2,2'-butyliminodiethanol

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

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

 

BEA (CAS 111-75-1)
- 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

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type

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
There is evidence that a link exists also between liver/ kidney damage observed in a number of studies with ethanolamines (as a consequence of a perturbed choline-homeostasis) and the reproductive toxicity. Ethanolamines DEA, DEEA and MMEA produced toxicity effects to male reproductive system, while TEA, MEA, MDEA and MMEA mainly reduced number of implantation sites and/or produced pre- or post-implantation losses. DMAE and MMEA did not produce reproductive effects: there were no treatment-related effects of treatment on gonads or on any gestational parameters, including pre- and post implantation loss or sex ratio. However, the authors (Zahniser et al., 1978) linked the deaths of pups from mothers fed choline deficient diets supplemented with DMAE or MMEA to perturbed choline uptake and phospholipid synthesis in new born rats leading to respiratory distress and deaths (please refer to the full version of read-across statement). In contrast, DBEA did not produced reproductive toxicity effect in the present study, although the reduction in testes and epididymitis weights and histopathological changes in the testes and epididymitis were observed. Although the findings with DBEA were assessed as treatment-related (due to the exposure type: head-nose) but not substance-related, their incidence was higher in the treatment groups. The systemic toxicity with the reproductive findings induced by ethanolamines is considered to be as a secondary consequence of choline-homeostasis perturbation. The results of studies with ethanolamines mentioned above indicate, that both choline and betaine are required by the embryo and both molecules play an important role and ensure embryonic survival (e.g. as osmolyte and methyl-group donor). Ethanolamines administration during pregnancy can perturb choline homeostasis affecting a lot of maturation systems of embryos i.e. cellular uptake of choline, its metabolism pathway, synthesis of aberrant phospholipids

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 and reproductive 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. Reproductive function was not impaired. 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 among butyl derivatives of ethanolamines and, if BDEA was tested in a reproductive/developmental screening test 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 or reproductive/developmental 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 including reproductive 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

Positive control:

None

Clinical signs:

no effects observed

Description (incidence and severity):

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.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no test substance-related or spontaneous mortalities in any of the groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Food consumption and 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.

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).

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 and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption and 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 efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Endocrine findings:

not specified

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

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:

not specified

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

increased weight of testes and epididymides

Reproductive performance:

no effects observed

Description (incidence and severity):

For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100 % in all groups including the controls.

Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter.

One low-concentration male (25 mg/m³ - No. 17) did not generate implants in the mated female (No. 117). The animal No. 17 showed some changes in testes and epididymides, which might have impaired fertility, even if animals with similar findings in the same group did produce offspring.

Even though, the male fertility index ranged between 90 % and 100 % without showing any relation to exposure concentration. This reflects the normal range of biological variation inherent in the strain of rats used for this study.

The female mating index calculated after the mating period for F1 litter was 100 % in all test groups.

The mean duration until sperm was detected (GD 0) varied between 2.1 and 3.1 days without any relation to exposure concentrations.

All sperm positive rats delivered pups or had implants in utero with the following exception:
- Low-dose female No. 117 (mated with male No. 17) did not become pregnant.

The fertility index varied between 90 % in test group 1, 100 % test groups 2, 3 and in control. These values reflect the normal range of biological variation inherent in the strain of rats used for this study.

The non-pregnant female did not have any relevant gross lesions .

The mean duration of gestation was similar in all test groups (i.e. between 21.8 and 22.1 days).

The gestation index were 100 % in all test groups as well as control.

Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the controls, taking normal biological variation into account (10.4 / 11.9 / 11.9 and 10.5 implants/dam in test groups 0-3 (0, 25, 75 and 225 mg/m³). There were no statistically significant differences in post-implantation loss between the groups (6.5 % / 7.7 % / 7.2 % / 9.7 %), and the mean number of F1 pups delivered per dam remained unaffected (9.7 / 11.0 / 11.0 and 9.4 pups/dam at 0, 25, 75 and 225 mg/m³).

The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 100 % (test group 1, 2 and control), 97.9 % (test group 3). Two stillborn pups (2.1 %) were only in test group 3 animals. However, this is within the normal range of biological variation inherent in the strain of rats used for this study.

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
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 FOOD CONSUMPTION (PARENTAL ANIMALS)
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).

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.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100 % in all groups including the controls.

Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter.

One low-concentration male (25 mg/m³ - No. 17) did not generate implants in the mated female (No. 117). The animal No. 17 showed some changes in testes and epididymides, which might have impaired fertility, even if animals with similar findings in the same group did produce offspring.

Even though, the male fertility index ranged between 90 % and 100 % without showing any relation to exposure concentration. This reflects the normal range of biological variation inherent in the strain of rats used for this study.

The female mating index calculated after the mating period for F1 litter was 100 % in all test groups.

The mean duration until sperm was detected (GD 0) varied between 2.1 and 3.1 days without any relation to exposure concentrations.

All sperm positive rats delivered pups or had implants in utero with the following exception:
- Low-dose female No. 117 (mated with male No. 17) did not become pregnant.

The fertility index varied between 90 % in test group 1, 100 % test groups 2, 3 and in control. These values reflect the normal range of biological variation inherent in the strain of rats used for this study.

The non-pregnant female did not have any relevant gross lesions .

The mean duration of gestation was similar in all test groups (i.e. between 21.8 and 22.1 days).

The gestation index were 100% in all test groups as well as control.

Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the controls, taking normal biological variation into account (10.4 / 11.9 / 11.9 and 10.5 implants/dam in test groups 0-3 (0, 25, 75 and 225 mg/m³). There were no statistically significant differences in post-implantation loss between the groups (6.5 % / 7.7 % / 7.2 % / 9.7 %), and the mean number of F1 pups delivered per dam remained unaffected (9.7 / 11.0 / 11.0 and 9.4 pups/dam at 0, 25, 75 and 225 mg/m³).

The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 100 % (test group 1, 2 and control), 97.9 % (test group 3). Two stillborn pups (2.1 %) were only in test group 3 animals. However, this is within the normal range of biological variation inherent in the strain of rats used for this study.


ORGAN WEIGHTS (PARENTAL ANIMALS)
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 (PARENTAL ANIMALS)
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.
The mating pair (Nos. 17/117), which did not produce offspring did not show relevant gross lesions.

HISTOPATHOLOGY (PARENTAL ANIMALS)
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.

OTHER FINDINGS (PARENTAL ANIMALS)
No treatment-related changes among hematological parameters were observed. 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.
- Functional Observational Battery (FOB) nd Motor activity measurement (MA): 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. No test substance-related or spontaneous findings were observed in male and female animals of all test groups (please refer to section 7.5.2. for detailed information).

Dose descriptor:

NOAEC

Effect level:

> 236.3 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect with regard to reproductive and developmental parameters.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no test substance related adverse clinical signs observed in any of surviving F1 generation pups of the different test groups.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

The mean number of delivered F1 pups per dam and the rates of liveborn were evenly distributed about the groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study. The low rate of stillborn pups in test group 3 was within the biological variation of this stain and was considered to be not related to the test substance.
The viability index indicating pup mortality during lactation (PND 0-4) varied between 98.5 % (test group 2), 99.1 % (test group 1) and 100 % (test group 3 and control) without showing any association to the treatment.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test compound-related influence on F1 pup body weights and pup body weight change were noted in all test groups.
Two female runts were seen in the control and two female runts in test group 3 (225 mg/m³). As there were as many runts in the control as in test group 3, this finding was considered to be incidental and not substance-related.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature .

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No findings were observed at gross necropsy in any male or female pups of all test groups. One male and one female pup of test group 2 (75 mg/m³) could not be assessed because they had been cannibalized. Because no pup was cannibalized in high concentration groups, this finding is considered as incidental.

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
The mean number of delivered F1 pups per dam and the rates of liveborn were evenly distributed about the groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study. The low rate of stillborn pups in test group 3 was within the biological variation of this stain and was considered to be not related to the test substance.
The viability index indicating pup mortality during lactation (PND 0 - 4) varied between 98.5 % (test group 2), 99.1 % (test group 1) and 100 % (test group 3 and control) without showing any association to the treatment.

CLINICAL SIGNS (OFFSPRING)
There were no test substance related adverse clinical signs observed in any of surviving F1 generation pups of the different test groups.

BODY WEIGHT (OFFSPRING)
No test compound-related influence on F1 pup body weights and pup body weight change were noted in all test groups.
Two female runts were seen in the control and two female runts in test group 3 (225 mg/m³). As there were as many runts in the control as in test group 3, this finding was considered to be incidental and not substance-related.

SEXUAL MATURATION (OFFSPRING)
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature .

GROSS PATHOLOGY (OFFSPRING)
No findings were observed at gross necropsy in any male or female pups of all test groups. One male and one female pup of test group 2 (75 mg/m³) could not be assessed because they had been cannibalized. Because no pup was cannibalized in high concentration groups, this finding is considered as incidental.

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

> 236.3 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effect with regard to reproductive and developmental parameters.

Reproductive 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.

 

Measurements concerning 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.

 

Clinical examinations and examination of reproductive performance

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.

 

Food consumption

In high concentration (225 mg/m³) food consumption during pre-mating wassignificantly 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 thehigh 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).

 

Body weight

- 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;

- 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;

- 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);

- Lactation: The mean body weights of F0 female animals were not statistically significantly different from the control group 0 during lactation period;

- After PND 4: The mean body weights of F0 female animals after PND 4 were not statistically different to the controls;

 

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.

 

Detailed clinical observation

The detailed clinical observations did not reveal any abnormalities in male and female animals of all test groups.

 

Functional Observation Battery (FOB) and motor activity measurement (MA)

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. No test substance-related or spontaneous findings were observed in male and female animals of all test groups (please refer to section 7.5.2. for detailed information).

 

Male reproduction data:

For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100 % in all groups including the controls. Fertility was proven for most of the F0 parental maleswithin the scheduled mating interval for F1 litter. One low-concentration male (25 mg/m³ - No. 17) did not generate implants in the mated female (No. 117).The animal No. 17 showed some changes in testes and epididymides, which might have impaired fertility, even if animals with similar findings in the same group did produce offspring. Even though, the male fertility index ranged between 90 % and 100 % without showing any relation to exposure concentration. This reflects the normal range of biological variation inherent in the strain of rats used for this study.

 

Female reproduction and delivery data:

The female mating index calculated after the mating period for F1 litter was 100 % in all test groups.

The mean duration until sperm was detected (GD 0) varied between 2.1 and 3.1 days without any relation to exposure concentrations.

All sperm positive rats delivered pups or had implants in utero with the following exception:

- Low-dose female No. 117 (mated with male No. 17) did not become pregnant.

The fertility index varied between 90 % in test group 1, 100 % test groups 2, 3 and in control. These values reflect the normal range of biological variation inherent in the strain of rats used for this study.

The non-pregnant femaledid not have any relevant gross lesions.

The mean duration of gestation was similar in all test groups (i.e. between 21.8 and 22.1 days).

The gestation index were 100 % in all test groups as well as control.

Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the controls, taking normal biological variation into account (10.4 / 11.9 / 11.9 and 10.5 implants/dam in test groups 0-3 (0, 25, 75 and 225 mg/m³). There were no statistically significant differences in post-implantation loss between the groups (6.5 % / 7.7 % / 7.2 % / 9.7 %), and the mean number of F1 pups delivered per dam remained unaffected (9.7 / 11.0 /11.0 and 9.4 pups/dam at 0, 25, 75 and 225 mg/m³). The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 100 % (test group 1, 2 and control), 97.9 % (test group 3). Two stillborn pups (2.1 %) were only in test group 3 animals. However, this is within the normal range of biological variation inherent in the strain of rats used for this study.

 

Haematology and clinical chemistry

No treatment-related changes among hematological and 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.

 

Pathology

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: 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 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. The test substance did not cause any adverse effect with regard to reproductive and developmental parameters.The male mating index was 100 % in all groups including the controls. The male fertility index ranged between 90 % and 100 % without showing any relation to exposure concentration. The female mating and fertility indices and duration of gestation were similar to controls. Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the controls, taking normal biological variation into account. There were no statistically significant differences in post-implantation loss between the groups and the mean number of F1 pups delivered per dam remained unaffected. The rate of liveborn pups was also not affected by the test substance. The mean number of delivered F1 pups per dam and the rates of liveborn were evenly distributed about the groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study. The viability index and sex ration of pups did not show any association to the treatment. There were no test substance related adverse clinical signs observed in any of surviving F1 generation pups of the different test groups. No test compound-related influence on F1 pup body weights and pup body weight change were noted in all test groups. No findings were observed at gross necropsy in any male or female pups of all test groups. Systemic toxicity effects were only transiently reduced food consumption, body weight and body weight gain in parental animals. In their histopathology, lesions of nasal epithelia were observed in animals exposed to 72.1 and 236.3 mg/m³ test substance. Thus, concerning systemic toxicity, reproductive and developmental parameters the NOAEC for 2-dibutylaminoethanol was determined to be 236.3 mg/m³ for male and female animals. Considering histopathological changes, the NOAEC for local effects was 20.6 mg/m³.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

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 repeated dose and reproductive toxicity screening study with BEA no significant adverse effects were noted in the female animals treated during with the test substance orally (gavage) up to the highest dose of 240 mg/kg bw/day. In male animals a reduction in body weight, body weight gain and food consumption was observed the highest dose of 240 mg/kg bw/day. No effects on male and female fertility and pup development were observed.
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 screening 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 belonging to the control, low and mid dose groups were normal throughout the study period.
Salivation and snuffles were observed in male and female rats of high dose group (see corresponding table in 'Any other information on results') . Salivation was considered toxicologically insignificant 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.
Please also refer to attached Tables below.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No treatment related mortality or morbidity was observed during the study period in control and test item treated groups.
Please also refer to attached Tables below.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males:
- The mean body weights for the low and mid dose groups were comparable with the control group. Statistically significantly lower mean body weight (<10 %) was observed in the high dose group on treatment days 22, 29, 36, 43 and 50 when compared with that of the control group. These observed changes were considered as treatment related due to the continuous reduction in body weight (please refer to corresponding table in 'Any other information on results').
- The mean body weight gain of the low and mid dose groups was comparable with the control group with the exception of statistically significant reductions in mean body weight gain observed in the low dose group during treatment days 15-22, in the mid dose group during treatment days 1-8 when compared with that of the control group. These were considered incidental and not treatment related, furthermore, changes remained below 5 % compared to concurrent control. animals
- Statistically significant reductions in mean body weight gain in the high dose group during treatment days 1-8, 15-22, 22-29 and 1-50, clearly exceeding a reduction by 10 % compared to control animals, was observed when compared with that of the control group. These observed changes in high dose group were considered as treatment related due to the continuous reduction in body weight gain.
(please refer to corresponding table in 'Any other information on results')
Please also refer to attached Tables below.

Females:
- The mean body weight of the low, mid and high dose groups was comparable with the control group.
- The mean body weight gain of the low, mid and high dose groups was comparable with the control group. Incidental reduction (statistically significant) in body weight gain in the high dose group during pre-mating day 1-8 was observed when compared with the control group.
Please also refer to attached Tables below.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males:
- The mean food consumption of the low and mid dose groups was comparable with that of the control group.
- A statistically significant reduction in food consumption was observed during treatment days 1-8, 8-15 and overall (days 1-50) in the high dose group when compared with the control group. These observed changes were considered as related to treatment in presence of notable change in body weight and body weight gain.
(please refer to corresponding table in 'Any other information on results')
Please also refer to attached Tables below.

Female:
- The mean food consumption of the low, mid and high dose groups was comparable with the control group.
- However, some incidental changes were observed, as in the mid and high dose group a statistically significant decrease in mean food consumption during pre-mating day 1-8 was observed when compared with the control group.
- A statistically significant decrease in food consumption was observed during gestation day 0-7 in the low and high dose groups when compared with the control group. Changes observed during pre-mating and gestation period were incidental in nature based on comparable food consumption during rest of treatment period. Please also refer to attached Tables below.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Test item treatment did not lead to any treatment related alteration in haematological parameters, beside of the following changes, which were in the end classified as unrelated to test item treatment:
- A statistically significant increase was noted in reticulocyte count in high dose group males. It was considered unrelated to test item treatment in absence of effects in other red blood cell related parameters (RBC, haemoglobin and haematocrit).
- A statistically significant increase was observed in lymphocytes in low dose group males which was not considered as related to test item treatment, due to absence of dose dependency.
Please also refer to attached Tables below.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Test item treatment did not lead to any treatment related alteration in clinical chemistry parameters, beside of the following:
- In high dose males, statistically significant decreases in total protein, globulin, sodium and chloride were noted. These alterations could be related to test item treatment and were not considered as adverse as values were in historical ranges except total protein in 1 male rat of the high dose group. Treatment related histopathological lesions were not observed in liver and kidneys which indicates that the effects were not adverse.
- Statistically significant increase was noted in urea and BUN in low dose females which was not considered as related to test item treatment, due to absence of dose dependency.
Please also refer to attached Tables below.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urinalysis did not reveal any test item related effect.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

NOB:
- Home Cage Observations: All rats from treatment and control groups revealed normal postures when 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.
- Handling Observations: NBO performed during handling of rats did not reveal any abnormality related to treatment. All rats revealed normal behavior during removal (very easy - animals sit quietly) and handling (easy - alert, limbs put against the body). None of the rats showed lacrimation, salivation or piloerection. Eyelids were wide open in all rats. Eye and skin examination of rats from all groups did not reveal any abnormality.
- Open Field Observations: All rats from treatment and control groups showed normal gait, mobility, arousal and respiration during the two minutes observation period. Clonic and tonic movements, stereotypy and bizarre behavior were absent. No treatment related significant changes were observed in vocalisation, rearing, and urination and defecation counts of male and female rats from treatment groups when compared with the control group. Some incidental changes were noted in male rats, including statistically significant decrease in rearing count at weeks 2, 3 and 4 in the high dose group and defecation count in the low dose group at week 7 when compared with the control group. Some incidental changes were noted in female rats, viz., a statistically significant decrease in rear count at week 3 in the high dose group and urination count at pre-treatment week in the low, mid and high dose group when compared with the control group. The findings were considered as incidental based on the lack of findings in other related parameters (motor activity). During 3 minutes of open field observation, snuffle in male rat No 91 and 94 at week 5, 6, 7, and last week of terminal sacrifice whereas in female rat No 119 at week 5, 6, and 7 was observed.
Snuffles were considered as test item related effect (as it was also observed during daily clinical signs observation, as well) but non-adverse in nature due to absence of effect in remaining rats and no gross observations in lung.
Please also refer to attached Tables below.

FOB:
- The motor activity counts of male and female rats from all treatment groups were comparable with the control group.
- Sensory reactivity parameters viz., approach response, touch response, click response, pupil response, tail pinch response and air righting reflex in all treatment groups were comparable with the control group.
- The hindlimb and forelimb grip strength values of rats from treatment groups were comparable with the control group.
- The hindlimb foot splay values of rats from treatment groups were comparable with the control group.
Please also refer to attached Tables below.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Treatment with the test item did not cause histopathological alterations in any of the organs, beside of the following:
- The microscopic lesions observed in some of the organs were at lower rate of occurrence in control and high dose group and with minimal to mild severity. Hence, those lesions were spontaneous or incidental in nature representing the normal physiological/metabolic or congenital changes and not treatment related. Moreover, in organs showing statistical alterations in organ weight (heart, adrenals, prostate, testes, seminal vesicles with coagulating gland and thymus), did not reveal any test item related lesions and observed lesions were incidental or spontaneous in nature.
Please also refer to attached Tables below.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

- Thyroid Hormone Analysis: Thyroid hormone i.e., T3, T4 and TSH serum levels were comparable in males, females and pups (PND 4 and 14) when compared with that of the control group.
- External examination of rats did not reveal any abnormality.
- Home Cage Observations and Handling Observations: no abnormalities related to treatment.
- Open Field Observations: some incidental changes (statistically significant) decrease in rear count. The findings were considered as incidental based on the lack of findings in other related parameters (motor activity). Snuffles were considered as test item related effect (as it was also observed during daily clinical signs observation, as well) but non-adverse in nature due to absence of effect in remaining rats and no gross observations in lung.
- Motor Activity, Sensory Reactivity Measurements, Grip Strength, Hindlimb Foot Splay: no effects observed.
Please also refer to attached Tables below.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

The mean number of oestrous cycles and mean number of oestrous cycle lengths were comparable between test item groups and the control group.
Please also refer to attached Tables below.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

Male fertility index, female fertility index, gestation index, parturition index, percentage of pregnant rats and mating index were comparable with the control group. The duration of the gestation days and pre-coital interval were comparable among all the test item treated groups with the control group. No statistically significant change in the duration of gestation days and pre-coital interval were observed between all test item treated groups and the control group.
The mean number of implants, pre and post-natal loss, mean percentage of pre and post-natal loss were comparable between all test item treated groups and the control group. The percent pre-natal loss was higher in high dose group compared to control group though it lacks statistical significance. This was due to all pups belonging to rat No 118 were found either dead or cannibalised on PND 0. The percent post-natal loss was higher in high dose group compared to control group though it lacks statistical significance. This was due to the complete litter loss by PND 1, of rat N° 108, belonging to high dose group. These changes were limited to one rat (and were not spread across the group), hence the observed changes were considered unrelated to treatment and incidental in nature.
Please also refer to attached Tables below.

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

120 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

240 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: systemic toxicity

Clinical signs:

no effects observed

Description (incidence and severity):

All pups belonging to the control and test item treated groups were normal throughout the study period.
Please also refer to attached Tables below.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

There was no treatment related pup mortality and mortality index observed in test item treated groups when compared with that of the control group. Except, a statistically significant increase in the mortality index of female pups belonging to high dose group was observed during post-natal day 0-4 when compared with the control group. This was due to the complete litter loss of 1 rat (rat N° 108) out of 11 rats of high dose group dams i.e., changes were limited to one rat (and were not spread across the group), hence the observed changes were considered unrelated to treatment and incidental in nature.
Please also refer to attached Tables below.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight and body weight gain of males, females and combined males and females were comparable between all test item treated groups and the control group.
Please also refer to attached Tables below.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

The ano-genital distance of male, female and composite of male and female pups was comparable with the control group.
Please also refer to attached Tables below.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No male pups belonging to either the control or test item treated groups showed retention of nipples.
Please also refer to attached Tables below.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Terminal body weight and thyroid gland weights (both absolute and relative) of PND 14 pups (male and female) were comparable with the control group.
Please also refer to attached Tables below.

Gross pathological findings:

not specified

Histopathological findings:

not specified

Other effects:

no effects observed

Description (incidence and severity):

Litter Size and Male Sex Ratio: No treatment related differences were observed in mean counts of male pups, female pups, and combined male and female pups when compared with the control group except for a statistically significant decrease in mean count of males on post-natal day 0 in high dose group. This change was considered as unrelated to treatment as combined male and female pups counts was comparable with that of the control group. The male/female sex ratio of test item treated groups was comparable to that of the control group.
External examination of pups did not reveal any abnormality
Please also refer to attached Tables below.

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

240 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

other: Post-natal growth

Reproductive effects observed:

no

Clinical signs

Clinical Sign of Toxicity	Sex	Number of Rats Showing Clinical Sign during Study
		Gl (N = 15)	G2 (N = 15)	G3 (N = 15)	G4 (N = 15)
Salivation	M	0	0	0	15
Snuffles	M	0	0	0	2
Salivation	F	0	0	0	9
Snuffles	F	0	0	0	2

 

A statistically significant decrease (9% decrease from control) was noted in terminal body weight of male rats of the high dose group.

% body weight compared to control - Male rats

Treatment Days	% body weight compared to control
	G2 vs Gl	G3 vs Gl	G4 vs Gl
1	0.14	0.67	-0.08
8	-0.55	-1.96	-3.86
15	-0.73	-2.04	-5.01
22	-2.53	-2.86	-6.74*
29	-3.65	-3.48	-8.59**
36	-3.35	-2.37	-8.87**
43	-4	-2.77	-9.71**
50	-3.77	-2.84	-9.48**

Note: * - Represent significant level at p≤0.05; ** - Represent significant level at p≤0.01

 

body weight gain compare to control - Male rats

Treatment Days	%body weight gain compared to control
	G2 vs Gl	G3 vs GI	G4 vs Gl
1-8	-18.20	-70.06*	-101.82**
8-15	-8.16	-5.54	-52.48
15-22	-67.63**	-32.29	-69.35**
22-29	-39.99	-23.70	-68.80*
29-36	15.27	67.43	-26.62
36-43	-30.98	-19.51	-44.57
43-50	6.75	-6.36	0.87
1-50	-23.59	-20.65	-57.19**

Note: * - Represent significant level at p ≤0.05; ** - Represent significant level at p ≤ 0.01

 

% food consumption compare to control - Male rats

Treatment Days	% food consumption compared to control
	G2 vs Gl	G3 vs Gl	G4 vs Gl
1-8	-2.05	-8.22	-12.33**
8-15	-4.37	-11.03	-17.36*
29-36	1.02	1.59	-4.29
36-43	-6.25	-3.55	-12.76
43-50	-3.91	-3.17	-9.82
1-50	-3.23	-5.04	-11.55*

Note: * - Represent significant level at p ≤ 0.05; ** - Represent significant level at p ≤ 0.01

 

Fertility : Male fertility index, female fertility index, gestation index, parturition index, percentage of pregnant rats and mating index were comparable with the control group. The duration of the gestation days and pre-coital interval were comparable among all the test item treated groups with the control group.

 

Pre and Post-natal Data: The mean number of implants, pre and post-natal loss, mean percentage of pre and post-natal loss were comparable between all test item treated groups and the control group. The percent pre-natal loss was higher in high dose group compared to control group though it lacks statistical significance. This was due to all pups belonging to rat No 118 were found either dead or cannibalised on PND 0.

The percent post-natal loss was higher in high dose group compared to control group though it lacks statistical significance. This was due to the complete litter loss by PND 1, of rat N° 108, belonging to high dose group. These changes were limited to one rat (and were not spread across the group), hence the observed changes were considered unrelated to treatment and incidental in nature

 

Live birth index of test item treated groups was comparable with the control group.

 

Survival index of test item treated groups was comparable with that of the control group. A statistically significant decrease in live birth index of males and composite of males and females were observed in high dose group when compared with the control group. A statistically significant decrease in survival index of females was observed during post-natal day 0-4 in high dose group when compared with the control group. These findings were due to complete litter loss of rat N° 108, hence these observed changes were considered as unrelated to treatment.

Conclusions:

- 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.
- NOAEL for systemic toxicity was 240 mg/kg bw/day for female rats based on no effect on systemic endpoints.
- NOAEL for reproductive and fertility toxicity was 240 mg/kg bw/day based on no effect on fertility and reproductive performance.
- NOAEL for developmental toxicity was 240 mg/kg bw/day based on no effect on mortality, clinical signs and postnatal growth.

Executive summary:

The study was conducted to evaluate the toxic characteristics, systemic toxicity, reproduction/developmental toxicity and neurotoxicity of N-Butylaminoethanol (BEA) when administered by gavage to male and female Wistar rats during pre-mating, mating, gestation, and lactation periods.

 

Methods: 15 rats / sex / group were treated with N-Butylaminoethanol receiving the following dose levels: Group No - 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, oestrous cycle, neurobehavioural observation (NBO), functional observational battery (FBO), pub observation, blood collection, clinical pathology, thyroid hormone, organ weights, histopathology. Males were sacrificed when ~80 % of females had delivered, females on LD15 and pups on PND 14.

 

Results: The results of dose formulation analyses were within an acceptable range of ± 10 % of the nominal concentration and the Coefficient of Variation (% CV) was less than 10. Thus, the prepared dose had an acceptable active ingredient (a.i.) concentration and were homogeneously prepared.

 

N-Butylaminoethanol at 60, 120 and 240 mg/kg b.wt./day

- No signs of toxicity and mortality were observed in male and female rats from low and mid dose groups. In the high dose group, salivation in all male and in 9 female rats and snuffles in 2 male and 2 female rats were observed. Salivation was toxicologically insignificant, hence considered as non-adverse in nature. Whereas, snuffles were considered as a treatment related effect but non-adverse in nature as this observation was limited to 2 of 15 rats only and in absence of gross observation in lungs.

- No treatment related effects on body weight, body weight gain and food consumption were observed in male and female rats from the low and mid dose groups. In high dose male rats, statististically significant treatment related effects were observed in body weight, body weight gain and food consumption when compared with that of the control group. Although body weights for these male rats were <10 % lower than control males, terminal body weight were <9 % lower than control males, the body weight gains were approximately 60 % lower than control. Overall, food consumption was about 12 % lower than control. Hence, changes are considered as treatment related adverse in nature.

- Similar findings were not seen in high dose female rats.

- In male and female rats, NBO (Neurobehavioral Observations) and FOB (Functional Observational Battery) parameters were comparable to control.

- No treatment related effects on pre-natal loss, percent pre-natal loss, post-natal loss, percent postnatal loss, number of implants, mating index, male fertility index, female fertility index, gestation index, parturition index, pre-coital interval, percent of pregnant females, duration of gestation, and the number of pup count (litter size) were observed.

- No effects on sex ratio, mean oestrus cycle length and mean number of oestrus cycles prior to mating were observed.

- In pups, no effects on body weight, body weight gain, AGD or nipple retention were observed.

- No treatment related effects on mortality index, live birth index and survival index were observed.

- Haematology, clinical chemistry and urine analyses values for the test item treated groups were comparable with that of the control group.

- Organ (absolute and relative) weights of rats (parent rats) and pups were comparable with that of the respective control group.

- External and internal examination of rats (all adult rats and pups) did not reveal any lesions

- No treatment related histopathological findings

 

Low and Mid dose: No signs of toxicity on clinical signs, mortality, body weight, body weight gain, feed consumption, oestrous cycle, NBO, FOB, clinical pathology, and organ weight (absolute and relative) were observed. No treatment related macroscopic and microscopic lesions were observed.

No treatment related effect on fertility index, mating index, gestation index, percent of pregnant female, duration of the gestation period, number of implants, prenatal and postnatal loss (number and percent) was observed. No effect on pup body weight, body weight gain, litter size and male sex ratio was observed in F1 pups. No effect on endocrine sensitive endpoints (viz., AGD, nipple retention and thyroid weight and hormone level).

 

High dose: No sign of toxicity on mortality, NBO, FOB, oestrous cycle, organ weight, clinical pathology (absolute and relative) was observed. No treatment related macroscopic and microscopic lesions were observed.

Salivation is 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 treatment related effect on fertility index, mating index, gestation index, percent of pregnant females, duration of the gestation period, number of implants, prenatal and postnatal loss (number and percent) was observed. No effect on pup body weight, body weight gain, litter size and male sex ratio was observed in F1 pups. No effect on endocrine sensitive endpoints (viz., AGD, nipple retention, thyroid weight and hormone level).

 

Conclusion:

Based on results of this study, it was concluded that:

- 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.

- No Observed Adverse Effect Level (NOAEL) for systemic toxicity was 240 mg/kg bw/day for female rats based on no effect on systemic endpoints.

- NOAEL for reproductive and fertility toxicity was 240 mg/kg bw/day based on no effect on fertility and reproductive performance.

- The NOAEL for developmental toxicity was 240 mg/kg bw/day based on no effect on mortality, clinical signs and postnatal growth.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

240 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP guideline study (Klimisch 1).

Effect on fertility: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

236.3 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP guideline study (Klimisch 1).

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

There are no reproductive 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.

 

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. The test substance did not cause any adverse effect with regard to reproductive and developmental parameters. The male mating index was 100 % in all groups including the controls. The male fertility index ranged between 90 % and 100 % without showing any relation to exposure concentration. The female mating and fertility indices and duration of gestation were similar to controls. Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the controls, taking normal biological variation into account. There were no statistically significant differences in post-implantation loss between the groups and the mean number of F1 pups delivered per dam remained unaffected. The rate of liveborn pups was also not affected by the test substance. The mean number of delivered F1 pups per dam and the rates of liveborn were evenly distributed about the groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study. The viability index and sex ration of pups did not show any association to the treatment. There were no test substance related adverse clinical signs observed in any of surviving F1 generation pups of the different test groups. No test compound-related influence on F1 pup body weights and pup body weight change were noted in all test groups. No findings were observed at gross necropsy in any male or female pups of all test groups. Systemic toxicity effects were only transiently reduced food consumption, body weight and body weight gain in parental animals. In their histopathology, lesions of nasal epithelia were observed in animals exposed to 72.1 and 236.3 mg/m³ test substance. Thus, concerning systemic toxicity, reproductive and developmental parameters the NOAEC for 2-dibutylaminoethanol was determined to be 236.3 mg/m³ for male and female animals. Considering histopathological changes, the NOAEC for local effects was 20.6 mg/m³.

 

An oral combined repeated dose study with reproductive screening according to OECD Guideline 422 was conducted to evaluate the toxic characteristics, systemic toxicity, reproduction/developmental toxicity and neurotoxicity of N-Butylaminoethanol (BEA) when administered by gavage to male and female Wistar rats 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, oestrous cycle, neurobehavioural observation (NBO), functional observational battery (FBO), pub observation, blood collection, clinical pathology, thyroid hormone, organ weights, histopathology. Males were sacrificed when ~80 % of females had delivered, females on LD15 and pups on PND 14. The results of dose formulation analyses were within an acceptable range of ± 10 % of the nominal concentration and the Coefficient of Variation (% CV) was less than 10. Thus, the prepared dose had an acceptable active ingredient (a.i.) concentration and were homogeneously prepared. No signs of toxicity and mortality were observed in male and female rats from low and mid dose groups. In the high dose group, salivation in all male and in 9 female rats and snuffles in 2 male and 2 female rats were observed. Salivation was toxicologically insignificant, hence considered as non-adverse in nature. Whereas, snuffles were considered as a treatment related effect but non-adverse in nature as this observation was limited to 2 of 15 rats only and in absence of gross observation in lungs. No treatment related effects on body weight, body weight gain and food consumption were observed in male and female rats from the low and mid dose groups. In high dose male rats, statistically significant treatment related effects were observed in body weight, body weight gain and food consumption when compared with that of the control group. Although body weights for these male rats were <10 % lower than control males, terminal body weight were <9 % lower than control males, the body weight gains were approximately 60 % lower than control. Overall, food consumption was about 12 % lower than control. Hence, changes are considered as treatment related adverse in nature. Similar findings were not seen in high dose female rats. In male and female rats, NBO (Neurobehavioral Observations) and FOB (Functional Observational Battery) parameters were comparable to control. No treatment related effects on pre-natal loss, percent pre-natal loss, post-natal loss, percent postnatal loss, number of implants, mating index, male fertility index, female fertility index, gestation index, parturition index, pre-coital interval, percent of pregnant females, duration of gestation, and the number of pup count (litter size) were observed. No effects on sex ratio, mean oestrus cycle length and mean number of oestrus cycles prior to mating were observed. In pups, no effects on body weight, body weight gain, AGD or nipple retention were observed. No treatment related effects on mortality index, live birth index and survival index were observed. Haematology, clinical chemistry and urine analyses values for the test item treated groups were comparable with that of the control group. Organ (absolute and relative) weights of rats (parent rats) and pups were comparable with that of the respective control group. External and internal examination of rats (all adult rats and pups) did not reveal any lesions. No treatment related histopathological findings.

Based on results of this study, it was concluded that:

- 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.

- No Observed Adverse Effect Level (NOAEL) for systemic toxicity was 240 mg/kg bw/day for female rats based on no effect on systemic endpoints.

- NOAEL for reproductive and fertility toxicity was 240 mg/kg bw/day based on no effect on fertility and reproductive performance.

- The NOAEL for developmental toxicity was 240 mg/kg bw/day based on no effect on mortality, clinical signs and postnatal growth.

Effects on developmental toxicity

Description of key information

DBEA (CAS 102-81-8)

- Oral, PNDT (OECD 414), rat: NOAEL (maternal) =  60 mg/kg bw/d, NOAEL (fetuses) = 60 mg/kg bw/d (highest dose examined)

 

BEA (CAS 111-75-1)
- Oral, PNDT (OECD 414), rat: NOAEL (maternal) = 140 mg/kg bw/d, NOAEL (fetuses) = 280 mg/kg bw/d 

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

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
There is evidence that a link exists also between liver/ kidney damage observed in a number of studies with ethanolamines (as a consequence of a perturbed choline-homeostasis) and the reproductive toxicity. Ethanolamines DEA, DEEA and MMEA produced toxicity effects to male reproductive system, while TEA, MEA, MDEA and MMEA mainly reduced number of implantation sites and/or produced pre- or post-implantation losses. DMAE and MMEA did not produce reproductive effects: there were no treatment-related effects of treatment on gonads or on any gestational parameters, including pre- and post implantation loss or sex ratio. However, the authors (Zahniser et al., 1978) linked the deaths of pups from mothers fed choline deficient diets supplemented with DMAE or MMEA to perturbed choline uptake and phospholipid synthesis in new born rats leading to respiratory distress and deaths (please refer to the full version of read-across statement).
Under the conditions of this prenatal developmental toxicity study, the oral administration of DBEA to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) caused evidence of distinct maternal toxicity, such as severe clinical findings (twitching, convulsions, tremor, abdominal position), a reduction of water and food consumption and a decrease in body weight/body weight gain. Due to the above-mentioned findings, treatment of the high-dose was stopped and the dams were euthanized without further examinations on GD 13/12/11 of the study. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is the mid-dose of 60 mg/kg bw/d. Examination of the low- and mid-dose fetuses was performed and showed no adverse findings. In conclusion, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 60 mg/kg bw/d. Under the conditions of this study the test item is not teratogenic.

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, treatment-related

Description (incidence and severity):

For almost all females of test group 3 (200 mg/kg bw/d) severe clinical findings were recorded during the treatment period (within the 2-hour examination interval after treatment):
twitching after treatment in 23 females from GD 7 onwards, convulsion after treatment in 6 females from GD 10 onwards, tremor after treatment in 3 females on GD 9 and 11, abdominal position after treatment in 17 females from GD 8 onwards.
As mentioned above, all females of test group 3 were sacrificed prematurely on GD 13/12/11.
Furthermore, most females (23 out of 25) of the high-dose group and 6 females of the middose group showed transient salivation during the treatment period. Salivation occurred in the respective animals within the 2-hour examination interval after treatment (i.e. 0-2h) and was initially observed on GD 7 (200 mg/kg bw/d) or GD 18 (60 mg/kg bw/d). Salivation occurred most probably due to the bad taste of the test substance, was assessed to be a local effect and, therefore, considered as not adverse.
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any female of test group 1 (20 mg/kg bw/d).
see attached document: summary of maternal clinical observation

Description (incidence):

Due to severe clinical findings in almost all animals of test group 3, the high-dose group of 200 mg/kg bw/d was sacrificed prematurely due to animal welfare reasons on GD 13/12/11 ) of the study.
There were no further test substance-related or spontaneous mortalities in any females of test groups 0-2 (0, 20 or 60 mg/kg bw/d).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see attached document: summary bw_bwc
From GD 8 onwards until premature sacrifice on GD 11-13 the mean body weights of the highdose dams (200 mg/kg bw/d) were lower in comparison to the concurrent control group, attaining statistical significance on GD 10 (-4%). The average body weight gain of the highdose dams (200 mg/kg bw/d) was distinctly and statistically significantly reduced during GD 6-10 showing a body weight loss during GD 6-8. These effects were assessed as treatment related and adverse.
In test group 2 (60 mg/kg bw/d) the mean body weights were generally comparable to the concurrent control group. The average body weight gain was statistically significantly reduced on GD 6-8 but recovered afterwards and was comparable to the control values again throughout the remaining study period. If calculated for the entire treatment period (GD 6-19), the mean body weight gain of the mid-dose dams was comparable to the concurrent control group.
The mean body weights and the average body weight gain of the low-dose dams (20 mg/kg
bw/d) were generally comparable to the concurrent control group throughout the entire study period.
Corrected (net) body weight gain
The corrected body weight gain of test groups 1 and 2 (20 and 60 mg/kg bw/d) revealed no difference of any biological relevance to the corresponding control group. Moreover, mean carcass weights remained unaffected by the treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

see attached document: summary food consumption
The mean food consumption of the high-dose dams (200 mg/kg bw/d) was statistically significantly reduced from GD 6 onwards until premature sacrifice on GD 11-13 (up to 31 % below control). This was assessed as treatment-related and adverse.
The mean food consumption of the mid- and low-dose dams (60 and 20 mg/kg bw/d) was generally comparable to the concurrent control group throughout the entire study period.

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

effects observed, treatment-related

Description (incidence and severity):

see attached document; summary maternal water consumption
The mean water consumption of the dams in test group 3 (200 mg/kg bw/d) was statistically significantly reduced from GD 6 onwards until premature sacrifice during GD 11-13 (up to 21 % in comparison to the concurrent control). This was assessed as treatment-related and adverse.
In test group 2 (60 mg/kg bw/d) the mean water consumption was statistically significantly reduced during GD 6-10 (up to 12 % below control) but recovered afterwards and was comparable to the control values again until scheduled sacrifice on GD 20.
The mean water consumption of the dams in test group 1 (20 mg/kg bw/d) was comparable to the concurrent control group throughout the entire study period.

Ophthalmological findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

see attached document: summary gravid uterine weitghts and bwc
The mean gravid uterus weights of the low- and mid-dose animals (20 and 60 mg/kg bw/d) were not influenced by the test substance. The differences between these groups and the control group revealed no dose-dependency and were assessed to be without biological relevance.
Absolute and relative organ weights
All mean absolute and relative weight parameters did not show significant differences when compared to the control group 0.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

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.

Other effects:

no effects observed

Description (incidence and severity):

See attached document: Incidence of microscopic findings
Thyroid hormones
At gestation day 20, in dams of test groups 1 and 2 (20 and 60 mg/kg bw/d) no treatment related alterations of T3, T4 and TSH were observed.
Gross lesions
No macroscopic findings of thyroid glands were noted in test groups 0 (control), 1 (20 mg/kg bw/d) and 2 (60 mg/kg bw/d).

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

A 100 % post-implantation loss was observed in one female each in test groups 1 and 2. In one lowdose dam (20 mg/kg bw/d) one early resorption was recorded after staining the empty uterus at C-section (i.e. pregnant by stain), whereas for one mid-dose dam (60 mg/kg bw/d) 9 early and 3 late resorptions were recorded (no viable fetuses). In the other dams of test groups 1 and 2 post-implantation losses and number of resorptions were comparable to the control. Since only one dam per test group was affected without relation to dose, these isolated findings were regarded as incidental and not treatment-related.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

see attached document: summary of reproduction data
There were no test substance-related and/or biologically relevant differences between the test groups 0-2 in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and post-implantation losses, the number of resorptions and viable fetuses. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age.

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

In one lowdose dam (20 mg/kg bw/d) one early resorption was recorded after staining the empty uterus at C-section (i.e. pregnant by stain), whereas for one dam in the mid-dose (60 mg/kg bw/d) 9 early and 3 late resorptions were recorded (no viable fetuses).

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

There were no test substance-related and/or biologically relevant differences between the test groups 0-2 in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and post-implantation losses, the number of resorptions and viable fetuses. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age.
Regarding pathology, no treatment-related findings were noted. 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.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

water consumption and compound intake

Fetal body weight changes:

no effects observed

Description (incidence and severity):

see attached document: summary placental and fetal bw
The mean fetal weights of test groups 1 and 2 (20 and 60 mg/kg bw/d) were not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses in test groups 1 and 2 (20 and 60 mg/kg bw/d) was comparable to the control fetuses.

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One low-dose male fetus an umbilical hernia was recordrd. Since the finding was not related to dose,
it was not assessed as treatment-related.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

For only one low-dose fetus a skeletal malformation was recorded: malpositioned and bipartite sternebra (unchanged cartilage). Since the finding was not related to dose, it was not assessed as treatment-related.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Two soft tissue variations were detected, i.e. short innominate and dilated renal pelvis, both in test groups 0 and 1. The incidences of these variations were neither statistically significantly, or dose-dependently increased in the treated groups. Therefore, they were not assessed as treatment-related.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Two unclassified external observations were recorded. A skin tag was seen in one low-dose fetus (20 mg/kg bw/d), furthermore, placentae fused were seen in one control litter. These findings were not considered biologically relevant, since they were single events without relation to dose.
For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared without a relation to dose The overall affected fetuses/litter incidences of skeletal variations were comparable to the historical control data.

Details on embryotoxic / teratogenic effects:

Soft tissue malformations did not occur in any fetus in this study. There were noted external and skeletal malformations in two fetuses of the low-dose group.. These observations in single fetuses were unrelated to dose and both can be found in the historical control data, thus they are considered as incidental events.
External variations did not occur in any fetus in this study. Two soft tissue variations and a range of skeletal variations were noted in all test groups including the controls. Two out of three incidences showed no relation to dosing. The skeletal variations are equally distributed about the different test groups, if normal biological variation is taken into account, and can be found in the historical control data at a comparable frequency.
Unclassified soft tissue observations did not occur in any of the fetuses in this study. A spontaneous origin is assumed for the unclassified external and skeletal cartilage observations which were observed in several fetuses of all test groups The distribution and type of these findings do not suggest any relation to treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: teratogenicity / embryotoxicity

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Treatment related:

no

Total soft tissue variations

 

		Test group 0 0 mg/kg bw/d	Test group 1 20 mg/kg bw/d	Test group 2 60 mg/kg bw/d
Litter Fetuses	N N	25 133	24 121	24 121
Fetal incidence	N (%)	3 (2.3)	3 (2.5)	0.0
Litter incidence	N (%)	3 (12)	2 (8.3)	0.0
Affected fetuses/litter	Mean %	2.0	2.5	0.0

 

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

Total fetal skeletal variations

 

		Test group 0 0 mg/kg bw/d	Test group 1 20 mg/kg bw/d	Test group 2 60 mg/kg bw/d
Litter Fetuses	N N	25 144	24 129	24 132
Fetal incidence	N (%)	138 (96)	127 (98)	129 (98)
Litter incidence	N (%)	25 (100)	24 (100)	24 (100)
Affected fetuses/litter	Mean %	95.9	98.6	97.9

 

mg/kg bw/d = milligram per kilogram body weight per day; N = number; % = per cent

 

 

 

Occurrence of statistically significantly increased fetal skeletal         

variations (expressed as mean percentage of affected fetuses/litter)

 

Finding	Test group 0 0 mg/kg bw/d	Test group 1 20 mg/kg bw/d	Test group 2 60 mg/kg bw/d	HCD Mean % (range)
Incomplete ossification of basisphenoid	10.4	32.8**	18.4	22.4 (5.3 - 43.2)
Incomplete ossification of supraoccipital; unchanged cartilage	11.8	17.2	23.6*	22.4 (3.0 - 51.2)
Incomplete ossification of skull; unchanged cartilage	3.5	9.2*	5.0	7.5 (2.1 - 13.4)

 

mg/kg bw/d = milligram per kilogram body weight per day; HCD = Historical control data; % = per cent

* = p≤0.05 (Wilcoxon-test [one-sided]) ** = p≤0.01 (Wilcoxon-test [one-sided])

 

Conclusions:

Under the conditions of this prenatal developmental toxicity study, the oral administration of Dibutylethanolamine (DBEA) to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) caused evidence of distinct maternal toxicity, such as severe clinical findings (twitching, convulsions, tremor, abdominal position), a reduction of water and food consumption and a decrease in body weight/body weight gain. Due to the above-mentioned findings, treatment of the high-dose was stopped and the dams were euthanized without further examinations on GD 13/12/11 of the study. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is the mid-dose of 60 mg/kg bw/d. Examination of the low- and mid-dose fetuses was performed and showed no adverse findings. In conclusion, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 60 mg/kg bw/d. Under the conditions of this study the test item is not teratogenic.

Executive summary:

In a prenatal developmental toxicity study, the test substance Dibutylethanolamine (DBEA) was administered to pregnant Wistar rats daily by gavage from implantation to one day prior to the expected day of parturition (GD 6-19) to evaluate its potential maternal and prenatal developmental toxicity. Analyses confirmed the correctness of the prepared concentrations and the stability of the test substance in the vehicle. Concerning clinical examination, strong signs of systemic maternal toxicity was observed at the highest test group of 200 mg/kg bw/d Dibutylethanolamine. Since severe clinical findings (twitching, convulsions, tremor, abdominal position) were observed in almost all animals, the high-dose group was sacrificed prematurely due to animal welfare reasons. Treatment of that test group was stopped and the dams were euthanized without further examinations on GD 13/12/11 of the study. The next lower dose of 60 mg/kg bw/d showed no adverse clinical findings. Water and food consumptions were statistically significantly reduced in high-dose dams during GD 6-13 (up to 21 % and 31 % below control, respectively). Furthermore, high-dose dams showed a statistically significant decrease in body weight (change) including a body weight loss during GD 6-8. The above-mentioned findings were assessed as treatment-related and adverse. Mid-dose dams showed a reduction in water consumption in the beginning of treatment (GD 6-10: up to 12% below control) but recovered afterwards to control levels. Food consumption was not affected in mid-dose dams. Body weight change was statistically significantly reduced  on GD 6-8 but recovered afterwards and body weight in general was comparable to the control values. These two marginal findings in the mid-dose group were only observed in the beginning of treatment but not thereafter. They were considered to be not sufficient to proof adversity and therefore, they were not assessed as treatment-related and adverse. In the low dose group (20 mg/kg bw/d), no adverse findings were observed. Concerning thyroid hormone levels, no treatment-related, adverse effect was observed up to a dose of the compound of 60 mg/kg bw/d. Regarding pathology, no treatment-related findings were noted. 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. No differences of toxicological relevance between the control and the treated groups (20 or 60 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and post-implantation loss. Similarly, no toxicologically relevant influence of the test substance on sex distribution and anogenital distance/index of the fetuses was noted at any dose. Overall, there was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose.

Reference 2

Endpoint:

developmental toxicity

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 oral (gavage) pre-natal developmental toxicity study with BEA decreased body weight gain and food consumption were observed in dams at the highest dose of 280 mg/kg bw/day. No effects on pup development were observed.
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 screening 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:

no effects observed

Description (incidence and severity):

No clinical sign of toxicity was observed up to a dose level of 140 mg/kg bw/day.
In 280 mg/kg bw/day dose group, salivation was observed approximately 10 to 15 minutes after dosing on a few days in 5 of 25 female rats and persisted for approximately 45 to 50 minutes. This finding was considered a response to the dose solution and toxicologically insignificant, hence considered as non-adverse in nature.
Please also refer to attached tables below.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

Please also refer to attached tables below.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weights and body weight changes of the pregnant female rats were comparable between the control and the test item treated groups up to a dose level of 140 mg/kg bw/day.

For the 280 mg/kg bw/day dose group, a statistically significant decrease in the mean body weight of the pregnant female rats was observed on gestation day 20. A statistically significant decrease in day 20 corrected body weight was also noted for this group. In addition, statistically significant decreases in mean body weight change were observed during the gestation days 5-8, 8-11, 17-20, and 5-20. Decreased mean body weight change for this group were also observed during the gestation period days 11-14 and 14-17 without statistical significance.
Please also refer to attached tables below.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The mean food consumption of the pregnant female rats was comparable between the control and the test item treated groups up to a dose level of 140 mg/kg bw/day.
Statistically significant decreased mean food consumption was seen for 280 mg/kg bw/day dose group for gestation days 5-8, 8-11, 11-14, 14-17, 17-20, and 5-20.
Decrease in the mean body weight and body weight change are correlated with the decreased mean food consumption and are considered as adverse effect of the test item.
Please also refer to attached tables below.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Test item treatment did not lead to any alteration in the absolute and relative organ weight of the thyroid gland.
Please also refer to attached tables below.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Internal (gross) examination of the terminally sacrificed control and test item treated female rats did not reveal any lesion of pathological significance.
Please also refer to attached tables below.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Histological examination of the thyroid gland revealed ultimobranchial cyst/s in 2 and 1 female rats of the control and 280 mg/kg bw/day dose group, respectively, and considered as spontaneous or incidental and therefore non-adverse in nature.
Please also refer to attached tables below.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

- Thyroid Hormone Analysis: Serum level of T3, T4, and TSH in female rats belonging to test item groups was comparable to the control group.
- External a examination of the terminally sacrificed control and test item treated female rats did not reveal any lesion of pathological significance.
Please also refer to attached tables below.

Number of abortions:

no effects observed

Description (incidence and severity):

Please also refer to attached tables below.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Please also refer to attached tables below.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

Please also refer to attached tables below.

Early or late resorptions:

no effects observed

Description (incidence and severity):

Please also refer to attached tables below.

Dead fetuses:

no effects observed

Description (incidence and severity):

Please also refer to attached tables below.

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Please also refer to attached tables below.

Changes in number of pregnant:

not examined

Description (incidence and severity):

Please also refer to attached tables below.

Other effects:

no effects observed

Description (incidence and severity):

The mean absolute and relative uterine weight of the pregnant female rats were comparable between the control and the test item treated groups.
Please also refer to attached tables below.

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: All examined parameters

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The mean weight of male, female and total foetuses (male + female) was comparable between the control and the test item treated groups up to the dose level of 140 mg/kg bw/day.
In the 280 mg/kg bw/day dose group, male foetus body weight was statistically significantly decreased (- 6.57 % than control group) when compared to the control group. The body weights for female foetuses and for a composite of foetuses of both sexes were also lower than control group (- 4.55 % and - 5.57 %, respectively) without statistical significance. These lower foetal body weights were consistent with decreased maternal body weight. Hence, these findings were considered as secondary to the decreased maternal body weight and non-adverse in nature.
Please also refer to attached tables below.

Reduction in number of live offspring:

not examined

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male sex ratio were comparable between the control and the test item treated groups.
Please also refer to attached tables below.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The mean count of male, female and total foetuses (male + female) was comparable between the control and the test item treated groups.
Please also refer to attached tables below.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

The mean anogenital distance of male and female foetuses and the male sex ratio were comparable between the control and the test item treated groups.
Please also refer to attached tables below.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

A total of 278, 264, 272, and 252 foetuses were examined in 0, 70, 140, and 280 mg/kg bw/day dose groups, respectively.
No external anomalies were observed in foetuses of the control and treatment groups up to the dose level of 280 mg/kg bw/day except one runt foetus (1/14) in the 280 mg/kg bwt/day dose group. This finding was comparable with that of the control group.
Please also refer to attached tables below.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

A total of 143, 137, 143, and 131 foetuses were observed for the skeletal evaluation in 0, 70, 140, and 280 mg/kg bw/day dose groups, respectively.
There were several incidences of the statistically significant differences in the skeletal variations in all test item treated groups (see Table 1, "Any other information on results"). These decreased skeletal variations were only because of higher number of incidences in control group compared to treatment and have no toxicological relevance. Due to the lack of dose dependency, the increase in number of foetuses and litters with Rib: Full supernumerary was considered as incidental and non-adverse (see Table 2, "Any other information on results"). In case of xiphisternum, the data indicate a delayed ossification associated with reduced foetal weights i.e., a delay in foetal development rather than a direct effect on bone tissue. In addition, this increase in the number of foetuses with Xiphisternum: Unossified was within the range of historical control data therefore considered as non-adverse in nature.
Please also refer to attached tables below.

Visceral malformations:

no effects observed

Description (incidence and severity):

A total of 135, 127, 129, and 121 foetuses were examined in 0, 70, 140, and 280 mg/kg bw/day dose groups, respectively.
No gross treatment related visceral findings were observed in foetuses of the control and the treatment groups up to the dose level of 280 mg/kg bw/day with the exception of dilated ureter seen in one foetus from the control group. There were no any other visceral findings in any dose groups.
Please also refer to attached tables below.

Other effects:

no effects observed

Description (incidence and severity):

The mean anogenital distance of male and female foetuses were comparable between the control and the test item treated groups.
Please also refer to attached tables below.

Dose descriptor:

NOAEL

Effect level:

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

Basis for effect level:

other: all examined parameters

Abnormalities:

no effects observed

Developmental effects observed:

no

Lowest effective dose / conc.:

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

Treatment related:

no

Table 1: Statistically significant decrease in skeletal variations

Group	G1	G2	G3	G4
Dose (mg/kg b. bw/day)	0	70	140	280
Total N° of Foetuses Examined	143	137	143	131
14th Rib: Extra ossification centre	63	35 (decrease)	38 (decrease)	34 (decrease)
Rib: Short supernumerary	33	17 (decrease)	19 (decrease)	18 (decrease)
Squamosal: Incomplete ossification	7	1 (decrease)	1 (decrease)	0 (decrease)
Total N° of Litters Examined	23	23	22	22
Interparietal: Incomplete ossification	6	2	1 (decrease)	0 (decrease)
Squamosal: Incomplete ossification	6	1 (decrease)	1 (decrease)	0 (decrease)

Table 2: Statistically significant increase in skeletal variations

Group	G1	G2	G3	G4
Dose (mg/kg bw/day)	0	70	140	280
Total N° of Foetuses Examined	143	137	143	131
Rib: Full supernumerary	1	3	10 (increase)	0
Xiphisternum: Unossified	0	5 (increase)	8 (increase)	13 (increase)
Total N° of Litters Examined	23	23	22	22
Rib: Full supernumerary	1	3	6 (increase)	0

Please also refer to attached additional tables below.

Conclusions:

Based on the result of the present study, it is concluded that the “No Observed Adverse Effect Level (NOAEL)” of N-butylaminoethanol (BEA) for maternal toxicity is 140 mg/kg bw/day and 280 for developmental toxicity under the conditions and procedures followed in the study. The LOAEL for maternal toxicity was 280 mg/kg/day based on adverse effects observed on body weight, body weight change, and food consumption.

Executive summary:

This study was performed to evaluate the prenatal developmental and maternal toxicity potential of N-butylaminoethanol (BEA), when administered orally through gavage to mated female rats.

 

Method: N-butylaminoethanol was administered orally from gestation days (GD) 5 to 19, through gavage, to 25 mated female rats per group at dose levels of 70, 140, and 280 mg/kg bw/day. The control group received the vehicle, reverse osmosis water, only. Rats were observed twice daily for mortality and clinical signs. Maternal body weights and food consumption were recorded throughout the gestation period. All rats were sacrificed on GD 20 and assessed for gross pathological changes. The uteri were excised, weighed, and examined for the numbers of implantation sites, early and late resorptions, and numbers of live and dead foetuses. The ovaries were excised and the number of corpora lutea counted. The foetuses were identified for sex, weighed, and examined for external findings. The anogenital distance was measured for all foetuses. Serum thyroid hormones T3 (liothyronine), T4 (levothyroxine), and TSH (thyroid stimulating hormone) were analysed from all female rats during the terminal sacrifice. At the time of terminal sacrifice, the weight of the thyroid gland was recorded from all female rats and preserved for histopathology. Detailed histological examination of the thyroid gland was performed in control and high dose group female rats. Following appropriated fixation, foetuses were examined for visceral abnormalities including razor sectioning of the head, and for skeletal abnormalities.

Dose formulations were analysed for the test item concentration and homogeneity, prior to the initiation of the treatment and once during the treatment period.

Results: The dose formulation analysis results revealed that the mean recovery was within the acceptance level of ± 10 % of nominal value and % CV was < 10. The dose formulation analysis demonstrated that the dose formulations were homogeneous.

No treatment related effects were seen in the 70 and 140 mg/kg bw/day groups for any evaluated parameter which include: Mortality and clinical signs of toxicity, mean body weight, body weight change, food consumption, and 20th day corrected body weight, Mean absolute and relative uterine weights, serum level of T3 (Liothyronine), T4 (Levothyroxine), and TSH (Thyroid Stimulating Hormone), external and internal (gross) examination of the terminally sacrificed female rats, absolute and relative weights of the thyroid gland, Histological examination of thyroid gland, mean numbers of corpora lutea, implantation sites, live foetuses, dead foetuses, resorptions (early, late, and total), pre-implantation loss, and post-implantation loss, mean percent of live foetuses, dead foetuses, pre-implantation loss, post-implantation loss, and total resorptions, mean foetal count and foetal body weight of male, female, and total foetuses (male + female), anogenital distance of male and female foetuses, male sex ratio, Incidences of malformation/variation for external, visceral, head razor and skeletal examination.

No treatment related adverse effects were seen in the 280 mg/kg b. wt./day group for following parameters: Mortality, salivation was observed approximately 10 to 15 minutes after dosing in 5 of 25 female rats and persisted for approximately 45 to 50 minutes. This finding is considered a response to the dose solution and toxicologically insignificant, hence considered as non-adverse in nature, Mean absolute and relative uterine weights, Serum level of T3, T4, and TSH, External and internal (gross) examination of the terminally sacrificed female rats, Absolute and relative weight of thyroid gland, histological examination of thyroid gland, mean numbers of corpora lutea, implantation sites, live foetuses, dead foetuses, resorptions (early, late, and total), pre-implantation loss, and post-implantation loss, the mean percent of live foetuses, dead foetuses, pre-implantation loss, post-implantation loss, and total resorptions, mean foetal count of male, female, and total foetuses (male + female), anogenital distance of male and female foetuses, statistically significant decrease in mean body weight of male foetuses (- 6.57 % than control group) was observed when compared to the control group. The body weights for female foetuses and for a composite of foetuses of both sexes were also lower than control group (- 4.55 % and - 5.57 %, respectively) without statistical significance. These lower foetus body weights were consistent with decreased maternal body weight. Hence, these findings were considered as secondary to the decreased maternal body weight and non-adverse in nature, Male sex ratio, Incidence of malformation/variation for external, visceral, head razor, and skeletal examination of foetuses.

The following treatment related adverse effects were seen in the 280 mg/kg bw/day group:

- Statistically significant decrease in the mean body weight of the pregnant female rats was observed on gestation day 20. Statistically significant decrease in 20th day corrected body weight of the pregnant female rats was observed. Statistically significant decrease in the mean body weight change of the pregnant female rats was observed during the gestation period 5-8, 8-11, 17-20, and 5-20. Decrease in the mean body weight change of the pregnant female rats was also observed during gestation period 11-14 and 14-17 without statistical significance.

- Statistically significant decrease in mean food consumption of the pregnant female rats was observed during the gestation period 5-8, 8-11, 11-14, 14-17, 17-20, and 5-20.

- Decrease in the mean body weight and body weight change are correlated with the decreased mean food consumption and are considered as adverse effect of the test item.

Conclusion: Based on the result of the present study, it is concluded that the “No Observed Adverse Effect Level (NOAEL)” of N-butylaminoethanol (BEA) for maternal toxicity is 140 mg/kg bw/day and 280 for developmental toxicity under the conditions and procedures followed in the study. The LOAEL for maternal toxicity was 280 mg/kg/day based on adverse effects observed on body weight, body weight change, and food consumption.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

60 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP guideline studies (Klimisch 1).

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

There are no developmental 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.

 

In a GLP-compliant prenatal developmental toxicity study according to OECD Guideline 414, the test substance Dibutylethanolamine (DBEA) was administered to pregnant Wistar rats daily by gavage from implantation to one day prior to the expected day of parturition (GD 6-19) to evaluate its potential maternal and prenatal developmental toxicity. Doses of 20, 60 and 200 mg/kg bw/d in the vehicle corn oil were applied. Analyses confirmed the correctness of the prepared concentrations and the stability of the test substance in the vehicle. Concerning clinical examination, strong signs of systemic maternal toxicity was observed at the highest test group of 200 mg/kg bw/d Dibutylethanolamine. Since severe clinical findings (twitching, convulsions, tremor, abdominal position) were observed in almost all animals, the high-dose group was sacrificed prematurely due to animal welfare reasons. Treatment of that test group was stopped, and the dams were euthanized without further examinations on GD 13/12/11 of the study. The next lower dose of 60 mg/kg bw/d showed no adverse clinical findings. Water and food consumptions were statistically significantly reduced in high-dose dams during GD 6-13 (up to 21 % and 31 % below control, respectively). Furthermore, high-dose dams showed a statistically significant decrease in body weight (change) including a body weight loss during GD 6-8. The above-mentioned findings were assessed as treatment-related and adverse. Mid-dose dams showed a reduction in water consumption in the beginning of treatment (GD 6-10: up to 12 % below control) but recovered afterwards to control levels. Food consumption was not affected in mid-dose dams. Body weight change was statistically significantly reduced on GD 6-8 but recovered afterwards and body weight in general was comparable to the control values. These two marginal findings in the mid-dose group were only observed in the beginning of treatment but not thereafter. They were considered to be not sufficient to proof adversity and therefore, they were not assessed as treatment-related and adverse. In the low dose group (20 mg/kg bw/d), no adverse findings were observed. Concerning thyroid hormone levels, no treatment-related, adverse effect was observed up to a dose of the compound of 60 mg/kg bw/d. Regarding pathology, no treatment-related findings were noted. 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. No differences of toxicological relevance between the control and the treated groups (20 or 60 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and post-implantation loss. Similarly, no toxicologically relevant influence of the test substance on sex distribution and anogenital distance/index of the fetuses was noted at any dose. Overall, there was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose. 

 

A GLP-compliant prenatal developmental toxicity study according to OECD Guideline 414 study was performed to evaluate the prenatal developmental and maternal toxicity potential of N-butylaminoethanol (BEA), when administered orally through gavage to mated female rats. N-butylaminoethanol was administered orally from gestation days (GD) 5 to 19, through gavage, to 25 mated female rats per group at dose levels of 70, 140, and 280 mg/kg bw/day. The control group received the vehicle, reverse osmosis water, only. Rats were observed twice daily for mortality and clinical signs. Maternal body weights and food consumption were recorded throughout the gestation period. All rats were sacrificed on GD 20 and assessed for gross pathological changes. The uteri were excised, weighed, and examined for the numbers of implantation sites, early and late resorptions, and numbers of live and dead foetuses. The ovaries were excised and the number of corpora lutea counted. The foetuses were identified for sex, weighed, and examined for external findings. The anogenital distance was measured for all foetuses. Serum thyroid hormones T3 (liothyronine), T4 (levothyroxine), and TSH (thyroid stimulating hormone) were analysed from all female rats during the terminal sacrifice. At the time of terminal sacrifice, the weight of the thyroid gland was recorded from all female rats and preserved for histopathology. Detailed histological examination of the thyroid gland was performed in control and high dose group female rats. Following appropriated fixation, foetuses were examined for visceral abnormalities including razor sectioning of the head, and for skeletal abnormalities. Dose formulations were analysed for the test item concentration and homogeneity, prior to the initiation of the treatment and once during the treatment period.

The dose formulation analysis results revealed that the mean recovery was within the acceptance level of ± 10 % of nominal value and % CV was < 10. The dose formulation analysis demonstrated that the dose formulations were homogeneous. No treatment related effects were seen in the 70 and 140 mg/kg bw/day groups for any evaluated parameter which include: Mortality and clinical signs of toxicity, mean body weight, body weight change, food consumption, and 20th day corrected body weight, Mean absolute and relative uterine weights, serum level of T3 (Liothyronine), T4 (Levothyroxine), and TSH (Thyroid Stimulating Hormone), external and internal (gross) examination of the terminally sacrificed female rats, absolute and relative weights of the thyroid gland, Histological examination of thyroid gland, mean numbers of corpora lutea, implantation sites, live foetuses, dead foetuses, resorptions (early, late, and total), pre-implantation loss, and post-implantation loss, mean percent of live foetuses, dead foetuses, pre-implantation loss, post-implantation loss, and total resorptions, mean foetal count and foetal body weight of male, female, and total foetuses (male + female), anogenital distance of male and female foetuses, male sex ratio, Incidences of malformation/variation for external, visceral, head razor and skeletal examination. No treatment related adverse effects were seen in the 280 mg/kg b. wt./day group for following parameters: Mortality, salivation was observed approximately 10 to 15 minutes after dosing in 5 of 25 female rats and persisted for approximately 45 to 50 minutes. This finding is considered a response to the dose solution and toxicologically insignificant, hence considered as non-adverse in nature, Mean absolute and relative uterine weights, Serum level of T3, T4, and TSH, External and internal (gross) examination of the terminally sacrificed female rats, Absolute and relative weight of thyroid gland, histological examination of thyroid gland, mean numbers of corpora lutea, implantation sites, live foetuses, dead foetuses, resorptions (early, late, and total), pre-implantation loss, and post-implantation loss, the mean percent of live foetuses, dead foetuses, pre-implantation loss, post-implantation loss, and total resorptions, mean foetal count of male, female, and total foetuses (male + female), anogenital distance of male and female foetuses, statistically significant decrease in mean body weight of male foetuses (- 6.57 % than control group) was observed when compared to the control group. The body weights for female foetuses and for a composite of foetuses of both sexes were also lower than control group (- 4.55 % and - 5.57 %, respectively) without statistical significance. These lower foetus body weights were consistent with decreased maternal body weight. Hence, these findings were considered as secondary to the decreased maternal body weight and non-adverse in nature, Male sex ratio, Incidence of malformation/variation for external, visceral, head razor, and skeletal examination of foetuses.

The following treatment related adverse effects were seen in the 280 mg/kg bw/day group:

- Statistically significant decrease in the mean body weight of the pregnant female rats was observed on gestation day 20. Statistically significant decrease in 20th day corrected body weight of the pregnant female rats was observed. Statistically significant decrease in the mean body weight change of the pregnant female rats was observed during the gestation period 5-8, 8-11, 17-20, and 5-20. Decrease in the mean body weight change of the pregnant female rats was also observed during gestation period 11-14 and 14-17 without statistical significance.

- Statistically significant decrease in mean food consumption of the pregnant female rats was observed during the gestation period 5-8, 8-11, 11-14, 14-17, 17-20, and 5-20.

- Decrease in the mean body weight and body weight change are correlated with the decreased mean food consumption and are considered as adverse effect of the test item.

Based on the result of the present study, it is concluded that the “No Observed Adverse Effect Level (NOAEL)” of N-butylaminoethanol for maternal toxicity is 140 mg/kg bw/day and 280 mg/kg bw/day for developmental toxicity under the conditions and procedures followed in the study. The LOAEL for maternal toxicity was 280 mg/kg/day based on adverse effects observed on body weight, body weight change, and food consumption.

 

Justification for classification or non-classification

The source substances DBEA and BEA did not cause any adverse effect with regard to reproductive and developmental parameters.

Based on these data, the target substance Butyldiethanolamine does not need to be classified and labelled for reproductive or developmental effects in accordance with Regulation (EC) No1272/2008.