Tris[2-[2-(2-methoxyethoxy)ethoxy]ethyl] orthoborate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

There are no fertility studies available with B-TEGME.

A testing proposal for an EOGRTS without extension of Cohort 1B, without Cohorts 2A and 2B, and without Cohort 3 in rats via oral application has been submitted and is currently being evaluated (ECHA Communication number TPE-D-2114397224-45-01/D).

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

As no fertility studies are available for B-TEGME, a testing proposal for an EOGRTS without extension of Cohort 1B, without Cohorts 2A and 2B, and without Cohort 3 in rats via oral application has been submitted. The testing proposal evaluation is currently in progress (ECHA Communication number TPE-D-2114397224-45-01/D).

Effects on developmental toxicity

Description of key information

The maternal/developmental NOAEL in a Charnoff & Kavlock rat study was determined to be at least 170 mg B-TEGME/kg bw/day based on a toxicity study with oral application of up to 1000 mg brake fluid/kg bw/day.

 

Prenatal developmental toxicity was further investigated in a prenatal development study in rats dosed at 30, 300 and 1000 mg/kg body weigtht B-TEGME. A further group of twenty-four time mated females was exposed to the vehicle only (Methyltriglycol or TEGME) to serve as a control. The oral administration of B-TEGME and TEGME by oral gavage in pregnant rats resulted in some delayed ossification effects in foetuses; however, these effects were not considered of toxicological relevance and are most probably secondary to maternal toxicity effects of TEGME that has been used as vehicle in concentrations from ca. 3200 mg/kg bw/d (high dose group) to ca. 4200 mg/kg bw/d (control/vehicle group). Developmental toxicity experiments conducted with TEGME indicate developmental effects at doses > 1,000 mg/kg/day. Effects observed in offspring from rats treated with 1250 mg/kg/day TEGME or rabbits treated with 1500 mg/kg/day TEGME during gestation included skeletal variants and decreased body weight gain (see attached OECD SIDS document for TEGME, CAS 112-35-6, August 2005).

 

In a prenatal developmental toxicity study in New Zealand White rabbits, B-TEGME was administered as pure substance without being formulated with a vehicle to groups of 25 inseminated female New Zealand White rabbits orally by gavage in doses of 0, 100, 250 and 500 mg/kg on gestation days (GD) 6 through 28. B-TEGME caused evidence of maternal toxicity at the high dose of 500 mg/kg bw/d, such as doe mortality, and abortion in the most sensitive individuals as well as slightly reduced food consumption. The NOAEL for maternal toxicity was 250 mg/kg bw/d. Reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, pre- and post-implantation loss as well as uterine weight, placental weight, fetal weight and sex distribution of the fetuses were unchanged. In the high-dose group (500 mg/kg bw/d) a number of fetuses from different litters were affected by visceral (multiple malformations of urogenital tract) or skeletal (malformations of axial skeleton) malformations, some of which exceeded their historical control ranges. In addition, significantly increased incidences of external, skeletal and cartilage variations, all above the historical control range, were noted. The total of these findings caused the significant increase of overall malformations and variations in the high-dose group, although these particular values were still within the historical control range of the test facility. Since there was evidence for treatment-related adverse effects of B-TEGME on fetal morphology at the high-dose of 500 mg/kg bw/d, the NOAEL for prenatal developmental toxicity was 250 mg/kg bw/d.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Oct 2017 - 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

22 January 2001

Deviations:

no

GLP compliance:

yes (incl. certificate)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, 55116 Mainz

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BASF, B756
- Expiration date of the lot/batch: 01 Feb 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: guaranteed by sponsor
- Solubility and stability of the test substance in the solvent/vehicle: no vehicle used

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no purification step before application to animals

FORM AS APPLIED IN THE TEST: pure, unformulated substance

Species:

rabbit

Strain:

New Zealand White

Remarks:

Crl:KBL(NZW)

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Breeder: Charles River Laboratories, France)
- Age at study initiation: 15-17 weeks
- Weight at study initiation: pregnant females between 3054 – 4070 g
- Fasting period before study: not applicable
- Housing: during acclimatization housed singly in Type 4X03B700CP cages (TECNIPLAST Deutschland GmbH, Hohenpeißen-berg, Germany; floor space 4264 mm², internal height 450 mm)
- Diet: ad libitum; pelleted Kliba maintenance diet (Provimi Kliba SA (new name Granovit AG), Kaiseraugst, Switzerland)
- Water: ad libitum, potable tap water in water bottles
- Acclimation period: 5 days before artificial insemination

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20±2
- Humidity (%): 45-65
- Air changes (per hr): 15 times
- Photoperiod (hrs dark / hrs light): 12/12 (6.00 h to 18.00 h / 18.00 h to 6.00 h)

IN-LIFE DATES: From: 2017-11-13 To: 2017-12-21

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: applied as pure substance without being formulated with a vehicle

DIET PREPARATION
- no diet applied

VEHICLE
- no vehicle used

Analytical verification of doses or concentrations:

no

Remarks:

no stability, homogeneity and concentration control analyses of test item formulations were conducted due to pure substance application

Details on mating procedure:

- Impregnation procedure: artificial insemination
- Day of insemination referred to as day 0 of pregnancy

Duration of treatment / exposure:

implantation to one day prior to the expected day of parturition (GD 6-28)

Frequency of treatment:

daily

Duration of test:

30d

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

250 mg/kg bw/day

Dose / conc.:

500 mg/kg bw/day

No. of animals per sex per dose:

25

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: Based on dose-range finding studies with non-pregnant (300 and 1,000 mg/kg bw/d for 21 d) and pregnant (300 and 600 mg/kg bw/d for GD 6 through GD 28) female New Zealand White rabbits the doses were selected. In the first study at 1,000 mg/kg bw/d, one out of three female animals were found dead on study day 12 with similar necropsy findings in all three animals. At 300 mg/kg bw/d no adverse effects were observed. In the second study at 600 mg/kg bw/d, one out of five female animals were killed moribund on study day 21 after showing reduced nutritional condition. At 300 mg/kg bw/d no adverse effects were observed.
- Rationale for animal assignment: There are historical control data available from the test facility for New Zealand White rabbits and the specific strain has been proven to be sensitive to substances with a teratogenic potential.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
- at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity; animals examined several times daily (GD 0-29) if signs occured
- during administration period (GD 6-28): all animals checked daily for abnormal clinical signs before administration as well as within 5 hours after

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 2, 4, 6, 9, 11, 14, 16, 19, 21, 23, 25, 28 and 29

FOOD CONSUMPTION: Yes
- Time schedule for examination: daily during GD 0-29

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- 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: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter (and the heads of any fetus which revealed severe findings during the external examina-tion, e.g. anophthalmia, microphthalmia or hydrocephalus)

Statistics:

DUNNETT-test (two-sided) for the hypothesis of equal means:
Food consumption ("mean of means" = values that allow a rough estimation of the total food consumption during different time intervals [pretreatment, treatment and entire study]), body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight

FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions:
Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings

WILCOXON-test (one-sided) for the hypothe-sis of equal medians:
Proportions of fetuses with malformations, variations and/or unclassified observations in each litter

For all:
* for p < 0.05; ** for p < 0.01

Indices:

The conception rate (in %):
(number of pregnant animals / number of fertilized animals) * 100


The preimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice:
([number of corpora lutea – number of implantations] / number of corpora lutea) * 100


The postimplantation loss (in %) for each individual pregnant animal which underwent scheduled sacrifice:
([number of implantations – number of live fetuses] / number of implantations) * 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- control: one female had blood in bedding before and after treatment on GD 16, reduced defecation was observed in four females
- 100 mg/kg bw/d: reduced defecation in one animal observed
- 250 mg/kg bw/d: reduced defecation in one animal observed
- 500 mg/kg bw/d: reduced defecation in six females observed, no defecation observed in three animals

- adverse effect is indicated by higher incidence of both findings in the high-dose group

Mortality:

mortality observed, treatment-related

Description (incidence):

- 500 mg/kg bw/d: one female were found dead on the day of scheduled sacrifice (GD 29), two females were sacrificed after abortion ahead of schedule (GD 29 and GD 28)
- simultaneous abortion/death of 3 high-dose does immediately before term points to maternal toxicity

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- no significant treatment related effect on body weights/body weight gain
- particularly no effect on carcass weights or corrected (net) body weight gain at all dose levels

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- the mean food consumption of the does in the 500 mg/kg bw/d group was reduced from GD 9 onwards
- this effect was statistical significant on GD 9-10 and GD 13-15
- afterwards (around GD 17) the effect recovered, being comparable to or exceeding the control values

- overall, the high-dose does consumed 7% less food than the control animals during the treatment period (GD 6-28)
- food consumption of the low- and mid-dose rabbits (100 and 250 mg/kg bw/d) was comparable to thecontrol

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry 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):

- gravid uterus weights were not significantly different from controls, differences between all groups were assessed to be without biological relevance and no dose-dependence was observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- spontaneous findings: noted in individual females of all groups, i.e. watery feces in one control and one high-dose doe, a blind ending uterine horn (left) in one high-dose doe, absence of uterine horn (left), absent kidney (left) and absent ureter (left) in one high-dose doe
- additional fiindings associated with unscheduled maternal death or sacrifice: findings after gavage error (thoracic cavity filled with blood) in one doe, watery feces in one doe (sacrificed after abortion on GD 29), an empty stomach in one doe No. 78 (died on GD 29), watery feces, stomach filled to distension with feed, very dry, hard feces in rectum in one doe

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Number of abortions:

no effects observed

Description (incidence and severity):

- no test substance-related and/or biologically relevant differences between the different test groups

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

- no test substance-related and/or biologically relevant differences between the different test groups

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

- no test substance-related and/or biologically relevant differences between the different test groups

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

- one dead fetus found at cesarean section of one 500 mg/kg bw/d doe
- it is considered a rare finding but may occur spontaneously in this rabbit strain

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

- conception rate: 88% in 100 mg/kg bw/d group , 96% in 250 and 500 mg/kg bw/d groups and 100% in the control group (0 mg/kg bw/d)

Details on maternal toxic effects:

All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age.

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

clinical signs

mortality

food consumption and compound intake

Dose descriptor:

LOAEL

Effect level:

500 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

clinical signs

mortality

food consumption and compound intake

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

- mean fetal weights of test groups 1, 2 and 3 were not influenced by the test substance, no biologically relevant differences in comparison to the control group

Changes in sex ratio:

no effects observed

Description (incidence and severity):

- distribution of the fetuses in test groups 100, 250 and 500 mg/kg bw/d was comparable to the control fetuses

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

- occurred in test groups 100 and 250 mg/kg bw/d), as listed in Tab. 1
- male fetus No. 31-02 had multiple soft tissue and skeletal malformations additionally
- male fetus No. 70-04 had an associated soft tissue malformation

The distribution of external malformations about the dose groups does not indicate an association to the treatment, no statistically significant differences between the groups were noted (Tab. 2).

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

- detected in single fetuses of test groups 100 and 500 mg/kg bw/d, as shown in Tab. 7
- male 100 mg/kg bw/d fetus No. 31-02 had multiple skeletal malformations affecting the vertebral column, ribs, sternum and pelvic girdle, furthermore, additional external and multiple visceral malformations, considered to be spontaneous in origin and not treatment-related
- in the 500 mg/kg bw/d group the incidences for all individual malformations as well the significantly increased litter incidence and mean% affected fetuses per litter with skeletal malformations (Tab. 8) were covered by the historical control ranges
- exception: the litter and affected fetuses/litter incidences for severely fused sternebrae (bony plate) exceeded their historical control ranges at 500 mg/kg bw/d

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

- occurred in fetuses of the test groups 100, 250 or 500 mg/kg bw/d, as listed in Tab. 4
- male low-dose fetus No. 31-02 had multiple visceral malformations, i.e. absent kidney, absent ureter, absent adrenals and a right-sided aortic arch, furthermore, additional external and multiple skeletal malformations
- male mid-dose fetus No. 70-04 had an additional external malformation
- three fetuses (out of two litters) of the 500 mg/kg bw/d group multiple visceral malformations affecting the urogenital tract were recorded

- no statistically significant differences in the overall incidences between the groups were noted (Tab. 5)
- the 500 mg/kg bw/d group litter incidence (9.5%; historical range 0 – 4.8%) as well as affected fetuses/litter incidence (mean% 1.5; historical range 0 – 0.6) for fetuses with multiple visceral malformations were above the historical control

Details on embryotoxic / teratogenic effects:

Fetal external variations:
- one external variation, i.e. paw hyperflexion, was recorded in six fetuses of four 500 mg/kg bw/d litters - resulted in an increased litter incidence (19% vs. 0 in control) and mean% of affected fetuses per litter (4.6 vs. 0 in control)
- the increased overall incidences of external variations are given in Tab. 3

Fetal soft tissue variations:
- broad variety of soft tissue variations, i.e. cystic dilatation of the brain, malpositioned carotid branches, short innominate, absent lung lobe (Lobus inferior medialis) and dilated renal pelvis in individual fetuses of test groups 0, 100, 250 or 500 mg/kg bw/d
- incidences were neither statistically significantly different from control nor dose-dependent and, therefore, not considered biologically relevant (Tab. 6)

Fetal skeletal variations:
- skeletal variations of different bone structures were observed in all test groups, with or without effects on corresponding cartilages
- observed skeletal variations were related to several parts of fetal skeletons and appeared in the majority of cases without a relation to dosing
- overall affected fetuses/litter incidences of skeletal variations were statistically significantly increased in all substance-treated groups (Tab. 9)
- this effects were above the historical control range (HCD: mean% 93.9 [83.2 - 100.0])
- all skeletal variations with statistically significant differences between the control and the treated groups are given in table 10
- two of the findings – supernumerary thoracic vertebra and supernumerary rib (13th, cartilage present) had higher incidences than the controls in all treated groups
- this increase was not statistically significant and hovered around the upper limit of the historical range in the low-dose group and not dose-related for the supernumerary rib (13th, cartilage present).

Fetal skeletal unclassified cartilage observations:
- the results are given table 11
- incidence ‘knobby rib cartilage’ was statistically significantly increased in test group 250 mg/kg bw/d (affected fetuses/litter, mean%: 0.0/1.9/1.2*/1.1 [p≤0.05])
- the vaue was within the historical control range (mean% 0.4 [0.0 - 1.3]) and there is no dose-response - no association to the treatment was assumed
- incidence ‘branched rib cartilage’ was statistically significantly increased in test group 500 mg/kg bw/d (affected fetuses/litter, mean%: 0.0/0.7/0.8/3.1* [p≤0.05])
- litter and affected fetus-es/litter incidences were outside the historical control range (mean% 0.0 [0.0 - 0.5])

Fetal external unclassified observations (table 12):
- two unclassified external observations, i.e. placentae discolored and placentae necrobiotic, were recorded in one fetus of test groups 250 or 500 mg/kg bw/d
- the effects were not considered to be related to treatment

Fetal soft tissue unclassified observations: (table 13)
- in one control fetus, three low-dose, seven mid-dose and three high-dose fetuses: a blood coagulum around urinary bladder observed
- this finding is not considered to be treatment-related

Dose descriptor:

LOAEL

Effect level:

500 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

visceral malformations

other: significantly increased incidences of external, skeletal and cartilage variations above the historical control range

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

visceral malformations

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: sternum

skeletal: rib

skeletal: vertebra

skeletal: pelvic girdle

visceral/soft tissue: urinary

Developmental effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

not specified

Table 1: Individual fetal external malformations

Test group	Doe No.-Fetus No., Sex	Finding
0 (0 mg/kg bw/d)	none
1 (100 mg/kg bw/d)	31-02 M^a)b)	acaudate
	46-01 F	umbilical hernia
2 (250 mg/kg bw/d)	70-04 M^a)	umbilical hernia
3 (500 mg/kg bw/d)	none

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

^a)fetus with additional soft tissue malformation (see Tab. 4)

^b)fetus with additional skeletal malformation (see Tab. 7)

Table 2: Total external malformations

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 250 mg/kg bw/d	Test group 3 500 mg/kg bw/d
Litter Fetuses	N N	25 226	22 192	24 196	21 160
Fetal incidence	N (%)	0.0	2 (1.0)	1 (0.5)	0.0
Litter incidence	N (%)	0.0	2 (9.1)	1 (4.2)	0.0
Affected fetuses/litter	Mean%	0.0	1.2	0.6	0.0

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

Table 3: Total external variations

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 250 mg/kg bw/d	Test group 3 500 mg/kg bw/d
Litter Fetuses	N N	25 226	22 192	24 196	21 160
Fetal incidence	N (%)	0.0	0.0	0.0	6 (3.8)
Litter incidence	N (%)	0.0	0.0	0.0	4 (19)*^Fi
Affected fetuses/litter	Mean%	0.0	0.0	0.0	4.6*^Wi

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

*^Fi= p ≤ 0.05 (Fisher’s exact test [one-sided])   *^Wi= p ≤ 0.05 (Wilcoxon-test [one-sided])

Table 4: Individual fetal soft tissue malformations 

Test group	Doe No.-Fetus No., Sex	Finding
0 (0 mg/kg bw/d)	none
1 (100 mg/kg bw/d)	31-02 M^a)b)	multiple visceral malformations
2 (250 mg/kg bw/d)	70-04 M^a)	malpositioned kidney
3 (500 mg/kg bw/d)	82-02 F, 82-04 F	multiple visceral malformations
	83-04 F	multiple visceral malformations

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

^a)fetus with additional external malformation (see Tab. 2)

^b)fetus with additional skeletal malformation (see Tab. 7)

Table 5: Total soft tissue malformations

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 250 mg/kg bw/d	Test group 3 500 mg/kg bw/d
Litter Fetuses	N N	25 226	22 192	24 196	21 160
Fetal incidence	N (%)	0.0	1 (0.5)	1 (0.5)	3 (1.9)
Litter incidence	N (%)	0.0	1 (4.5)	1 (4.2)	2 (9.5)
Affected fetuses/litter	Mean%	0.0	0.6	0.6	1.5

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

 

Table 6: Total soft tissue variations

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 250 mg/kg bw/d	Test group 3 500 mg/kg bw/d
Litter Fetuses	N N	25 226	22 192	24 196	21 160
Fetal incidence	N (%)	10 (4.4)	3 (1.6)	5 (2.6)	8 (5.0)
Litter incidence	N (%)	7 (28)	3 (14)	4 (17)	3 (14)
Affected fetuses/litter	Mean%	4.9	1.8	3.2	4.5

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

Table 7: Individual fetal skeletal malformations

Test group	Doe No.-Fetus No., Sex	Finding
0 (0 mg/kg bw/d)	none
1 (100 mg/kg bw/d)	31-02 M^a)	multiple skeletal malformations
	37-06 M	misshapen thoracic vertebra
2 (250 mg/kg bw/d)	none
3 (500 mg/kg bw/d)	80-12 F	sternebrae severely fused (bony plate)
	87-09 F	severely malformed vertebral column and/or ribs
	88-03 M	misshapen lumbar vertebra
	92-09 F	thoracic hemivertebra, sternebrae severely fused (bony plate)

mg/kg bw/d = milligram per kilogram body weight per day; No. = number; M = male; F = female

^a)fetus with additional external and soft tissue malformations (see Tabs. 1 and 4)

Table 8: Total skeletal malformations

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 250 mg/kg bw/d	Test group 3 500 mg/kg bw/d
Litter Fetuses	N N	25 226	22 192	24 196	21 160
Fetal incidence	N (%)	0.0	2 (1.0)	0.0	4 (2.5)
Litter incidence	N (%)	0.0	2 (9.1)	0.0	4 (19)*^Fi
Affected fetuses/litter	Mean%	0.0	1.0	0.0	2.2*^Wi

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

*^Fi= p ≤ 0.05 (Fisher’s exact test [one-sided])   *^Wi= p ≤ 0.05 (Wilcoxon-test [one-sided])

 

Table 9: Total fetal skeletal variations

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 250 mg/kg bw/d	Test group 3 500 mg/kg bw/d
Litter Fetuses	N N	25 226	22 192	24 196	21 160
Fetal incidence	N (%)	210 (93)	189 (98)	194 (99)	160 (100)
Litter incidence	N (%)	25 (100)	22 (100)	24 (100)	21 (100)
Affected fetuses/litter	Mean%	92.1	97.3*	99.1**	100.0**

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

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

Table 10: 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 1 100 mg/kg bw/d	Test group 2 250 mg/kg bw/d	Test group 3 500 mg/kg bw/d	HCD Mean % (range)
Supernumerary thoracic vertebra	19.4	27.6	39.9**	46.9**	17.9 (7.8 - 25.6)
Supernumerary lumbar vertebra	0.7	0.6	1.5	7.2*	0.3 (0.0 - 2.4)
Unossified sternebra; unchanged cartilage	11.6	18.7*	21.9**	45.4**	15.4 (7.7 - 27.5)
Misshapen sternebra; unchanged cartilage	6.4	3.6	6.3	18.2**	9.8 (3.1 - 17.7)
Supernumerary rib (13^th); cartilage present	56.8	69.9	77.0**	70.7**	60.4 (50.0 - 70.9)
Incomplete ossification of talus; cartilage present	1.5	0.6	2.8	14.2**	2.1 (0.0 - 4.8)
Unossified talus; cartilage present	0.4	1.0	2.3	16.7**	0.9 (0.0 - 2.6)

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])

Table 11: Total unclassified cartilage observations

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 250 mg/kg bw/d	Test group 3 500 mg/kg bw/d
Litter Fetuses	N N	25 226	22 192	24 196	21 160
Fetal incidence	N (%)	62 (27)	35 (18)	37 (19)	59 (37)
Litter incidence	N (%)	23 (92)	16 (73)	16 (67)	19 (90)
Affected fetuses/litter	Mean%	30.5	18.7	17.1	34.3

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

 

Table 12: Total external unclassified observations 

		Test group 0 0 mg/kg bw/d	Test group 1 100 mg/kg bw/d	Test group 2 250 mg/kg bw/d	Test group 3 500 mg/kg bw/d
Litter Fetuses	N N	25 226	22 192	24 196	21 160
Fetal incidence	N (%)	0.0	0.0	1 (0.5)	1 (0.6)
Litter incidence	N (%)	0.0	0.0	1 (4.2)	1 (4.8)
Affected fetuses/litter	Mean%	0.0	0.0	0.8	0.7

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

Table 13: Total soft tissue unclassified observations

				Test group 0 0 mg/kg bw/d		Test group 1 100 mg/kg bw/d		Test group 2 250 mg/kg bw/d		Test group 3 500 mg/kg bw/d
Litter Fetuses		N N		25 226		22 192		24 196		21 160
Fetal incidence		N (%)		1 (0.4)		3 (1.6)		7 (3.6)		3 (1.9)
Litter incidence		N (%)		1 (4.0)		2 (9.1)		2 (8.3)		2 (9.5)
Affected fetuses/litter		Mean%		0.3		1.7		2.8		1.4

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

 

Conclusions:

The oral administration of B-TEGME to pregnant New Zealand White rabbits from implantation to one day prior to the expected day of parturition (GD 6-28) caused evidence of maternal toxicity at the high-dose level of 500 mg/kg bw/d, such as abortions/mortality and reduced food consumption. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 250 mg/kg bw/d.

Since there was evidence for treatment-related adverse effects of the test substance on fetal morphology at the high-dose of 500 mg/kg bw/d, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 250 mg/kg bw/d.

Executive summary:

B-TEGME was tested for its prenatal developmental toxicity in New Zealand White rabbits according to OECD 414.

The test substance was administered as pure substance without being formulated with a vehicle to groups of 25 inseminated female New Zealand White rabbits orally by gavage in doses of 100, 250 and 500 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 28. The control group, consisting of 25 females, was dosed with drinking water (same quantity as the high-dose animals) in parallel. At terminal sacrifice on GD 29, 21-25 females per group had implantation sites.

Food consumption and body weight of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day. On GD 29, all females were sacrificed and assessed by gross pathology (including weight determinations of the unopened uterus and placentas). For each doe, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for any external, soft tissue and skeletal (inclusive cartilage) findings.

The following test substance-related adverse effects/findings were noted:

Test group 3 (500 mg/kg bw/d):

Dams

- Death or abortion in 3 females immediately before term (GD 28/29), corroborative necropsy findings indicating enteropathy

- Lower food consumption (7% below control)

Fetuses

- Increased incidence of fetuses showing visceral (urogenital tract) or skeletal (axial skeleton) malformations, some above historical range

- Increased incidences of external, skeletal and cartilage variations, all above historical control range

- Increased incidences of total malformations and variations

Test group 2 (250 mg/kg bw/d):

- No test substance-related adverse effects on does, gestational parameters or fetuses.

Test group 1 (100 mg/kg bw/d):       

- No test substance-related adverse effects on does, gestational parameters or fetuses.

Under the conditions of this prenatal developmental toxicity study, the oral administration of B-TEGME to pregnant New Zealand White rabbits from implantation to one day prior to the expected day of parturition (GD 6 -28) caused evidence of maternal toxicity at the high-dose level of 500 mg/kg bw/d, such as abortions/mortality and reduced food consumption. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 250 mg/kg bw/d.

Since there was evidence for treatment-related adverse effects of the test substance on fetal morphology at the high-dose of 500 mg/kg bw/d, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 250 mg/kg bw/d.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

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

BASF SE, 40R0618/11R275, 2018

B-TEGME was tested for its prenatal developmental toxicity in New Zealand White rabbits according to OECD 414.

The test substance was administered as pure substance without being formulated with a vehicle to groups of 25 inseminated female New Zealand White rabbits orally by gavage in doses of 100, 250 and 500 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 28. The control group, consisting of 25 females, was dosed with drinking water (same quantity as the high-dose animals) in parallel. At terminal sacrifice on GD 29, 21-25 females per group had implantation sites.

Food consumption and body weight of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day. On GD 29, all females were sacrificed and assessed by gross pathology (including weight determinations of the unopened uterus and placentas). For each doe, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for any external, soft tissue and skeletal (inclusive cartilage) findings.

The following test substance-related adverse effects/findings were noted:

Test group 3 (500 mg/kg bw/d):

Dams:

- Death or abortion in 3 females immediately before term (GD 28/29), corroborative necropsy findings indicating enteropathy

- Lower food consumption (7% below control)

Fetuses:

- Increased incidence of fetuses showing visceral (urogenital tract) or skeletal (axial skeleton) malformations, some above historical range

- Increased incidences of external, skeletal and cartilage variations, all above historical control range

- Increased incidences of total malformations and variations

Test group 2 (250 mg/kg bw/d):

- No test substance-related adverse effects on does, gestational parameters or fetuses.

Test group 1 (100 mg/kg bw/d):       

- No test substance-related adverse effects on does, gestational parameters or fetuses.

B-TEGME caused death and abortions in 3 high-dose females (500 mg/kg bw/d), in addition reduced or no defecation was observed at a higher frequency at this dose level. Necropsy observations in the decedents comprised watery feces, an empty stomach or a stomach filled to distension with feed combined with very dry, hard feces in rectum. These clinical observations indicate that 500 mg/kg bw/d of the test item were maternally toxic to sensitive individuals in the present study.

The mean food consumption of the high-dose dams (500 mg/kg bw/d) was significantly below the concurrent control shortly after the beginning of the treatment and recovered afterwards, however, overall these animals consumed about 7% less food than the control group. There was no significant treatment related effect on body weights/body weight gain, and particularly on carcass weights or corrected (net) body weight gain at all dose levels.

No differences of toxicological relevance between the control and the treated groups (100, 250 or 500 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. All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age.

One dead fetus was found at cesarean section of high-dose doe No. 84 (500 mg/kg bw/d) which is a rare finding but may occur spontaneously in this rabbit strain.

Similarly, no influence of the test substance on uterine weight, placental weight, fetal weight and sex distribution of the fetuses was noted at any dose.

Finally, fetal examinations revealed that there is no adverse effect of the compound on the respective morphological structures up to the mid-dose tested (250 mg/kg bw/d).

In the high-dose group (500 mg/kg bw/d) a number of fetuses from different litters were affected by visceral (multiple malformations of urogenital tract) or skeletal (malformations of axial skeleton) malformations, some of which exceeded their historical control ranges. In addition, significantly increased incidences of external, skeletal and cartilage variations, all above the historical control range, were noted. The total of these findings caused the significant increase of overall malformations and variations in the high-dose group, although these particular values were still within the historical control range of the test facility.

Thus, for the high-dose (500 mg/kg bw/d) the collective spectrum of observed anomalies may suggest beginning developmental toxicity.

Under the conditions of this prenatal developmental toxicity study, the oral administration of B-TEGME to pregnant New Zealnd White rabbits from implantation to one day prior to the expected day of parturition (GD 6 -28) caused evidence of maternal toxicity at the high-dose level of 500 mg/kg bw/d, such as abortions/mortality and reduced food consumption. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 250 mg/kg bw/d.

Since there was evidence for treatment-related adverse effects of the test substance on fetal morphology at the high-dose of 500 mg/kg bw/d, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 250 mg/kg bw/d.

Shell, 41204726, 2015

In a supporting prenatal developmental toxicity study, the test item B-TEGME (89% pure) was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, between Days 5 and 19 of gestation at dose levels of 30, 300, and 1000 mg/kg bw/day (Shell, 2015). A further group of twenty-four time mated females was exposed to the vehicle only (Methyltriglycol or TEGME) to serve as a control; B-TEGME was also used as vehicle in the dosed groups.

Clinical signs and necropsy findings did not indicate any effect of treatment in parental females at any treatment level. Body weight development and food consumption in treated groups did not show any significant intergroup differences when compared to the concurrent control females. However, statistical analysis of the control (TEGME) group against a background control group using a standard vehicle did reveal a reduction in body weight development and food consumption. Therefore, a true effect of B-TEGME on maternal toxicity may have been masked by the concurrent control using TEGME.

The predominant external finding detected throughout the 1000 mg/kg bw/day dose group was an increase in the percent of observed small foetuses. At this treatment level a number of litters had a high incidence of foetuses observed to be small. Lower mean foetal weight was evident at 1000 mg/kg bw/day with the majority of the small foetuses also having the lower body weights. Visceral foetal examinations did not show an overall increase in foetuses with findings and therefore no treatment effects. An observed, statistically significant increase in foetuses with small/no renal papilla development was not dosage related and was therefore considered insufficient evidence of a treatment related effect. Skeletal examinations showed an increase in the overall number of foetuses with skeletal findings in litters from the 1000 and 300 mg/kg bw/day treatment groups. At 1000 mg/kg bw/day delayed ossification of the sternebra, post lumbar vertebral centra and arches were evident together with a reduced number of metacarpals and an increased incidence of a short 13th rib. These findings all achieved statistical significance. There were other observations of delayed ossification that did not achieve statistical significance but are of biological relevance. An increase incidence of incomplete ossification was evident in one, or more than one cranial bone plus increased numbers of foetuses with no ossification of one thoracic vertebral centra and incomplete ossification in more than one lumbar arch. The overall incidence of the skeletal findings detected were not restricted to the identified small foetuses or to litters containing small foetuses but were equally distributed throughout the 1000 mg/kg bw/day treatment group. Although in the skeletal developmental parameters which showed a statistical significance, the mean control values were also outside of the background control values, therefore suggesting that the control vehicle (TEGME) that is also present in the test item may also contribute to the effects detected in the treated litters. The findings were enhanced in the treated groups and these findings are indicative of an overall delay in offspring development. The pattern of foetal development differences does indicate that this is a non-specific treatment effect on foetal growth and development rather than a direct effect upon the development of a body system. Although there was no clear correlation of lower ossification to smaller/lighter fetuses, the higher number of fetuses per litter observed in the high dose group is considered to have contributed to these findings.

At 300 mg/kg bw/day, lower mean foetal weight was evident and skeletal examinations showed that development had been slightly delayed as dosage related trends could be observed for the same skeletal parameters as has been seen at 1000 mg/kg bw/day albeit to a lesser extent.

No effect on post implantation losses were evident at 1000 or 300 mg/kg bw/day therefore the test item does not affect embryo lethality.

No effects were evident at 30 mg/kg bw/day.

The study is considered to be reliable with restrictions as TEGME (2-(2-(2-methoxyethoxy)ethoxy)ethanol, CAS 112-35-6) has been used as vehicle in concentrations from 3200 mg/kg bw/d (high dose group) to 4200 mg/kg bw/d (control/vehicle group). Developmental toxicity experiments conducted with TEGME indicate developmental effects at doses > 1,000 mg/kg/day. Effects observed in offspring from rats treated with 1,250 mg/kg/day TEGME or rabbits treated with 1,500 mg/kg/day TEGME during gestation included skeletal variations and decreased body weight gain (see attached OECD SIDS document for TEGME, CAS 112-35-6; August 2005). Therefore, the vehicle TEGME might have masked maternal toxicity in this study and/or might have contributed to the observed developmental effects (slight delay in development).

Shell, SBGR.92.233, 1993

A screening developmental toxicity was performed in rats with a brake fluid containing 17% B-TEGME. Doses administered were 4, 25 and 170 mg B-TEGME/kg bw/day, based upon doses of 25, 150 and 1000 mg brake fluid/kg bw/day. This study was applied according to a Charnoff & Kavlock (CKA) design and in compliance with GLP, which was considered to be adequate, reliable and relevant. This study can therefore support further developmental toxicity testing. Dams were allowed to litter and the pups observed and weighed on days 1 and 5 post-partum, at which point the study was terminated. As there were no skeletal and visceral examinations in this study, it was considered as a supporting study for a final prenatal developmental toxicity study in rats. In this screening study, no treatment related effects on birth, live birth, or viability indices were found, neither were there treatment-related clinical effects on dams or pups, or gross abnormalities seen in the pups. The NOAEL for this study was at least 170 mg B-TEGME /kg bw/day based on the NOAEL of 1000 mg brake fluid/kg bw/day. It must be taken into consideration that the brake fluid also contained other borated and non-borated glycol ethers and other components.

Justification for classification or non-classification

The developmental effects observed in a prenatal developmental toxicity study in rats with B-TEGME and TEGME (slight delay in development) are not considered to be sufficient to classify B-TEGME for developmental toxicity. Furthermore, the vehicle TEGME might have masked maternal toxicity and/or might have contributed to the observed developmental effects (slight delay in development). Therefore, the results of this study are considered inconclusive for classification.

The findings of a prenatal developmental toxicity study in rabbits demonstrate clear maternal toxicity at the high dose tested (i.e. 500 mg/kg bw/d): B-TEGME caused death or abortion in 3 females immediately before term (GD 28/29), and corroborative necropsy findings indicating enteropathy. Furthermore, lower food consumption (7% below control) was observed at this dose level. Fetal examinations revealed that there was no adverse effect of B-TEGME on the respective morphological structures up to the mid-dose tested (250 mg/kg bw/d). In the high-dose group (500 mg/kg bw/d), several fetuses from different litters were affected by visceral (multiple malformations of urogenital tract) or skeletal (malformations of axial skeleton) malformations, some of which exceeded their historical control ranges. In addition, significantly increased incidences of external, skeletal and cartilage variations, all above the historical control range, were noted. Although the total of these findings caused a significant increase of overall malformations and variations in the high-dose group (500 mg/kg bw/d), the particular values were still within the historical control range of the test facility.

Conclusion: As in a prenatal developmental toxicity study in rabbits for the high-dose (500 mg/kg bw/d) the collective spectrum of observed anomalies may suggest beginning developmental toxicity, but the effects were observed only at a dose level which caused toxicity (including lethality) in maternal animals, classification with Repro. 2, H361d under Regulation (EC) No. 1272/2008 is warranted.