2-(2-aminoethoxy)ethanol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Test (OECD 471): negative (BASF, 2019)

HPRT Test (OECD 476): negative (BASF, 2019)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09.03.2018 - 09.11.2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Name of test substance: 2-(2-aminoethoxy)ethanol
CAS No.: 929-06-6
Identity: confirmed
Purity/composition: 99.4 area % corrected with the water content
Date of production: 14 Mar 2018

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

The phenobarbital/ß-naphthoflavone induced rat liver S9 fraction was obtained from Moltox Molecular Toxikology, Inc.; Boone, NC 28607; USA. The 5 mL portions of S9 fraction were stored at -70°C to -80°C.

Test concentrations with justification for top dose:

33 μg - 5000 μg/plate
In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate were generally selected as maximum test dose

Vehicle / solvent:

Due to the good solubility of the test substance in water, water was used as vehicle.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitro-o-phenylenediamine (NOPD)

Remarks:

sterility control: Additional plates were treated with soft agar, S9 mix, buffer, vehicle and the test substance but without the addition of tester strains

Details on test system and experimental conditions:

Standard plate test
The experimental procedure of the standard plate test (plate incorporation method) was based on the method of Ames et al.
• Salmonella typhimurium Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.
• Escherichia coli
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (trp+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.

Preincubation Test
The experimental procedure was based on the method described by Yahagi et al. and Matsushima et al..
0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) were incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar was added and, after mixing, the samples were poured onto the agar plates within approx. 30 seconds.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.

Rationale for test conditions:

Dose Selection:
According to the current OECD Guideline No. 471 the maximum concentration should be 5000 µg/plate, unless limited by toxicity or solubility of the test item.
In the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. The pre-experiment met the acceptance criteria, thus, it is reported as experiment I. The concentration range included two logarithmic decades.
The following concentrations were tested in experiment II:
33; 100; 333; 1000; 2500; and 5000 µg/plate

Evaluation criteria:

Acceptance criteria
Generally, the experiment was considered valid if the following criteria were met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain
• The sterility controls revealed no indication of bacterial contamination
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies compatible with the range of the historical positive control data or above
• Fresh bacterial culture containing approximately 109 cells per mL were used.

Assessment criteria
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

A weak bacteriotoxic effect (slight decrease in the number of his+ revertants) was observed in the standard plate test using test strain TA 1537 and in the preincubation assay using TA 1537 and TA 98 without S9 mix at a concentration of 5000 μg/plate, only.
No test substance precipitation was found with and without S9 mix.

Conclusions:

A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system.

Executive summary:

Under the experimental conditions chosen here, it is concluded that 2-(2 - aminoethoxy)ethanol is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 2018 - January 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

Name of test substance: 2-(2-aminoethoxy)ethanol
CAS No.: 929-06-6
Identity: Confirmed
Molecular weight: 105.14 g/mol
Physical state, appearance: Liquid, colorless, clear
Purity: 99.4 area%

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

The CHO (Chinese hamster ovary) cell line is a permanent cell line derived from the Chinese hamster and has a
- high proliferation rate (doubling time of about 12 - 16 hours)
- high plating efficiency (about 90%)
- karyotype with a modal number of 20 chromosomes.
Stocks of the CHO cell line (1-mL portions) are maintained at -196°C in the gas phase above the liquified nitrogen using 7% (v/v) DMSO in culture medium as a cryoprotectant. Each batch used for mutagenicity testing was checked for mycoplasma contamination

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix

The S9 fraction was prepared according to Ames et al. at BASF SE in an AAALAC-approved laboratory in accordance with the German Animal Welfare Act and the effective European Council Directive.
At least 5 male Wistar rats [Crl:WI(Han)] (200 - 300 g; Charles River Laboratories Germany GmbH) received 80 mg/kg b.w. phenobarbital i.p. and β-naphthoflavone orally (both supplied by Sigma-Aldrich, 82024 Taufkirchen, Germany) each on three consecutive days. During this time, the animals were housed in polycarbonate cages: central air conditioning with a fixed range of temperature of 20 - 24°C and a fixed relative humidity of 45 - 65%. The day/night rhythm was 12 hours: light from 6 am – 6 pm and darkness from 6 pm – 6 am. Standardized pelleted feed and drinking water from bottles were available ad libitum.
24 hours after the last administration, the rats were sacrificed and the livers were prepared using sterile solvents and glassware at a temperature of +4°C. The livers were weighed and washed in a weight-equivalent volume of a 150 mM KCl solution and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at +4°C, 5 mL portions of the supernatant (S9 fraction) were stored at -70°C to -80°C.

The concentrations of the cofactors in the S9 mix were:
− MgCl2 8 mM
− KCl 33 mM
− glucose-6-phosphate 5 mM
− NADP 4 mM
− phosphate buffer (pH 7.4) 15 mM

Test concentrations with justification for top dose:

1st Experiment
- without S9 mix: 0; 68.8; 137.5; 275.0; 550.0; 1100.0 μg/mL
- with S9 mix: 0; 68.8; 137.5; 275.0; 550.0; 1100.0 μg/mL

2nd Experiment
- with S9 mix: 0; 187.5; 375.0; 550.0; 750.0; 1100.0 μg/mL

The highest tested concentration was based on the molecular weight of the compound (105.14 g/mol).

Vehicle / solvent:

Due to the good solubility of the test substance in water, aqueous culture medium (Ham's F12) was selected as vehicle

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

culture media is used as solvent

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6-thioguanine-containing medium for another week.
Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted.

Expression period
The exposure period was completed by rinsing several times with HBSS. This was directly followed by the 1st passage in which 2x10^6 cells were seeded in 20 mL medium (in 175 cm2 flasks). The flasks were left to stand in the incubator for about 3 days at 37°C, relative humidity of ≥ 90% and 5% (v/v) CO2 atmosphere. After about 3 days, the cells were passaged a 2nd time in 175 cm2 flasks with 2x10^6 cells. After an entire expression period of 7 – 9 days the cells were transferred into selection medium (3rd passage).

Selection period
For selection of the mutants, two 175 cm2 flasks with 2x10^6 cells each from every treatment group, if possible, were seeded in 20 mL selection medium ("TG" medium) at the end of the expression period. The flasks were returned to the incubator for about 6 – 7 days. Only the cells resistant to 6-thioguanine that were assumed to be deficient of HPRT survived. At the end of the selection period, the medium was removed and the remaining colonies were fixed with methanol, stained with Giemsa and counted.

Rationale for test conditions:

according to guideline

Evaluation criteria:

Acceptance criteria
The HPRT assay is considered valid if the following criteria are met:
• The absolute cloning efficiencies of the negative/vehicle controls should not be less than 50% (with and without S9 mix).
• The background mutant frequency in the negative/vehicle controls should be within our historical negative control data range (95% control limit). Weak outliers can be judged acceptable if there is no evidence that the test system is not “under control”.
• Concurrent positive controls both with and without S9 mix should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase in mutant frequencies compared with the concurrent negative/vehicle control

Assessment criteria
A test substance is considered to be clearly positive if all following criteria are met:
• A statistically significant increase in mutant frequencies is obtained.
• A dose-related increase in mutant frequencies is observed.
• The corrected mutation frequencies (MFcorr.) exceeds both the concurrent negative/vehicle control value and the range of our laboratory’s historical negative control data (95% control limit) Isolated increases of mutant frequencies above our historical negative control range or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

A test substance is considered to be clearly negative if the following criteria are met:
• Neither a statistically significant nor dose-related increase in the corrected mutation frequencies is observed under any experimental condition.
• The corrected mutation frequencies in all treated test groups is close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95% control limit)

Statistics:

An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a possible dose-related increase of mutant frequencies. The used model is one of the proposed models of the International Workshop on Genotoxicity Test procedures Workgroup Report.
The dependent variable was the corrected mutant frequency and the independent variable was the concentration. The trend was judged as statistically significant whenever the one-sided p-value (probability value) was below 0.05 and the slope was greater than 0. In addition, a pair-wise comparison of each test group with the vehicle control group was carried out using one-sided Fisher's exact test with Bonferroni-Holm correction. The calculation was performed using R (9).
If the results of these tests were statistically significant compared with the respective vehicle control, labels (s p ≤ 0.05) are printed in the tables.
However, both, biological and statistical significance are considered together.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Remarks:

culture medium is vehicle

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

- In this study, in the absence and the presence of S9 mix, no precipitation in culture medium was observed up to the highest applied test substance concentration.
- After 4 hours treatment neither in the absence nor presence of metabolic activation, the cell morphology and attachment of the cells was adversely influenced (grade > 2) in any test group tested for gene mutations
- There was no decrease in the number of colonies as described by the relative survival in the presence and absence of S9 mix up to the highest evaluated concentrations for gene mutation
-

Based on the data and the observations from the pre-test and taking into account the current guidelines, the following doses were selected in this study.

Dose selection of the 1st Experiment:

Without/With S9 mix (4-hour exposure):

68.8 μg/mL, 137.5 μg/mL, 275.0 μg/mL, 550.0 μg/mL 1100µg/mL

For clarification of the results from the 1st Experiment, a 2nd Experiment was performed in the presence of S9 mix using the following concentrations.

Dose selection of the 2nd Experiment

With S9 mix (4-hour exposure)

187.5 μg/mL, 375.0 μg/mL, 550.0 μg/mL, 750.0 μg/mL, 1100.0 μg/mL

At least four concentrations were evaluated to describe a possible dose-response relationship.

Conclusions:

not mutagenic under the experimental conditions chosen

Executive summary:

According to the results of the present in vitro study, in two experiments performed independently of each other the test substance 2-(2-aminoethoxy)ethanol did not lead to a biologically relevant or dose-dependent increase the number of mutant colonies, either without S9 mix or after the addition of a metabolizing system.
The increased value obtained in the 1st Experiment in the presence of S9 mix is considered as irrelevant since it was within the historical negative control data range and could not be reproduced in a further experiment.
The mutation frequencies of the vehicle control groups were within our historical negative control data range (95% control limit) and, thus, fulfilled the acceptance criteria of this study.
The proficiency of the laboratory to perform the HPRT assay in CHO cells was demonstrated by the laboratory’s historical control database on vehicle and positive controls and by X-bar chart to identify the variability of the vehicle control data.
The increase in the frequencies of mutant colonies induced by the positive control substances EMS and DMBA clearly demonstrated the sensitivity of the test method and/or of the metabolic activity of the S9 mix employed. The values were within the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study

Thus, in the absence and the presence of metabolic activation, 2-(2-aminoethoxy)ethanol is not a mutagenic substance in the HPRT locus assay using CHO cells under the experimental conditions chosen

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Micronucleustest (OECD 474): negativ (Huntsman, 2001)

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 March 2001 - 28 May 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

Due to mortality in the high dose group (250 mg/kg) assigned to both the 48-hour harvest and the replacement animal groups, only four animals were available for harvest. This deviation had no impact on the integrity of this study.

GLP compliance:

yes

Type of assay:

micronucleus assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): Diglycolamine (DGA)
- Physical state: transparent colorless liquid
- Analytical purity: responsability of the Sponsor
- Lot/batch No.: Lot 8043-70, recieved on 2001-03-28
- Storage condition of test material: ambient

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: CD-1 (ICR) BR strain
- Source: Charles River Laboratories, Kingston, NY (dose range finding study) + Charles River Laboratories, Raleigh, NC (definitive study)
- Age at study initiation: young adult mice, 7 weeks
- Weight at study initiation: 20-40g (weight variation did not exceed 20% of the mean weight of each sex). 28.4 -33.5 g in the final study.
- Assigned to test groups randomly, by a computer program
- Fasting period before study: no
- Housing: sanitary, polycarbonate cages containing Sani-Chips hardwood Chip Laboratory bedding.
- Housed separated by gender, up to 5 animals per cage during acclimation, and full dose group after randomization.
- Diet (e.g. ad libitum): commercial diet, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 64-79 °F
- Humidity (%): 30-70%
- Air changes (per hr): at least 10 per hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark, 12 hrs light


IN-LIFE DATES: From: 17 April 2001 To: 08 May 2001

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: 0.5 % CMC (carboxymethyl cellulose)

Details on exposure:

one single intraperitoneal dose per mouse

Duration of treatment / exposure:

The animals were treated once and samples of bone marrow were taken 24 h and 48 h after the treatment.

Frequency of treatment:

The animals were treated once.

Post exposure period:

1st dose ranging study: 24h
2nd dose ranging study: 48h
final study: 24h and 48h

Dose / conc.:

62.5 mg/kg bw (total dose)

Dose / conc.:

125 mg/kg bw (total dose)

Dose / conc.:

250 mg/kg bw (total dose)

No. of animals per sex per dose:

6 male animals per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide

- Cyclophosphamide, dissolved in sterile deionized water
- Route of administration: oral, gavage
- Doses / concentrations: 80 mg/kg

Tissues and cell types examined:

Bone marrow of femur.
At least 2000 PCEs per animal were analyzed for the frequency of mironuclei. Cytotoxicity was assessed by scoring the numer of PCEs and normochromic erythrocytes (NCEs) in at least 500 erythrocytes for each animals.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
based on first dose range finding study with following doses: 500, 1000, 2000 mg/kg bw: all animals were found dead after 24h.
a second dose range finding study was performed with following doses: 62.5, 125, 250 mg/kg bw (3 animals per dose): animals were observed immediately after dosing, one hour after dosing and daily (for 2 days). 2 animals of the highest dose group died. Based on these results, the maximum tolerated dose was estimated to be 250 mg/kg bw.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Only males were used because there were no substantial differences in between the sexes in the dose range finding study.
Animals were dosed on April 30, 2001. Per dosing group (62.5, 125, 250 mg/kg, vehicle control and positive control), cells were harvested at 24 h after dosing. For both the 250 mg/kg and the vehicle control group, cells were harvested at 48 h after dosing in an extra group of 6 mice.
Animals were euthanized by CO2 inhalation followed by incision of the diaphragm.

DETAILS OF SLIDE PREPARATION:
Hind limb bones (tibias) were removed for marrow extraction. For each animal, the marrow flushed from the bones was combined in an individual centrifuge tube containing 3 to 5 mL fetal bovine serum (one tube per animal).
Following centrifugation to pellet the tissue, the supernatant was removed by aspiration and portions of the pellet were spread on slides and air dried. The slides were fixed in methanol, stained in May-Grünwald solution followed by Giemsa, and protected by permanently mounted coverslips.

METHOD OF ANALYSIS:
Slides were scored for micronuclei and the PCE to NCE cell ratio. The micronucleus frequency (expressed as percent icronucleated cells) was determined by analysing the number of micronucleated PCEs from at least 2000 PCEs per animal. The PCE: NCE ratio was determined by scoring the number of PCEs and NCEs observed scoring at least the first 500 erythrocytes per animal.

Evaluation criteria:

The criteria for the identification of micronuclei were those of Schmid (1976). Micronuclei were darkly stained and generally round, although amond- and ring-shaped micronuclei occasionally occurred. Micronuclei were sharp bordered and generally between 1/20th and 1/5th the size of the PCEs. The unit of scoring was the micronucleated cell, not the micronucleus.
The criteria for a positive response was the detection of a statistically significant increase in micronucleated PCEs for at least one dose level, and a statistically significant dose-related response. A test article that did not induce both of these responses as considered negative. Statistical significance was not the only determinant of a positive response. Biological relevance of the results were also considered in the final evaluation.

Statistics:

Assay data analysis was performed using an analysis of variance (Winer, 1971) on untransformed proportions of cells with micronuclei per animal and on untransformed PCE:NCE ratios when the variances were homogenous. Ranked proportions were used for heterogeneous variances. If the analysis of variance was statistically significant (p <= 0.05), a Dunnet's t-test was used to determine which groups, if any, were statistically significantly different from the vehical control. Analyses were performed separately for each sampling time.

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

signs of toxicity and mortality

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF first RANGE-FINDING STUDY
- Dose range: 500, 1000, 2000 mg/kg
- Clinical signs of toxicity in test animals: animals were found dead within 24h

RESULTS OF second RANGE-FINDING STUDY
- Dose range: 62.5, 125, 250 mg/kg
- Clinical signs of toxicity in test animals: no clinical signs in the lowest dosing groups (62.5 and 125 mg/kg). In the highest dosing group, slightly hypoactive 1h after dosing, rough haircoat 24h after dosing (+ 1/6 animals was found dead), clear discharge from eyes, hunched, labored respiration, hypoactive 48h after dosing.
- the maximum tolerated dose was estimated to be 250 mg/kg.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no induction
- % micronucleated PCEs (mean of 2000 per animal +/- S.E.):
62.5 mg/kg (24h harvest): 0.06 +- 0.02
125 mg/kg (24h harvest): 0.06 +- 0.02
250 mg/kg (24h harvest): 0.07 +- 0.03
250 mg/kg (48h harvest): 0.21 +- 0.08
vehicle control (24h harvest): 0.09 +- 0.04
vehicle control (48h harvest): 0.12 +- 0.03
positive control (24h harvest) 1.17 +- 0.12

- Appropriateness of dose levels and route: appropriate

- Statistical evaluation: the test article showed a statistically significant decrease in the PCE:NCE ratio at the 250 mg/kg dose level for the 24h harvest timepoint (cytotoxic to bone marrow). A statistically significant increase in micronucleated PCEs was not observed at any dose level or harvest timepoint. The positive control induced statistically significant increases in micronucleated PCEs as compared to that of the vehicle controls with a mean standard error of 1.17 +- 0.12 %.

Micronucleus Data Summary Table

 

Treatment	Dose	Harvest Time	% Micronucleated PCEs mean of 2000^a Per animals ± S.E. males	Ratio PCE:NCE Mean ± S.E. Males
Controls
Vehicle	0.5 % CMC	24 hr	0.09 ± 0.04	0.67 ± 0.09
		48 hr	0.12 ± 0.03	0.51 ± 0.02
Positive	CP 80 mg/kg	24 hr	1.17 ± 0.12*	0.55 ± 0.06
Test article	62.5 mg/kg	24 hr	0.06 ± 0.02	0.61 ± 0.05
	125 mg/kg	24 hr	0.06 ± 0.02	0.48 ± 0.05
	250 mg/kg	24 hr	0.07 ± 0.03	0.41 ± 0.04**
		48 hr	0.21 ± 0.08	0.41 ± 0.05

 

 

*Significantly greater than the corresponding vehicle control, ps0.01.

** Significantly less than the corresponding vehicle control, ps0.05.

^aOne animal from the 24-hour vehicle control group and two animais from the 62.5 mg/kg dose goup were scored out of >2000 PCE/animal. See individual animal data, Table 2.

CMC = Carboxymethy1 cellulose

CP = Cyclophosphamide

PCE = Polychromatic erythrocyte

NCE = Normochromatic erythrocyt

Conclusions:

The test article, Diglycolamine (DGA), was evaluated as negative in the mouse bone marrow micronucleus assay under the conditions of this assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro studies:

The test substance 2-(2-aminoethoxy)ethanol was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium ( TA 1535, TA 100, TA 1537, TA 98) and Escherichia coli ( E. coli WP2 uvrA), in a reverse mutation assay with concentrations up to 5000 µg/plate. A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system. (BASF, 2019)

 

Another AMES-test with 2 -(2 -aminoethoxy)ethanol was evaluated for mutagenicity in the Salmonella/microsome preincubation assay using a standard protocol approved by the National Toxicology Program. Doses of 0, 12.5, 25, 50, 100, 500, 2500, 5000 µg/plate were tested in four Salmonella typhimurium strains (TA98, TAl00, TAl535 and TAl537) in the presence and absence of Aroclor-induced rat or hamster liver S9. These tests were negative and the highest ineffective dose level tested in all four Salmonella tester strains under all treatment conditions was 2500 µg/plate (Zeiger et al. 1988).

A further AMES-test with 5 strains (TA98, TA100, TA 1535, TA1537 and TA1538) confirmed the negative results (Huntsman, 1982). Several other AMES-tests could not be taken into consideration because the test substance was a unknown mixture containing 2-(2-aminoethoxy)ethanol and several other substances (composition confidential, concentration of test substance unclear): In these assays the test substance showed ambiguous results (Chemfirst 1992-1997)

 

Furthermore, the substance 2-(2-aminoethoxy)ethanol was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. The test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other (BASF, 2019).

 

In an in vitro mammalian cell transformation assay using Mouse BALB/3T3 Cells, both in the absence and presence of metabolic activation (S9 mix) no dose response relationship was observed and transformed foci were not considered to be significant over control dose ranges (Huntsman, 1982).

 

In an unscheduled DNA damage and repair assay with male F344 rat hepatocytes, according to the OECD Guideline 482, no genotoxic potential could be observed (Huntsman, 1982).

 

In vivo studies:

In an in vivo MNT the test result was negative for chromosomal aberration (Huntsman, 2001).

Conclusion:
All endpoints are considered addressed. Reliable experimental data are available with the test substance in the Ames Assay, the HPRT Assay, in vitro cell transformation assay (BALB/3T3 Assay), in vitro Unscheduled DNA Synthesis (UDS) Assay and the in vivo Mouse Micronucleus Assay. In each case, a guideline method was followed under GLP conditions which resulted in negative findings.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Several bactieral reverse mutation assay, an HPRT, an in vitro mammalian cell transformation assay, an in vitro unscheduld DNA damara and repair assay and an in vivo mouse micronucleus assay with the test substance were negative. As a result the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the fourteenth time in Regulation (EC) No. 2020/217.