Ethanol, 2-ethoxy-, manufacture of, by-products from

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

2 900 mg/kg bw/day

Additional information

There is no reproductive toxicity study available for the UVCB substance itself or its major components. There is a 2 -generation reproductive toxicity study available for the related analogue 2 -(2 -ethoxyethoxy)ethanol.  A full and detailed justification for a category approach for meeting the individual data requirements for glycol ethers is included as an attachment in chapter 13 to the dossier submitted by the lead registrant.  There is clear data to show that within the glycol ethers family and for all reprotoxicity end points, toxicity decreases markedly as the lengths of the alkyl and alkoxy chains increase. For reproductive toxicity, the most sensitive effect is on the male testes, which manifest as testicular atrophy and arrestment of spermatogenisis.  All reproductive effects are caused by the alkoxyacetic acid metabolite rather than the parent glycol ether itself. From the available data, the most toxic member of the category is methoxyethanol. The trend towards manifest reproductive toxicity in the short chain glycol ethers is at least in part related to the trend for slower elimination of these metabolites with decreasing alkyl chain length and decreasing numbers of EO units in the molecule.

A detailed analysis of all the available data indicates that for substances with no data, extrapolation from a source substance with data that is to the left and/or upwards of the target glycol ether within the glycol ether family matrix is valid and robust This extrapolation as used here is shown in the table below:

Glycol ethers family
Number of EO units	Alcohol chain length
	Methyl	Ethyl	Propyl	Butyl	Pentyl	Hexyl
Mono	R	R
Di		Source
Tri		Target A
Tetra		Target B

 Note: R=only members of the family showing reproductive toxicity. Target A and Target B are the two major components of this UVCB substance.

The two-generational study on the source substance 2 -(2 -ethoxyethoxy)ethanol investigated the effects in reproduction and fertility of concentrations of 0%, 0.25%, 1.25%, and 2.5% in drinking water. Male and female CD-1 mice were continuously treated for 1 week prior to mating and for a 14 week breeding period followed by a 21 day holding period when they were separated and housed individually. There were two deaths among the male F0 animals treated at high dose and small decreases in the mean body weights. The body weights of the F1 offspring exposed to 2.5% level were slightly depressed at birth, at weaning and at 74 +/-10 days. 2 -(2 -ethoxyethoxy)ethanol did not have adverse effects on fertility and reproductive performance despite a 34% decrease in caudal epididymal sperm motility in the F1 males at 2.5%. The highest dose also increased liver weight and decreased brain weight in both sexes of the F1 generation. The NOAEL for F0 and F1 generations can be established at 1.25% 2 -(2 -ethoxyethoxy)ethanol based on systemic and reproductive (sperm motility) effects respectively (equivalent to 2200mg/kg), and 2.5% for the F2 generation. The substance 2 -(2 -ethoxyethoxy)ethanol is an analogue of the test substance used in the study and this result can be considered predictive of the toxicity of higher members of the series. Calculated pro-rata for molecular weight (based on the lowest major component of the UVCB substance) the no effect level would be ~2900mg/kg.

In conclusion, the available evidence shows that it is scientifically justifiable and acceptable to read across from 2-(2-ethoxyethoxy)ethanol to the higher molecular weight members in the series of the ethyl series that comprise the UVCB substance that is the subject of this registration and that the latter does not have any adverse reproductive effects.

Effects on developmental toxicity

Description of key information

RAT predicted NOAEL >678mg/kg.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

key study

Study period:

1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study. Rationale for weight of evidence approach using read across substances is described in the overall section summary.

Qualifier:

according to guideline

Guideline:

EPA OTS 798.4900 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)BR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage Michigan.
- Age at study initiation: (females) 71 days old; (males) 73 days old
- Weight at study initiation: (females) 174 – 227 g; (males) 238 – 342 g
- Housing: Female rats were housed individually in wire-bottomed stainless-steel cages suspended above absorbent paper liners, except during cohabitation.
- Diet: Certified Rodent Chow #5002 (Ralston Purina) ad libitum
- Water: Local water passes through a reverse osmosis membrane was available ad libitum from an automatic watering system.
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature: 70 - 78°F
- Humidity: 35 - 65%
- Air changes: minimum 10 changes/hr 100% fresh air that had been passed through 99.97% HEPA filters (Airo Clean~ room).
- Photoperiod: 12 hour light/12 hour dark cycle.

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on exposure:

Test material was administered as received. The control group rats were administered R.O. deionized water at a dosage volume (4.8 mL/kg) that was equivalent to that given to the highest dosage group.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Test material was administered as received.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: (5 days) Afternoon of September 19, 1989 to the morning of September 24, 1989.
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy:sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

Gestation days 6-15

Frequency of treatment:

Once daily on gestation day 6 -15. Each daily dosage given to the rats was administered at approximately the same time each day [between 1242 and
1542 hours EDT (1142 and 1442 hours EST)].

Duration of test:

Animals were sacrificed on day 20 of gestation.

Dose / conc.:

625 mg/kg bw/day

Dose / conc.:

1 250 mg/kg bw/day

Dose / conc.:

2 500 mg/kg bw/day

Dose / conc.:

5 000 mg/kg bw/day

No. of animals per sex per dose:

25

Control animals:

yes

Details on study design:

- Dose selection rationale: All dosages were adjusted daily on the basis of individual body weights recorded immediately prior to intubation.
- Rationale for animal assignment (if not random): The rats were assigned to the five dosage groups using a computer-generated (weight-ordered) randomization procedure based on day 0 of gestation body weights.
- Other: Animals were chosen for evaluation because it is one mammalian rodent species standardly accepted for use in developmental toxicity, this strain of rat had been demonstrated to be sensitive to developmental toxicants, it has been used throughout industry for nonclinical studies of developmental toxicity, historical control data and experience exist and it is the rodent species specified in the TGME Testing Consent Order.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily. Viability checks were made twice daily.

BODY WEIGHT: Yes
- Time schedule for examinations: Once weekly prior to mating. Day 0 of presumed gestation and daily during the dosage and postdosage periods.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Day 0 of presumed gestation and daily during the dosage and postdosage periods.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: uterus, ovaries.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: Yes
- Other: Gravid uterine weights were inadvertently not recorded for 8, 5, 4, 5 and 7 dams in the O(Control), 625, 1250, 2500, and 5000 mg/kg/day dosage groups, respectively. As sufficient numbers of values were available to allow adequate interpretation of these data, this deviation did not adversely affect the outcome of the study.

Fetal examinations:

- External examinations: Yes
- Soft tissue examinations: Yes: half per litter.
- Skeletal examinations: Yes: half per litter.
- One-half of fetuses in each litter were examined for soft tissue alterations using an adaptation of Wilson’s sectioning technique. The remaining fetuses in each litter were cleared, stained with alizarin red S and examined for skeletal alterations.

Statistics:

Maternal and foetal incidence were analyzed using the Variance Test for Homegeneity of the Binomial Distribution. Maternal body weight and feed consumption data, organ weight data, and litter averages for percent male fetuses, percent dead or resorbed conceptuses per litter, fetal body weights, fetal ossification sites, and percent fetal alterations were analyzed using Bartlett’s Test of Homogeneity of Variances and the Analysis of Variance, when appropriate. If the Analysis of Variance was significant (P ≤ 0.05), Dunnett’s test was used to identify the statistical significance of individual groups. If the Analysis of Variance was not appropriate, the Kruskal-Wallis Test was used when less than or equal to 75% ties were present; when more than 75% ties were present, the Fisher’s Exact Test was used. In cases where the Kruskal-Wallis Test was statistically significant (P ≤ 0.05), Dunn’s Method of Multiple Comparisons was used to identify the statistical significance of individual groups. All other Caesarean-sectioning data were evaluated using the procedure previously described for the Kruskal-Wallis Test.

Indices:

Gross external or soft tissue variations in the fetuses.

Historical control data:

No further data available.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
One non-pregnant high dosage group rat was found dead on day 13 of presumed gestation. Ataxia, decreased motor activity, and lost righting reflex occurred for this rat, as well as persistent weight loss and reduced feed consumption beginning after the initial dosage. Necropsy revealed distention of the stomach and intestines with gas; observations that were probably secondary to moderate autolysis. No other deaths occurred. The 5000 mg/kg/day dosage of TGME caused significant numbers (P≤ 0.01l) of rats to have decreased motor activity, excess salivation, ataxia, and impaired righting reflex, as compared with the control group numbers. Lost righting reflex, urine-stained abdominal fur, and rales occurred for a few high dosage group rats. The 5000 mg/kg/day dosage group rat that was found dead (13364) had gaseous distention of the stomach and intestines-that may have been caused by autolysis or anorexia. As described in the following information, all other gross lesions were considered unrelated to the test substance and occurred at incidences that were not dosage dependent. One 1250 mg/kg/day dosage group rat (13310) had common urinary tract lesions. Another 1250 mg/kg/day dosage group rat (13312) had a mass, in the region of the mammary gland that was probably a galactocele C. Significant reductions (P ≤ 0.01) in maternal body weight gains occurred for the high dosage group during the dosage period (calculated as days 6 to 16 of gestation). As a result the mean maternal body weights were significantly reduced (P ≤ 0.05 to P ≤ 0.01) for the high dosage group on days 9, 12 and 16 of gestation, as well as during the post-treatment period, on days 18 and 20 of gestation. The mean gravid uterine weight was significantly lower (P ≤ 0.05) for the high dosage group, and the mean maternal body weight on day 20e (day 20 of gestation body weight corrected for the gravid uterine weight) tended to be lower for the high dosage group, as compared with the control group mean. Reflecting these events, maternal body weight changes from days 6 to 20e and 0 to 20e of gestation were significantly decreased (P ≤ 0.05 to P ≤ 0.01) for the high dosage group. Maternal body weights were not affected by treatment with TGME for any of the other dosage groups. Groups given 2500 and 5000 mg/kg/day of TGME had statistically significantly reduced feed consumption for the entire dosage period (calculated as days 6 to 16 of gestation), as compared with the control group. The 2500 mg/kg/day dosage group had significantly reduced (P ≤ 0.05 to P ≤ 0.01) relative maternal feed consumption from days 6 to 16, 6 to 18, and 12 to 16 of gestation, and the 5000 mg/kg/day dosage group had significantly reduced (P ≤ 0.01) mean absolute and relative maternal feed consumption throughout the entire dosage period. Feed consumption was significantly increased (P ≤ 0.05 to P ≤ 0.01) for the high dosage group during the post dosage period, as compared with the control group values. Despite these rebound phenomena, absolute and relative maternal feed consumption values were significantly decreased (P ≤ 0.01) for the high dosage group on days 6 to 20 of gestation and for the entire gestation period (days 0 to 20 of gestation).

Dose descriptor:

NOEL

Effect level:

625 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

1 250 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Small, significant increases (P ≤ .05 to P ≤ 0.01) n embryo-fetal lethality occurred in the 5000 mg/kg/day dosage group (litter averages for total resorptions, late resorptions, percentage of resorbed conceptuses and dams with at least one resorption), as compared with the control group values. None of these effects were seen for the other dosage groups. Dosages of 1250 mg/kg/day and higher reduced fetal body weights. However, this effect was statistically significant (P ≤ 0.01) for only the 2500 and 5000 mg/kg/day dosage groups. Dosages of TGME as high as 5000 mg/kg/day did not cause gross external, internal soft tissue or skeletal malformations in the fetuses. These dosages also did not result in increased incidences of gross external or soft tissue variations in the fetuses. There were no dosage-dependent, statistically significant differences in the litter or fetal incidences of these parameters. Groups given 1250 mg/kg/day and higher dosages of TGME had significant increases (P ≤ 0.05 to P ≤ 0.0l) in the litter and/or fetal incidences of reversible delays in fetal ossification, common observations in fetuses with reduced body weights. The 2500 and 5000 mg/kg/day dosage groups also had significant increases (P ≤ 0.05 to P ≤ 0.0l) in the litter and/or fetal incidences of cervical ribs, a common variation in rats.

Dose descriptor:

NOEL

Effect level:

625 mg/kg bw/day

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: developmental effects

Abnormalities:

not specified

Developmental effects observed:

not specified

The death of one nonpregnant high dosage group (5000 mg/kg/day) rat was considered treatment-related. This rat had clinical signs of treatment with the test substance (ataxia, decreased motor activity, lost righting reflex, persistent weight loss, and reduced feed consumption). At necropsy, gaseous distention of the stomach and intestines may have been interrelated with anorexia and/or moderate autolysis. No other deaths occurred in this study, and no other gross lesions seen during necropsy examination were considered to be effects of TGME.

Conclusions:

The results from this study indicate that TGME is not a developmental toxicant and does not present a unique hazard to development of rat conceptuses. There were no increases in the incidence of external, internal, soft tissue, or skeletal malformations or in the incidences of external or internal soft tissue variations at doses as high as 5000 mg/kg/day of TGME.

Executive summary:

In a guideline (TSCA) and GLP study, the developmental toxicity of TGME was evaluated in rats following oral (gavage) administration at dosages up to 5,000 mg/kg bw/day on Days 6-15 of gestation. The 1250 mg/kg/day dose level slightly reduced relative feed consumption during the dosing period, but the absolute feed consumption at this level and the average maternal body weights and body weight gains at the 1250 and 2500 mg/kg/day levels were not affected by treatment with TGME. Thus, 1250 mg/kg/day is considered a NOAEL, and 625 mg/kg/day is considered a NOEL for maternal toxicity.

There were no increases in the incidence of external, internal soft tissue, or skeletal malformations or in the incidences of external or internal soft tissue variations at doses as high as 5000 mg/kg/day of TGME. Reversible delays in fetal ossification and slightly lower fetal body weight (not statistically different from the control weight) occurred in the 1250 mg/kg/day dosage group. Thus, 1250 mg/kg/day is considered very near the NOAEL for TGME developmental toxicity, and 625 mg/kg/day is considered the NOEL for developmental toxicity.

For TGEE, the lowest member of the homologous series in this UVCB substance, this would be equivalent in molecular weight terms to 678 and 1357mg/kg/day respectively.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

678 mg/kg bw/day

Additional information

There are no full developmental toxicity studies available using the UVCB substance itself as the test substance but there is data available for a close structural analogue. A full and detailed justification for a category approach for meeting the individual data requirements for glycol ethers is included as an attachment in chapter 13 of the IUCLID dossier submitted by the lead registrant  There is clear data to show that within the glycol ethers family and for all reprotoxicity end points, including developmental effects, toxicity decreases markedly as the lengths of the alkyl and alkoxy chains increase. All reproductive effects are caused by the alkoxyacetic acid metabolite rather than the parent glycol ether itself. From the available data, the most toxic member of the category is methoxyethanol. The trend towards manifest reproductive toxicity in the short chain glycol ethers is at least in part related to the trend for slower elimination of these metabolites with decreasing alkyl chain length and decreasing numbers of EO units in the molecule.

The ideal situation in terms of read across and extrapolation would be to have data on all of the lower molecular weight members until a ‘boundary’ was evident delineating the point at which toxicity decreased to a negligible level, at which point it would be valid to extrapolate in any direction to the right or downwards on the category map to fill an empty data gap. Extrapolation from a substance with data that is to the left and upwards of the target glycol ether is valid and robust. Using data from 2-(2-(2 -methoxyethoxy)ethoxy) ethanol can be considered as meeting these requirements since, according to the trend analysis, if the latter is not reprotoxic, then neither will be substances of higher molecular weight or longer alcohol chain length in the series of ethylene glycol ethyl ethers, such as the components of the target substance.

The positions of the source and target substance in the glycol ethers family matrix are shown below:

Number of EO units	Alcohol chain length
	Methyl	Ethyl	Propyl	Butyl	Pentyl	Hexyl
Mono	R1	R1
Di	R2
Tri	Source	Target A
Tetra		Target B

 Note: R=only members of the family that show developmental toxicity, R1=category 1B, R2=category 2. Target A and Target B are the two major components of this UVCB substance.

Details of the available data on the source substance are described below:

In a guideline (TSCA) and GLP study, the developmental toxicity of TGME was evaluated in rats following oral (gavage) administration at dosages up to 5,000 mg/kg bw/day on Days 6-15 of gestation. The 1250 mg/kg/day dose level slightly reduced relative feed consumption during the dosing period, but the absolute feed consumption at this level and the average maternal body weights and body weight gains at the 1250 and 2500 mg/kg/day levels were not affected by treatment with TGME. Thus, 1250 mg/kg/day is considered a NOAEL, and 625 mg/kg/day is considered a NOEL for maternal toxicity. There were no increases in the incidence of external, internal soft tissue, or skeletal malformations or in the incidences of external or internal soft tissue variations at doses as high as 5000 mg/kg/day of TGME. Reversible delays in fetal ossification and slightly lower fetal body weight (not statistically different from the control weight) occurred in the 1250 mg/kg/day dosage group. Thus, 1250 mg/kg/day is considered very near the NOAEL for TGME developmental toxicity, and 625 mg/kg/day is considered the NOEL for developmental toxicity. For TGEE, the lowest member of the homologous series in this UVCB substance, this would be equivalent in molecular weight terms to 678 and 1357mg/kg/day respectively

Overall, the available evidence indicates that developmental toxicity is not a significant property of this substance.

Justification for classification or non-classification

There is no indication of significant reproductive toxicity effects, even at doses well in excess of 1000mg/kg bw, the normal limit dose used. There is no indication that reproductive toxicity is a characteristic of this substance and that classification is warranted for this end point.