*: p <= 0.05, **: p <= 0.01

 

Table 5: Relative increase of relative weights in females

Relative weights	Females
Group (mg/m³)	1 (25)	2 (75)	3 (225)
Thymus	129 %*	96 %	107 %

*: p <= 0.05, **: p <= 0.01

 

Weight changes in thymus, spleen and liver were regarded as incidental as no dose response relationship and/or histopathological correlates were detected.

The decrease in epididymis and testis weights was considered to be treatment-related even in the absence of a clear dose response relationship or, in the case of the testis, no changes in relative weights as there was a histopathological correlate.

All other mean absolute or relative weight parameters did not show significant differences when compared to the control group 0.

 

Histopathology

Treatment-related findings were observed in level I of larynx, and level I and II of nasal cavity, in males and females as well as in epididymis and testis in male animals with incidences and grading shown in the table below:

 

Larynx

 Treatment – related findings in the larynx were epithelial alteration characterized by slight modification of epithelial cells (i.e., three to four cell layers, focally flattened and stratified) indicating beginning metaplastic transformation and minimal to slight squamous metaplasia (characterized by three to four cell layers of flattened, stratified epithelium with no signs of keratinization and only affecting the epiglottis when graded minimal and up to five cell layers of flattened, stratified epithelium, occasionally minimal focal keratinization, with/without focal desquamation of superficial cells when graded slight).

 

Table 6: Incidence and grading of histological findings in larynx

	Male animals				Female animals
Test group (mg/m³)	0 (0)	1 (25)	2 (75)	3 (225)	0 (0)	1 (25)	2 (75)	3 (225)
No. of animals	10	10	10	10	9	10	10	10
Epithelial alteration	1	6	8	5	1	6	8	8
Grade 1	1	6	8	4	1	6	4	8
Grade 2				1			4
Metaplasia, squamous			2	5			1	2
Grade 1			2	4			1	2
Grade 2				1

 

Nasal cavity, level I

The following treatment- related findings were noted in the nasal cavity:

Degeneration / regeneration of transitional and respiratory epithelium was characterized by variable vacuolation, presence of few apoptotic bodies, minimal infiltrates of inflammatory cells, increased size and basophilia of nuclei and minimal disorganization of cells.

Metaplasia, squamous was characterized by flattened epithelial cells with variably present minimal keratinization.

 

Table 7: Incidence and grading of histological findings in nasal cavity

	Male animals				Female animals
Test group (mg/m³)	0 (0)	1 (25)	2 (75)	3 (225)	0 (0)	1 (25)	2 (75)	3 (225)
No. of animals	10	10	10	10	9	10	10	10
Degeneration /regeneration transitional epithelium				2			1	8
Grade 1				2			1	7
Grade 2								1
Metaplasia, squamous, transitional epithelium				1				3
Grade 1				1				3
Degeneration /regeneration respiratory epithelium				2
Grade 1				2

 

Nasal cavity, level II:

One male test group 3 (225 mg/m³) animal (No 134) showed minimal degeneration / regeneration of the olfactory epithelium, in level I it showed degeneration/regeneration of the transitional epithelium)

 

Testes

Tubular degeneration was observed in a higher incidence in treated test groups. This finding was characterized by randomly affected (not stage specific) tubules with sloughed spermatogenic cells, vacuolation of the spermatogenic epithelium or missing germ cell layers.

 

Table 8: Incidence and grading of histological findings in testes

	Male animals
Test group (mg/m³)	0 (0)	1 (25)	2 (75)	3 (225)
No. of animals	10	10	10	10
Degeneration, tubular	4	6	7	8
Grade 1	3	2	2	3
Grade 2		3	3	4
Grade 3	1	1	2	1
Sperm plug				1
present				1

 

Epididymides

Debris in the epididimydes was characterized by sloughed spermatogenic cells and noted with increased incidence in treated animals.

 

Table 9: Incidence and grading of histological findings in epididymides

	Male animals
Test group (mg/m³)	0 (0)	1 (25)	2 (75)	3 (225)
No. of animals	10	10	10	10
Debris	2	4	4	7
present	2	4	4	7

 

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

 

Fertility

The male partner of the mating pair (Nos. 17/117), which did not produce offspring showed testicular degeneration and debris in the epididymis which might have impaired fertility even if animals with similar findings in the same group did produce offspring.

Conclusions:

Inhalation exposure to the target substance BDEA is predicted to be of the same or even weaker potency than that of DBEA, if a study with BDEA with the same dose levels was conducted. The same conclusion is thus drawn for BDEA: dibutylethanolamine to maximum of 236.3 mg/m³ during 2 weeks premating, mating, gestation period did not cause any adverse effect with regard to reproductive and developmental parameters, but only transiently reduced food consumption, body weight and body weight gain. No adverse parental or pup findings were evident at any concentration. In histopathology lesions of nasal epithelia was observed in animals exposed to 72.1 and 236.3 mg/m³ test substance. Thus, concerning reproductive and developmental parameters the no observed adverse effect concentration (NOAEC) for dibutylethanolamine was determined to be >236.3 mg/m³. Considering histopathological changes the NOAEC for dibutylethanolamine was 20.6 mg/m³.

Executive summary:

To evaluate the toxicity profile of Dibutylethanolamine (DBEA) after inhalation exposure, groups of ten male and ten female Wistar rats (F0 animals) per test group were exposed nose-only to dynamic atmosphere of DBEA for 6 hours per day on each day. The duration of treatment covered a 2-week pre-mating and 2-week mating period in both sexes, 1 day post-mating in males, and the entire gestation period of the females. After the lactation period and after necropsy of the pups total all parental females were exposed to the test substance on 4 consecutive days.

 

The target concentrations were 25, 75 and 225 mg/m³ (correspond to measured concentrations 20.6, 72.1 and 236.3 mg/m³, respectively). A concurrent control group was exposed to conditioned air. For adaptation to the experimental conditions all animals were kept in glass restraining tubes identical to those used in the study and were exposed nose-only to fresh air on two days before start of the exposure period.

 

After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm was detected in the vaginal smear. F0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of post-implantation loss for all F0 females.

 

A detailed clinical observation (DCO) was performed in all animals before initial test substance exposure and, as a rule, thereafter at weekly intervals. Clinical observation was performed at least three times on exposure days and once a day during the other days.

 

Food consumption of the F0 parents was determined once weekly during premating. In dams food consumption was determined for gestation days 0 - 7, 7 - 14, 14 - 20 and lactation days 1 - 4. During the 4 exposure days after necropsy of the pups the food consumption was determined also.

 

Body weights of F0 parents were determined once a week, in males throughout the study and in females during premating. During gestation and lactation period, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20,on the day after parturitionpostnatal day [PND] 1) and on PND 4. After the pups are sacrificed the females that were exposed for 4 consecutive days were weight once before the exposure period and once on the last exposure day.

 

A functional observational battery (FOB) was performed and motor activity was measured in 5 parental males and females per group. The FOB of the female animals was on study day 12. The male animals were performed at the end of the exposure period on study day 26.

 

The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1 and on PND 4. Their viability was recorded. At necropsy on PND 4, all pups were sacrificed with CO₂, under isoflurane anesthesia, and examined macroscopically for external and visceral findings.

 

Clinico-chemical and hematological examinations were performed in 5 animals per sex and group towards the end of the exposure period.

 

A complete necropsy including gross pathological evaluation and weighing of selected organs was performed. Organs and tissues were examined histopathologically as required by the corresponding test guidelines.

 

Results

Test group 3 (225 mg/m³)

 

Significantly decreased food consumption during pre-mating (p < 0.05) in male animals between study days 0-7 (-9 %) and 7-14 (-11 %) and in female animals between study days 0-7 (-7 %) and 7-14 (-5^%);

Significantly decreased food consumption during gestationin female animals during the whole period (-11 %,p < 0.05);

Significantly lowered mean body weights F0 females on gestation days (GD) 7 (-5.7 %, p < 0.05) and 14 (5.2 %, p < 0.05);

Significantly reduced body weight gain of the F0 male animals during premating period (-12.9 g versus -1.8 g in the control, p < 0.05);

Significantly lowered mean body weight gain of F0 female animals from gestation day 0 to 7 (-37 %, p < 0.01).

 

Nasal cavity, level I

 

Degeneration /regeneration transitional epithelium in 2/10 male (graded minimal) and 8/10 female animals (graded minimal in 7/10 and slight in 1/10 animals);

Metaplasia, squamous, transitional epitheliumgraded minimal in 1/10 male and 3/10 female animals.

Degeneration /regeneration respiratory epithelium in 2/10 male animals graded minimal;

 

Nasal cavity, level II

 

Degeneration /regeneration olfactory epithelium graded minimal in one male animal.

 

Test group 2 (75 mg/m³)

Significantly decreased food consumption during gestation in female animals between study days 0 - 7 (-9 %, p < 0.05).

 

Nasal cavity, level I

 

Degeneration /regeneration transitional epithelium in one female animal (graded minimal)

 

Test group 1 (25 mg/m³)

 

No adverse effect observed

 

Conclusion

Inhalation exposure to dibutylethanolamine to maximum of 236.3 mg/m³ during 2 weeks premating, mating, gestation period did not cause any adverse effect with regard to reproductive and developmental parameters, but only transiently reduced food consumption, body weight and body weight gain. No adverse parental or pup findings were evident at any concentration.In histopathology lesions of nasal epithelia was observed in animals exposed to 72.1 and 236.3 mg/m³ test substance. Thus, concerning reproductive and developmental parametersthe no observed adverse effect concentration (NOAEC) for dibutylethanolamine was determined to be >236.3 mg/m³. Considering histopathological changes the NOAEC fordibutylethanolaminewas 20.6 mg/m³.

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

20.6 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP guideline study on a structurally similar chemical.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

There are no repeated dose toxicity studies available for Butyldiethanolamine. Therefore, the data on its structural analogues Dibutylethanolamine (DBEA, CAS 102-81-8) and Butylethanolamine (CAS 111-75-1) are used to evaluate this endpoint.

 

Repeated dose toxicity: oral

 

Key information:

In a GLP-compliant 90-day study according to OECD Guideline 408 Dibutylethanolamine (DBEA) was administered orally by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0 mg/kg bw/day (test group 0), 15 mg/kg bw/d (test group 1), 50 mg/kg bw/d (test group 2) and 150 mg/kg bw/d (test group 3) over a period of 3 months. Corn oil served as vehicle. Control animals were dosed daily with the vehicle only. With regard to clinical examinations, signs of general systemic toxicity were not observed even at a dose level of 150 mg/kg bw/d of DBEA. In addition, no test substance-related effects on estrous cycle length and the number of cycles were obtained. Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose of 150 mg/kg bw/d. The unique potassium increase observed for male animals of test group 3 (150 mg/kg bw/d) was considered to be potentially treatment-related but regarded to be non-adverse (ECETOC Technical Report No. 85, 2002). A relation to the kidney-related findings observed in these male animals of the test group 3 was excluded. Regarding pathology, neither treatment-related weight changes nor gross lesions were observed. The target organ were the kidneys. In 8 out of 10 males and 1 out of 10 females of test group 3, macrovesicular vacuolation of the epithelial cells of the collecting ducts was observed. No signs of necrosis, degeneration or inflammation were present. Therefore, under the conditions of the present study the NOAEL was 50 mg/kg bw/d for male and female Wistar rats.

 

In a GLP-compliant 90-day study according to OECD Guideline 408 N-Butylaminoethanol (BEA) was administered orally by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0, 60, 120 and 240 mg/kg bw/d over a period of 3 months. RO water served as vehicle. Control animals were dosed daily with the vehicle only. Mortality was observed in one high dose female rat on day 85. However, there were no treatment related changes observed in body weight, food consumption, clinical pathology, gross and microscopic examination, in female rats of high dose group. Hence, this death was considered not to be related to treatment. Mild salivation in male and female rats and gasping in male rats of high dose treated group were observed on multiple days of study. Salivation observed in high dose group was considered as a response to the dose solution and considered as non-adverse in nature. Gasping was not supported with any microscopic change observed in respiratory organs. Hence, it was considered as possibly related an irritancy response to the test material and non-adverse in nature. During this study, there was no treatment related adverse change observed in the body weight, food consumption, clinical pathology, and histopathology, up to dose level 240 mg/kg bw/day. Based on above results, it is concluded that BEA did not produce any relevant toxicity or test-item related adverse effect up to 240 mg/kg bw/day when administered orally through gavage for 90 consecutive days in Wistar rats. Therefore, the No Observed Adverse Effect Level (NOAEL) of BEA is 240 mg/kg bw/day under the conditions and procedure followed in this study.

 

Supporting information:

In the 28-day study (according to OECD 407; Ministry of Health, Labour and Welfare, Japan, 2004), the NOAEL for Dibutylethanolamine (DBEA) was 100 mg/kg bw/day for both males and females and the LOAEL was set to 400 mg/kg bw due to mortality and severe findings in nervous system, liver and kidney. There were significant increases in relative liver weights in males and females of the highest dose group. There were also significant increases in relative kidney weight for males as well as in actual and relative adrenal weights for females. In animals that died on study and in those that survived until scheduled necropsy, increases in liver and kidney sizes were noted. Microscopically, centrilobular hypertrophy of hepatocytes and vacuolization of the epithelial cells of the collecting ducts of kidneys were observed. The other target organs were adrenals, spleen, lungs and thymus. Concerning parameters of clinical chemistry, males and females in the 400 mg/kg bw group showed a significant decrease in chlorine level. In the 400 mg/kg bw group, the sodium level significantly decreased for males, while females developed tendencies toward significantly increased levels of total cholesterol and glucose as well as declined cholinesterase activity in which further data about inhibition of choline esterase activity by butyl ethanolamines are presented) . At the same time, females in the 100 mg/kg bw group showed a significant increase in triglyceride level, and also females in the 400 mg/kg bw group had a tendency toward a high value. For the cases autopsied at the end of the recovery test period, there were no other significant changes, comparing the control with each of the other administration groups.

 

In a supporting 30-day feeding study there was no evidence of a cumulative effect (equivalent or similar to OECD 407; Cornish et al., 1969) of Dibutylethanolamine (DBEA). Initial weight losses varied directly with the concentration of the test substance in the water. Once an adjustment of water and food intake had been made, animals gained weight at an approximate normal rate over the remainder of the 30-day period. All haematological data was in the normal range, although there was considerable variation in the white cell count among the various groups of treated animals. The significant increase in kidney to body weight ratios at the high dose level cannot be directly interpreted as an effect on kidney as histologic examination of the tissues including kidney, indicate that these organs were essentially normal at the 30-day feeding period. The kidney weights of treated and control groups were comparable at the end of the thirty day feeding period, however, treated animals had not regained their initial weight, thus the kidney to body weight ratios remained elevated. Thus, the NOAEL was set to 430 mg/kg bw/day for male animals and to 330 mg/kg bw/day for female animals (highest dose tested).

 

In a GLP-compliant Combined repeated dose study with reproductive toxicity screening according to OECD Guideline 422 N-Butylaminoethanol (BEA) was administered by gavage to male and female Wistar rats over 28 days and beyond (during pre-mating, mating, gestation, and lactation periods). 15 rats / sex / group were treated with N-Butylaminoethanol receiving the following dose levels: G1 (control): 0 mg/kg bw/day; G2 (low dose): 60 mg/kg bw/day; G3 (mid dose): 120 mg/kg bw/day; G4 (high dose): 240 mg/kg bw/day. Males were treated two weeks prior to mating during the mating (two weeks) and until approximately 73 % of the females had delivered. Total number of treatment days was 50. Females were treated two weeks prior to mating, the variable time to conception, the duration of pregnancy and fourteen days after delivery. The following parameters were analysed: mortality/morbidity, clinical signs, body weight, food consumption, neurobehavioural observation (NBO), functional observational battery (FBO), blood collection, clinical pathology, thyroid hormone, organ weights, histopathology. Males were sacrificed when ~80 % of females had delivered, females in LD15 and pups on PND 14. In the low and mid dose group no signs of toxicity on clinical sign, mortality, body weight, body weight gain, feed consumption, NBO, FOB, clinical pathology, and organ weight (absolute and relative) were observed. No treatment related macroscopic and microscopic lesions were observed. In the high dose group no sign of toxicity on mortality, NBO, FOB, organ weight, clinical pathology (absolute and relative) was observed. No treatment related macroscopic and microscopic lesions were observed. Salivation was toxicologically insignificant, hence considered as non-adverse in nature. Snuffles were considered as test item related effect but non-adverse in nature due to absence of effect in remaining rats and no gross observations in lung. In male rats, treatment related effects were observed on body weight (< 10 %), terminal body weight (< 9 %), overall body weight gain (~< 60 %), and overall food consumption (< 12 %). These endpoints were consistently decreased during treatment period. Hence, changes were considered as treatment related adverse in nature. No effect on endocrine sensitive endpoints. 

Based on results of this study, it was concluded that the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was 120 mg/kg bw/day for males based on effects on body weight, body weight gain and food consumption in male rats receiving 240 mg/kg bw/day. The 240 mg/kg bw/day was the NOAEL for female rats based on no effect on systemic endpoints.

 

Repeated dose toxicity: inhalation

 

Key information:

To evaluate the toxicity profile of Dibutylethanolamine (DBEA) after inhalation exposure, groups of ten male and ten female Wistar rats (F0 animals) per test group were exposed nose-only to vapours of DBEA at target concentrations of 25, 75 and 225 mg/m³ (correspond to measured concentrations 20.6, 72.1 and 236.3 mg/m³, respectively) for 6 hours per day on each day (BASF SE, 2013, Report No.87R0286/05I017, GLP, OECD 422).The duration of treatment covered a 2-week pre-mating and 2-week mating period in both sexes, 1 day post-mating in males, and the entire gestation period of the females. After the lactation period and after necropsy of the pups total all parental females were exposed to the test substance on 4 consecutive days. The total exposure amounts to 28 and 50 day in males and females, respectively. 

After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm was detected in the vaginal smear. F0 animals were examined for their reproductive performance including determination of the number of implantation sites and the calculation of post-implantation loss for all F0 females. A detailed clinical observation (DCO) was performed in all animals before initial test substance exposure and, as a rule, thereafter at weekly intervals. Clinical observation was performed at least three times on exposure days and once a day during the other days. Food consumption of the F0 parents was determined once weekly during premating. In dams food consumption was determined for gestation days 0 - 7, 7 - 14, 14 - 20 and lactation days 1 - 4. During the 4 exposure days after necropsy of the pups the food consumption was determined also. Body weights of F0 parents were determined once a week, in males throughout the study and in females during premating. During gestation and lactation period, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, on the day after parturition postnatal day [PND] 1 and on PND 4. After the pups are sacrificed the females that were exposed for 4 consecutive days were weight once before the exposure period and once on the last exposure day. A functional observational battery (FOB) was performed and motor activity was measured in 5 parental males and females per group. The FOB of the female animals was on study day 12. The male animals were performed at the end of the exposure period on study day 26. The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1 and on PND 4. Their viability was recorded. At necropsy on PND 4, all pups were sacrificed with CO2, under isoflurane anaesthesia, and examined macroscopically for external and visceral findings. Clinico-chemical and haematological examinations were performed in 5 animals per sex and group towards the end of the exposure period. A complete necropsy including gross pathological evaluation and weighing of selected organs was performed. Organs and tissues were examined histopathologically as required by the corresponding test guidelines. 

No relevant clinical signs of toxicity were observed in exposed parental animals during the premating and mating phases in all test groups. In the highest dose group, food consumption was reduced slightly in male and female animals during premating and mating period. Consistently to the reduced food consumption, slightly reduced body weight gain was observed. During the whole gestation period, food consumption was significantly reduced in the highest dose group (225 mg/m³) and in the mid dose group (75 mg/m³) during the first week of gestation. The mean body weights of the highest dose group females were significantly reduced on gestation day 7 and 14. These unspecific findings might be an indirectly consequence of the local irritation in the nasal cavity. They are considered to be treatment-related and adverse. The detailed clinical observations did not reveal any abnormalities in male and female animals of all test groups. No test substance-related or spontaneous findings were observed in male and female animals of all test groups during FOB and MA. Regarding haematology and clinical chemistry, no treatment-related, adverse effects were observed up to a dose of the compound of 225 mg/m³.

Generally, no toxicologically relevant reproductive or developmental difference was observed between animals exposed to measured concentrations of 20.6, 72.1 and 236.3 mg/m³ and controls. These concentrations of dibutylethanolamine did not adversely impact the reproduction of these rats, nor did treatment impact delivery and pup viability. Furthermore, none of the F1 generation pups showed any evidence of developmental toxicity in response to dibutylethanolamine.

At necropsy, target organs were the level I of the larynx, and level I and II of the nasal cavity, in males and females as well as epididymis and testis in male animals. Laryngeal epithelial alteration in many male and female animals of all treated test groups (25, 75, 225 mg/m³, correspond to treated groups 1, 2 and 3) and minimal to slight squamous metaplasia without inflammation in male and female animals of test groups 2 and 3 were regarded as non-adverse according to the literature (Kaufmann et al., 2009). Degeneration / regeneration of nasal epithelia in level I in 2/10 males (transitional and/or olfactory) and 8/10 females (transitional) as well as squamous metaplasia of transitional epithelium in one male and 3/10 female animals of test group 3 (225 mg/m³) and in one female of test group 2 (transitional) (75 mg/m³) as well as degeneration / regeneration of the olfactory epithelium in level II noted in one test group 3 (225 mg/m³) male was regarded as adverse. 

Tubular degeneration of the testis and resulting debris in the epididymides were observed in control and treated animals of all test groups. The incidence of these findings was, however, higher in treated animals correlating with decreased absolute and relative epididymides weights and absolute testis weights. (Tubular degeneration: control [4/10] test group 1 [6/10] test group 2 [7/10] test group 3 [8/10]; Debris in the epididymides: control [2/10] test group 1 [4/10] test group 2 [4/10] test group 3 [7/10]). This effect in the testis is likely due to the technical exposure scenario (heat [e.g. Brock WJ et al., 1996] and possibly evading movements by the animals leading to pressing backwards in tubes), rather than being a direct effect of the test substance as this finding is also found in control animals. These findings did generally not affect fertility with one possible exception in one group 1 male animal (No. 17). All findings in testes and epididymides were assessed as treatment - related and adverse but not substance-related. 

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

In conclusion, inhalation exposure to dibutylethanolamine to maximum of 236.3 mg/m³ during 2 weeks premating, mating, gestation period did not cause any adverse effect with regard to reproductive and developmental parameters, but only transiently reduced food consumption, body weight and body weight gain. No adverse parental or pup findings were evident at any concentration. In histopathology lesions of nasal epithelia was observed in animals exposed to 72.1 and 236.3 mg/m³ test substance. Thus, concerning reproductive and developmental parameters the no observed adverse effect concentration (NOAEC) for dibutylethanolamine was determined to be >236.3 mg/m³. Considering histopathological changes the NOAEC for dibutylethanolamine was 20.6 mg/m³.

 

Supporting information:

In a supporting study, male rats exposed for 6 months to 22 ppm (= 156 mg/m³) of 2-dibuthylaminoethanol (DBEA) (Cornish et al., 1969). The animals were comparable to controls throughout the exposure period (Cornish et al., 1969). No animal died. No untoward symptoms were noted in the animals during the exposure period. By the end of the 4th week of exposure, total body weight gain was comparable to the controls. Serum bilirubin levels were higher than controls in 3 of 5 rats in the animals that were sacrificed after 4 weeks of exposure. Kidney to body weight ratios were somewhat elevated at the end of the first week on this schedule. No further significant changes occurred in the animals throughout the six month exposure period, and at sacrifice all biological measurements were comparable to those of the control group. Histological sections of the organs from animals exposed for 6 months were not different from the controls. No further details were given. The NOAEC was determined to be 156 mg/m³.

 

In another supporting study, which was designed as a range-finding study, exposure to high levels (70 ppm, 495 mg/m³) of 2-dibutylaminoethanol (DBEA) produced tremors and convulsive seizures in rats (Cornish et al., 1969). In addition, visual signs of eye and nasal irritation were evident in those animals exposed to approximately 70 ppm, 6 hours a day, for a 5 day period. One animal died and great weight losses occurred in these animals. A one week exposure to 33 ppm (234 mg/m³) resulted in a lack of growth, but no other significant findings. Slightly elevated bilirubin levels in some, but not all rats, were noted at the end of 5 days of exposure to 70 ppm. A similar elevation was not evident in the 33 ppm group. There was no histological evidence of tissue damage in these groups of animals. A NOAEC of 33 ppm and a LOAEC of 70 ppm were determined.

Justification for classification or non-classification

Based on these data, the substance does not need to be classified and labelled for STOT-RE according to Regulation (EC) No1272/2008.