(tris[2-(2-hydroxyethoxy)ethyl] borate )

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Read across approach adequate to support 7.6.1 according to "Guidance on information requirements and chemical safety assessment Chapter R.6: QSARs and grouping of chemicals" (ECHA 2008) and Read-Across Assessment
Framework (RAAF) (ECHA 2017)

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

Read across approach adequate to support 7.6.1 according to "Guidance on information requirements and chemical safety assessment Chapter R.6: QSARs and grouping of chemicals" (ECHA 2008) and Read-Across Assessment
Framework (RAAF) (ECHA 2017)

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

no guideline followed

Species:

rat

Strain:

other: CD (SD ) BR

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on study design:

Groups of 25 mated female rats (203 to 256 g) were given daily dosages of 4.8 ml of deionized water (control), or 0.6, 1.2, 2.4, and 4.8 ml/kg/day of triethylene glycol monomethyl ether (TGME) by gavage. These doses corresponded to 0, 625, 1250, 2500 or 5000 mg/kg/day. All doses were adjusted daily according to body weights recorded immediately prior to intubation.
Rats were observed at least twice daily during the dosage and postdosage periods for clinical signs, signs of resorption, premature deliveries and death. Body weight and feed consumption were recorded on Day 0 of presumed gestation and from days 6 through 20 of gestation. Rats were euthanized on Day 20 of presumed gestation, and the thoracic and abdominal viscera were examined for gross lesions. The uterus was excised from each rat and weighed. The number and placement of implantations were recorded and sites were categorized as early or late resorptions, or
live or dead fetuses. Each ovary was examined for the number of corpora lutea. Fetuses were weighed, sexed, and examined for external alterations. One-half were examined for soft tissue alterations, and the remaining half were examined for skeletal alterations. Dams that were found dead were necropsied on day of death and subjected to the same procedures described for scheduled termination.
Maternal and fetal incidence data were analyzed using the variance test for homogeneity 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 data were homogeneous). If the analysis of variance was significant, Dunnett's Test was used to identify the statistical significance of individual groups. If data were not homogeneous, 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, 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 procedures previously described for the Kruskal-Wallis Test.

Details on results:

One nonpregnant animal in the high dose group (5000 mg/kg/day) was found dead on day 13 of presumed gestation. This was considered by the authors to be treatment-related. Significant numbers of rats treated with the high dose exhibited decreased motor activity, excess salivation, ataxia, and impaired righting reflex. Food consumption of high dose animals were reduced over the entire dosage period. Average maternal body weight gains of rats in the high dose group were
reduced on days 6-9, 6-12, 12-16, and 6-16 of gestation. Consequently, average body weights of these animals were reduced on days 9, 12, and 16. Average gravid uterine weights of high-dose animals were also reduced.
Food consumption of rats receiving 2500 mg/kg/day was reduced during days 6-16, 6-18, and 12-16. Food consumption and average maternal body weights and body weight gains in rats receiving 1250 mg/kg/day were not significantly different from controls. Therefore, 1250 mg/kg/day was considered by study personnel to be the NOAEL for maternal toxicity.

Details on maternal toxic effects:

There was no effect of TGME on the number of pregnant dams or number of copora lutea, implantations, live litter size or fetal sex ratios.

Dose descriptor:

NOAEL

Effect level:

1 250 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

mortality

organ weights and organ / body weight ratios

Details on embryotoxic / teratogenic effects:

Foetal data (live/dead, sex, external defects, soft tissue and skeletal defects):
Significant increases in embryo-fetal lethality (litter averages for total resorptions (1.6 versus 0.6 in control), late resorptions (0.3 versus 0 in controls), percentage of resorbed con ceptuses (12.0 versus 4.4 in control) and dams with at least one resorption (81.8% versus 47.8 in control) occurred in the
5000 mg/kg group. Fetuses from rats treated with 2500 or 5000 mg/kg had lower body weights than controls (3.04 and 2.56 g (respectively) versus 3.32 in controls).
There was no effect of TGME on the incidences or types of gross external or internal soft tissue malformations. Groups given 1250 mg/kg/day and higher doses of TGME had significant increases
in the litter and/or fetal incidences of reversible delays in fetal ossification. Fetuses from rats given
2500 or 5000 mg/kg/day also had a significant increase in the incidence of cervical ribs. The NOAEL for developmental toxicity was considered by study personnel to be 625 mg/kg/day.

Dose descriptor:

NOAEL

Effect level:

625 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

reduction in number of live offspring

fetal/pup body weight changes

changes in litter size and weights

skeletal malformations

other: tissue malformation and fetal ossification

Developmental effects observed:

no

625 mg/kg bw/day is a conservative NOAEL based on specific doses used in the study.

The authors remarked that the skeletal variations noted were common observations in fetuses with reduced body weights. Since only reversible delays in fetal ossification were observed in the 1250 mg/kg/day group, the actual NOAEL may be close to this concentration

Cross-reference 2

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Justification for type of information:

Read across approach adequate to support 7.6.1 according to "Guidance on information requirements and chemical safety assessment Chapter R.6: QSARs and grouping of chemicals" (ECHA 2008) and Read-Across Assessment
Framework (RAAF) (ECHA 2017)

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Name of test material: Boric acid
- Analytical purity: > 99.7 %
- stability: Stable
- Lot/batch No.: 872703

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hazleton Research Products Inc., Denver, PA, USA
- Age at study initiation: 5 months of age
- Weight at study initiation: 2690-4380 g

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

VEHICLE
- Concentration in vehicle: 55 mg/mL boric acid
- Amount of vehicle: 5 mg/mL boric acid

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

no data

Details on mating procedure:

Impregnation procedure: Artificial insemination; designated Day 0

Duration of treatment / exposure:

Groups of 30 rabbits were used treated on Day 6 - 19 post-mating

Frequency of treatment:

no data

Duration of test:

terminate on day 30 of gestation

Remarks:

0, 62.5, 125 or 250 mg/kg bw boric acid equivalent to 0, 10.9, 21.8 and 43.5 mg B/kg bw

No. of animals per sex per dose:

30 females/group

Control animals:

yes

Details on study design:

no data

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 30
- Organs examined: Uterus

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 early resorptions: No data
- Number of late resorptions: No data
- Other:
Uteri were stained with ammonium sulphide as no implantations were visible.
Litter size, number of dead foetuses and foetal weight were assessed.

Fetal examinations:

- External examinations: Yes, including assessment for cleft palate
- Soft tissue examinations: Yes by dissection (Staples) and sex determined
- Skeletal examinations: Yes, all foetuses were skinned and cleaned and stained with alcian blue/alizarin red S
- Head examinations: Yes

Statistics:

no data

Indices:

no data

Historical control data:

no data

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Pregnant does exhibited no overt symptoms attributable to boric acid toxicity except in the high dose group. A decreased food intake (30 % reduction vs. controls during exposure period) and decreased maternal bodyweight were observed. Vaginal bleeding was noted at 43.5 mg B/kg bw between gestational days 19 - 30. All high-dose animals with vaginal bleeding had no live foetuses at sacrifice. At mid dose, increased body weight gain not clearly adverse. The authors considered 43.5 mg B/kg bw as the LOAEL for pregnant does and 21.8 mg B/kg bw as the NOAEL for maternal toxicity.

Dose descriptor:

LOAEL

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity - Based on reduced food intake, reduced body weight gain and abortions.

Dose descriptor:

NOAEL

Effect level:

125 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

LOAEL

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity - Based on increased resorptions and CVS malformations in surviving foetuses.

Dose descriptor:

NOAEL

Effect level:

125 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Dose descriptor:

LOAEL

Effect level:

43.5 mg/kg bw/day

Based on:

element

Basis for effect level:

other: maternal toxicity - Based on reduced food intake, reduced body weight gain and abortions.

Dose descriptor:

NOAEL

Effect level:

21.8 mg/kg bw/day

Based on:

element

Basis for effect level:

other: maternal toxicity

Dose descriptor:

LOAEL

Effect level:

43.5 mg/kg bw/day

Based on:

element

Basis for effect level:

other: developmental toxicity - Based on increased resorptions and CVS malformations in surviving foetuses.

Dose descriptor:

NOAEL

Effect level:

21.8 mg/kg bw/day

Based on:

element

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
At the highest dose in this study of 250 mg/kg bw boric acid (43.5 mg B/kg bw/day), 90 % of implants/litter were resorbed compared to 6 % for controls, and 73 % had complete litter loss (0 % in controls). In the mid and low dose groups, no difference in percentage resorptions per litter was seen, compared to controls.
Average foetal bodyweight per litter was 92 % of the controls at the high dose (43.5-mg B/kg bw) but even at this exposure, it did not reach statistical significance possibly due to the low number of pups surviving (14 fetuses from 6 litters).
An increased incidence of malformed live foetuses/litter was observed at 43.5 mg B/kg bw, primarily due to cardiovascular defects (72 % for major defects of heart and/or great vessel in the high-dose group vs. 3 % in controls). In the mid and low dose groups, there was no increase in malformations per litter or total malformations. There were no variations between any groups concerning the incidence of skeletal malformations.
The only skeletal variations of interest was a dose related reduction in the incidence of extra ribs on Lumbar I which the authors did not consider to be toxicologically important.
Since no definitive developmental effects were observed in animals exposed to either 62.5 or 125 mg/kg bw boric acid (10.9 or 21.8 mg B/kg bw/day), the authors concluded that 125 mg/kg boric acid per day (21.8 mg B/kg bw/day) was the NOAEL for developmental toxicity.

Dose descriptor:

NOAEL

Effect level:

>= 21.8 mg/kg bw/day (nominal)

Based on:

element

Sex:

male/female

Basis for effect level:

other: Since no definitive developmental effects were observed in animals exposed to 62.5 or 125 mg/kg bw boric acid (10.9 or 21.8 mg B/kg bw/day), it is concluded that 125 mg/kg boric acid per day (21.8 mg B/kg bw/day) was the NOAEL for developmental toxicity.

Dose descriptor:

NOAEL

Effect level:

>= 125 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Since no definitive developmental effects were observed in animals exposed to 62.5 or 125 mg/kg bw boric acid (10.9 or 21.8 mg B/kg bw/day), it is concluded that 125 mg/kg boric acid per day (21.8 mg B/kg bw/day) was the NOAEL for developmental toxicity.

Abnormalities:

not specified

Developmental effects observed:

not specified

Maternal effects

Parameter	Study control data	Low dose	Medium dose	High dose
Number of dams examined	30	30	30	30
Clinical findings during application of test substance				reduced food and bodyweight
Mortality of dams (%)	0	1	1	0
Abortions	0	0	0	3
Body weight gain               day 6-19                                              day 0-30	93 357	132 493	97 543	-137* 226
Food consumption g/kg/day  day 0-6                                                 day 6-19                                                  day 19-25	48.1 38.8 36.9	48.0 40.0 37.0	48.9 38.7 40.0	46.4 26.6* 44.9
Pregnancies  % pregnant at sacrifice	75	89	87	96
Necropsy findings in dams dead before end of test		gavage error lungs	stomach damage

* P < 0.05.

 

Litter response (Caesarean section data)

Parameter	Control data	Low dose	Medium dose	High dose
Corpora lutea                        number                                                  ±SEM mean No. per dam	12.2 0.7	10.7 0.5	11.5 0.4	10.0* 0.7
Implantations sites per litter     number                                                      ±SEM total/number of dams	9.5 0.8	8.4 0.6	8.3 0.5	8.6 0.7
Resorptions % per litter               % total/number of dams          ±SEM	6.3 2.4	5.9 1.9	7.7 2.1	89.9 5.0
total number of foetuses	159	175	153	14
total number of litters	18	23	20	6
live foetuses / litter                 number state ratio                               ±SEM	8.8 0.8	7.6 0.6	7.7 0.5	2.3* 0.8
dead foetuses / litter        % state ratio	0	2.8	0.4	0
foetus weight (mean)                   weight (g)                                                        ±SEM	44.8 1.5	46.5 1.4	45.7 1.2	41.1 2.7
Foetal sex ratio    % male per litter                                    ±SEM	50 5	51 4	55 4	69 10

* P<0.05

 

 

Examination of the foetuses

Parameter	Control data	Low dose	Medium dose	High dose
External malformations^* % foetuses per litter  ±SEM	0.8 0.8	1.4 1.0	1.0 1.0	11.1* 8.2
External malformations    No. of foetuses	1	2	1	2
Skeletal malformations^* % foetuses per litter ±SEM	19.9 5.4	19.9 4.0	24.3 6.4	38.9 20.0
Skeletal malformations     No. of foetuses	30	39	44	4
Visceral malformations^* % foetuses per litter  ±SEM	7.3 1.9	5.9 2.0	7.4 2.0	80.6* 16.3
Visceral malformations      No. of foetuses	13	11	12	11
% foetuses with cardiovascular       % malformations                               ±SEM	2.7 1.6	3.1 1.5	4.2 1.3	72.2* 16.5

*P<0.05

Conclusions:

The highest dose was very toxic to dams and 90 % of implants were resorbed at the highest dose level, and 72 % of surviving foetuses had cardiac or great vessel malformations or increase in resorptions were reported in the mid and low dose groups.

Cross-reference 3

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

developmental toxicity

Type of information:

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

Remarks:

Structural analogy validated with Qsar Toolbox version 4.0

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study is well documented and performed according to Charnoff and Kavlock screening method. In combination with the 28-day toxicity study , first indications on reproductive toxicity are avaialable.

Justification for type of information:

Read across approach adequate to support 7.6.1 according to "Guidance on information requirements and chemical safety assessment Chapter R.6: QSARs and grouping of chemicals" (ECHA 2008) and Read-Across Assessment
Framework (RAAF) (ECHA 2017)

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: Charnoff and Kavlock protocol

Deviations:

not applicable

Remarks:

10 dams per group - dosing GD 7-17, pups followed to post partum day 5 of lactation.

Principles of method if other than guideline:

Charnoff, N. and Kavlock, R.J. (1981). A potential in vivo screen for the determination of teratogenic effects in mamals. Teratology, 21, 34A.
The method uses smaller numbers of litters, and no skeletal or visceral examination, but follow pups post-partum to day 5 of lation at which time dams and pups are necropsied for gross pathological lesions.

GLP compliance:

yes

Specific details on test material used for the study:

Constituent

Species:

rat

Strain:

not specified

Route of administration:

oral: gavage

Vehicle:

not specified

Frequency of treatment:

daily

Duration of test:

lactation day 5

Remarks:

0,25,150,1000 mg/kg bw/day

No. of animals per sex per dose:

10 rats per group

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Behaviour (functional findings):

no effects observed

Other effects:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

changes in pregnancy duration

food consumption and compound intake

mortality

number of abortions

total litter losses by resorption

Reduction in number of live offspring:

no effects observed

Changes in litter size and weights:

no effects observed

Other effects:

no effects observed

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

reduction in number of live offspring

changes in litter size and weights

Developmental effects observed:

no

Conclusions:

the investigation concluded that the tested material is unlikely to be a teratogen

Cross-reference 4

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Justification for type of information:

Read across approach adequate to support 7.6.1 according to "Guidance on information requirements and chemical safety assessment Chapter R.6: QSARs and grouping of chemicals" (ECHA 2008) and Read-Across Assessment
Framework (RAAF) (ECHA 2017)

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

not specified

Species:

rat

Strain:

other: Sprague-Dawley Crl: CD(SD) IGS BR

Route of administration:

oral: gavage

Vehicle:

other: Ethanol, 2-[2-(2-methoxyethoxy)ethoxy]-

Duration of treatment / exposure:

Exposure days: 15 days (Day 5 to Day 19 of gestation)
Post-exposure observation period: None

Remarks:

0 (control group) ,30,300,1000 mg/kg bw/day

No. of animals per sex per dose:

8 animals in each group

Control animals:

yes

Details on study design:

The study was designed to evaluate the effects of the test item on embryonic and foetal development. Females were euthanized before delivery, on gestation Day 20. All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded. The ovaries and uteri of pregnant animals were removed, examined and recoded with number of corpora lutea, number, position and type of intrauterine implantation, foetal sex, external foetal appearance, foetal weight, placental weight and gravid uterus weight.

Details on maternal toxic effects:

Mortality and Time to Death
All animals survived to the scheduled necropsies.

Effects on Dams
No signs of clinical toxicity were detected. No treatment related abnormalities were noted during the macroscopic examination of the pregnant animals at termination on Day 20 of gestation.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Basis for effect level:

mortality

Details on embryotoxic / teratogenic effects:

Effects on Foetus
No treatment-related effects were noted for foetuses in treated animals when compared to control animals.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Basis for effect level:

other: no effects specified

Developmental effects observed:

no

Conclusions:

The No Observed Effect Level (NOEL) was established as 1000 mg/kg bw/day in this study, based on the absence of treatment-related effects at any of the doses administered.

Data source

Reference

Reference Type:

publication

Title:

Developmental toxicity study with diethylene glycol dosed by gavage to CD rats and CD-1 mice

Author:

Ballantyne, B. and Snellings W.M.

Year:

2005

Bibliographic source:

Food and Chemical Toxicology 43 (2005) 1637–1646

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-(2-hydroxyethoxy)ethan-1-ol

Cas Number:

111-46-6

Molecular formula:

C4H10O3

IUPAC Name:

2-(2-hydroxyethoxy)ethan-1-ol

Details on test material:

Constituent
Smile  OCCOCCO

Specific details on test material used for the study:

- obtained from Union Carbide Corporation, Hahnville, LA
- identification number: TFJ-44408 PL
- Purity : 99.87%.
- ethylene glycol content: 0.02 % , triethylene glycol: 0.11 %.

Test animals

Species:

mouse

Strain:

other:

Remarks:

Crl: CD-1 (ICR) BR

Details on test animals or test system and environmental conditions:

- Virgin male and virgin female [Crl: CD-1 (ICR) BR] outbred mice were obtained from Charles River Breeding Laboratories, Inc., Portage, MI.
- They were 42 days on arrival with males weighing 25–30 g, and females 23–25 g.
- During a 2-week acclimation period, they were housed one to three in stainless steel wire mesh cages.
- Food (Prolab Certified Ground Rodent Chow RMH-3220, Agway Inc., St. Marys, OH) and water were available ad libitum.
- Females were supplied water using water bottles having stainless steel sipper tubes to allow measurement of water consumption.
- Animals were assigned a unique number and identified by toe clipping and ear notching.

- Environmental temperature was maintained at 18–22 C, relative humidity at 40– 56%, and a 12-h photoperiod was used.
- Mice were mated, one male to one female, in stainless steel wire mesh cages.
- The day a dropped copulatory plug was found was designated gestation day (gd) 0.
- Thirty plug-positive females were assigned to each treatment group of the definitive study by a randomization procedure based on stratification on gd 0 body weights.
- The average body weight range of females at the start of the study was 25.07–25.27 g.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on mating procedure:

Mice were mated, one male to one female, in stainless steel wire mesh cages. The day a dropped copulatory plug was found was designated gestation day (gd) 0. Thirty plug-positive females were assigned to each treatment group of the definitive study by a randomization procedure based on stratification on gd 0 body weights.

Duration of treatment / exposure:

Groups of 30 timed-pregnant CD-1 mice were dosed daily by gavage over gd 6–15 with undiluted DEG at dosages of 0.0 (controls), 0.5, 2.5 or 10.0 ml/kg/day (equivalent to 0, 559, 2795 and 11,180 mg/kg/day). Controls received 10 ml/kg/day of deionized (Milli-Q) water.

Frequency of treatment:

daily

Duration of test:

GD 6-15

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.5 other: mL/Kg bw/day

Remarks:

equivalento to 559 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

2.5 other: mL/Kg bw/day

Remarks:

equivalento to 2795 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

10 other: mL/Kg bw/day

Remarks:

equivalento to 11180 mg/kg bw/day

No. of animals per sex per dose:

30 mice per group

Control animals:

yes, concurrent vehicle

Examinations

Maternal examinations:

- All females were examined daily for any clinical signs of toxicity.
- They were weighed on gd 0, 6, 9, 12, 15 and 18, and body weight changes calculated for the interweighing periods.
- Food and water consumption was measured at sequential 3-day intervals over gd 0–18.
- All surviving females were sacrificed on gd 18 by CO2 asphyxiation.
- Midline thoracolaparotomy was performed, and the gravid uterus, ovaries, cervix, vagina, and abdominal and thoracic cavities examined grossly.

Ovaries and uterine content:

- Corpora lutea were counted.
- Gravid uterine, liver, and kidney weights were recorded.
- The uterus was dissected longitudinally and all live and dead fetuses and resorption sites were recorded.
- Uteri that appeared non-gravid were placed in 10% ammonium sulfide for detection of early resorptions.

Fetal examinations:

- Live fetuses were weighed, sexed, and examined for any external malformations or variations.
- All live fetuses in each litter were examined for thoracic and abdominal visceral abnormalities.
- One-half of the fetuses from each litter were decapitated and heads fixed in Bouins solution for examination of craniofacial structures by sectioning.- All fetuses (50% intact, 50% decapitated) were eviscerated, fixed in ethanol, processed for skeletal staining and examined for skeletal malformations and variations

Statistics:

The unit of comparison was the pregnant female or litter (Weil, 1970). Results of quantitative continuous variables were intercompared for the DEG groups vs. the controls by Levenes test for equal variances (Levene, 1980), analyses of variance (ANOVA), and t-tests with Bonferroni probabilities for pairwise comparisons. When Levenes test indicted homogeneous variances, and the ANOVA was significant, a pooled t-test was used. When Levenes test indicated heterogeneous variances, all groups were compared with an ANOVA for unequal variances (Brown and Forsythe, 1974), followed by the separate variance t-test. Nonparametric data were statistically analyzed by the Kruskall–Walliss test, followed by the Mann–Whitney U-test. Incidence data were compared using Fishers Exact Test (Sokal and Rohlf, 1969). A probability value of p < 0.05 (two-tailed) was used as the critical level of significance for all tests.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

There was maternal toxicity at 11180 mg/kg bw/day (mortality, signs, increased water consumption) and at 2795 mg/kg/day (increased water consumption).

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

ca. 559 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

clinical signs

mortality

water consumption and compound intake

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Implantations were comparable across all groups. Fetal body weights were significantly reduced at 11180 mg/kg bw/day. There were no increases in variations or malformations, either total, by category, or individually.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

ca. 2 795 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

not specified

Lowest effective dose / conc.:

11 180 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

not specified

Dose response relationship:

not specified

Relevant for humans:

no

Any other information on results incl. tables

developmental effects as secondary non specific consequence of maternal toxicity effects

Under the conditions of these studies, the no-observed-adverse effect-level (NOAEL) for DEG given by gavage over gd 6–15 was 559 mg/kg/day maternal toxicity, and 2795 mg/kg/day with mice for developmental toxicity (fetotoxicity).

There were no indications of embryotoxicity or teratogenic effects at any dosage.

Applicant's summary and conclusion

Conclusions:

There were no indications of embryotoxicity or teratogenic effects at any dosage.